CHANGES IN THE ENDOCANNABINOID YSTEM

CHANGES IN THE ENDOCANNABINOID SYSTEM

IN THE BRAIN FOLLOWING BILATERAL VESTIBULAR DAMAGE

Jean-Ha Baek

A thesis submitted for the degree of

Doctor of Philosophy

at the University of Otago, Dunedin

New Zealand.

December 2010

ii

Dedication iii

To my parents,

who are my mentors and my inspiration

Fear of the LORD is the beginning of wisdom.

Knowledge of the Holy One results in understanding.

<Proverbs 9:10>

Abstract iv

ABSTRACT

Numerous studies have shown that bilateral vestibular deafferentation (BVD) results in

spatial memory deficits and hippocampal dysfunction in rats and humans. These deficits

appear to be long-lasting, suggesting long-term adverse effects on the hippocampus, a brain

region known for its role in learning and memory. Interestingly, the endocannabinoid system

has also been demonstrated to have a role in modulating hippocampal synaptic

neurotransmission and cognitive function. Not only are the cannabinoid receptors highly

expressed in the hippocampus, the activation of these receptors by either the endogenous

ligands (that is, endocannabinoids) or exogenous cannabinoid drugs results in the impairment

in a variety of learning and memory tasks. As it has been demonstrated that vestibular damage

causes learning and memory deficits and that the endocannabinoid system is critically

involved in the mechanism of learning and memory, the aim of this study was to investigate

whether the hippocampal endocannabinoid system plays a role in the functional changes seen

after vestibular damage.

By manipulating the available cues in the foraging task, 14 months post-BVD animals

were unable to use available visual cues and execute piloting as their navigating strategy. In

addition, their spatial navigation ability became worse in darkness. Importantly, it appears that

these cognitive impairments due to the vestibular damage are highly likely to be permanent. It

was expected that the administration of the cannabinoid receptor agonist would exacerbate the

observed spatial memory deficits in the BVD animals as it has been shown to have disruptive

effects on spatial learning and memory in both animals and humans. In the present study, it

was found that 1 mg/kg WIN55212-2 (WIN), a cannabinoid receptor agonist, was sufficient to

produce object recognition memory impairments in adult rats. However, when the same drug

was given to both the BVD and the sham animals, its effect on spatial memory was complex.

It was shown that WIN, at 2 mg/kg, significantly improved the performance in the dark in

BVD, but not sham animals, suggesting that BVD might have resulted in changes in the

brain‟s endocannabinoid system. However, the pre-treatment with AM251, a cannabinoid

receptor inverse agonist, further indicated the complexity of involvement of the

endocannabinoid system in the cognitive deficits following BVD.

Since cannabinoid cannabinoid 1 (CB1) receptors are well known to regulate synaptic

plasticity in the hippocampus, whether BVD resulted in changes in CB1 receptor expression

Abstract v

and affinity in the rat hippocampus at 1, 3 and 7 days post-surgery was investigated, using a

combination of western blotting and radioligand binding. It was found that the CB1 receptor

down-regulates in the CA3 region of the hippocampus following BVD, but with no changes

in the affinity of the CB1 receptor for WIN.

Overall, the results from the present study suggest that cognitive deficits following

BVD may be permanent and provide some preliminary evidence for future research on the

role of the endocannabinoid system in the observed cognitive deficits following vestibular

damage in order to further elucidate the potential mechanism(s) that may underlie this

impairment.

Acknowledgements vi

ACKNOWLEDGEMENTS

Throughout the course of this PhD study, I have been very fortunate to have so many

people who have guided me, and have enabled me to reach where I am now. I am forever

grateful for their encouragement and support.

Most of all, I would like to give my sincere gratitude to each of my supervisors, Prof.

Paul Smith, Dr. Yiwen Zheng, and Assoc. Prof. Cynthia Darlington. Aristotle once said,

“Those who wish to succeed must ask the right preliminary questions.” Each of my

supervisors has taught me exactly that – how to ask the right questions at in order to reveal

what is unknown.

I thank Paul deeply for being a fantastic supervisor and a good mentor. Paul was always

been there when I needed guidance. Paul has shown me how to approach research problems in

different ways and how to be both critical and philosophical about my work. Paul‟s

enthusiasm towards research will always serve as a reminder to me of how to enjoy science. I

also thank Paul for his ability to pick out the difference between when to use a comma and

when to use a semi-colon. His help in editing my writing has been tremendous. Huge thanks

are also due to Yiwen, whose contribution has been more than I can write on this page. She

has taught me almost all of my experimental skills, and she is an amazing teacher. Her depth

of knowledge is beyond measure. Yiwen always had answers to my “inexperienced” questions,

whether they were science-related or not. Thank you also to Cynthia, who has been a warm

influence during my PhD. She always had confidence in me when I doubted myself, and

brought out the good things in me. Cynthia has also taught me how to indulge myself in

research while keeping everything “cool”.

I am extremely thankful to Dr. Lisa Geddes who has helped me enormously to tie the

individual pieces together to make a “story” out of this thesis. I cannot imagine what it would

have been like without her objective and critical inputs towards this work. I am thankful that I

had a chance to get to know her in the last few months of PhD. Many thanks also to my PhD

buddy, Sangeeta Balabhadrapatruni, for her Tender Loving Care and for being a good listener.

I am very grateful for our friendship. Thank you also to my wonderful colleagues in the

Smith/Darlington/Zheng research group (also known as The Centre of Excellence in

Vestibular Research); Shaeza Begum, Irene Cheung, Emma Hamilton, Emily McNamara,

Acknowledgements vii

Lucy Stiles, Shweta Vagal, Georgina Wilson, and Jun Yoon for making my life in the lab

joyful and exciting. Many thanks are due to everyone who has contributed towards this work

in their own special ways, including people in the Department of Pharmacology and

Toxicology, many friends from Korean Evergreen Church in Dunedin, and from Saeronam

Church in Korea.

Sincere thanks to Dad, who has given me priceless advice on all of my experiments and

on my attitude towards scientific research. Despite his unexpected illness, he continued to

encourage me to do my best. Dad has shown me how to persevere in tough times, to be

humble, and to work diligently. Not only has he been a perfect Dad but also an amazing

scientist. Although God took Dad away towards the very end of this study, I know from the

bottom of my heart that God still loves me and that His amazing grace will continue to be

with me as it has been throughout the course of this study. Many thanks to Mum for all her

prayers, and for always giving me hope and strength to carry on with this study. I also thank

my brother for our valuable and memorable times that we had together in Dunedin.

List of Publications viii

LIST OF PUBLICATIONS

1) Baek, J.H., Zheng, Y., Darlington, C.L. & Smith, P.F. (2008) Cannabinoid CB2 receptor

expression in the rat brainstem cochlear and vestibular nuclei. Acta Otolaryngol, 128,

961-967.

2) Baek, J.H., Zheng, Y., Darlington, C.L. & Smith, P.F. (2009) The CB1 receptor agonist,

WIN 55,212-2, dose-dependently disrupts object recognition memory in adult rats.

Neurosci Lett, 464, 71-73.

3) Baek, J.H., Zheng, Y., Darlington, C.L. & Smith, P.F. (2010) Evidence that spatial memory

deficits following bilateral vestibular deafferentation in rats are probably permanent.

Neurobiol Learn Mem, 94, 402-413.

4) Baek, J.H., Zheng, Y., Darlington, C.L. & Smith, P.F. (2010, in press) Cannabinoid CB1

receptor expression and affinity in the rat hippocampus following bilateral vestibular

deafferentation. Neurosci Lett.

5) Smith, P.F., Geddes, L.H., Baek, J.-H., Darlington, C.L., and Zheng, Y. (2010) Modulation

of memory in humans by vestibular lesions and galvanic vestibular stimulation. Front

Neurology, 1(141): 1-8 (doi: 10.3389/fneur.2010.00141).

Table of Contents ix

TABLE OF CONTENTS

Dedication ······································································································································ iii

ABSTRACT ······································································································································ iv

ACKNOWLEDGEMENTS·················································································································· vi

LIST OF PUBLICATIONS················································································································ viii

TABLE OF CONTENTS ····················································································································· ix

LIST OF FIGURES AND TABLES ···································································································· xiv

LIST OF ABBREVIATIONS ·············································································································· xx

CHAPTER 1: INTRODUCTION ········································································································· 1

1.1 THE VESTIBULAR SYSTEM ······························································································· 3

1.1.1 Anatomical Structure and Function ································································· 3

1.1.2 Vestibular Pathways ························································································ 7

1.1.3 Peripheral Vestibular Deafferentation ····························································· 9

1.1.4 Vestibular-Hippocampal Connections ··························································· 11

1.2 THE ENDOCANNABINOID SYSTEM ·················································································· 19

1.2.1 Cannabinoid Receptors and Endocannabinoids ············································ 19

1.2.2 Neuromodulatory Role of the Endocannabinoid System ································· 25

1.2.3 The Role of Endocannabinoid System in Cognitive Function ························· 27

1.3 THE ASSOCIATION BETWEEN THE VESTIBULAR AND ENDOCANNABINOID SYSTEMS ······ 30

1.4 RESEARCH GOALS ········································································································· 31

CHAPTER 2: THE EFFECT OF THE CANNABINOID RECEPTOR AGONIST, WIN 55212-2, ON

OBJECT RECOGNITION MEMORY IN RATS ················································································· 32

2.1 INTRODUCTION ·············································································································· 33

2.2 MATERIALS AND METHODS···························································································· 36

2.2.1 Animals ········································································································· 36

2.2.2 Drugs ············································································································ 36

2.2.3 Object Recognition Memory Test ··································································· 36

2.2.4 Statistical Analysis ························································································ 37

Table of Contents x

2.3 RESULTS························································································································· 39

2.3.1 Total Exploration Time ·················································································· 39

2.3.2 Exploration Time by Objects ········································································· 40

2.3.3 Discrimination Index ····················································································· 41

2.4 DISCUSSION···················································································································· 42

CHAPTER 3: THE EFFECT OF CANNABINOIDS ON SPATIAL MEMORY FOLLOWING BILATERAL

VESTIBULAR DEAFFERENTATION IN RATS ················································································· 44

3.1 INTRODUCTION ·············································································································· 45


3.2.1 Animals ········································································································· 49

3.2.2 Peripheral Vestibular Lesion Surgery ···························································· 49

3.2.3 Drugs ············································································································ 50

3.2.4 Foraging Task Apparatus ·············································································· 50

3.2.5 Foraging Task Procedure ·············································································· 53

3.2.6 Data Acquisition ··························································································· 54

3.2.7 Statistical Analysis ························································································ 56

3.3 RESULTS························································································································· 58

3.3.1 General Behavioural Observations ······························································· 58

3.3.2 Pre-Training ································································································· 58

3.3.3 Light Probe Trial ··························································································· 60

3.3.4 Dark Training ······························································································· 64

3.3.5 AM251 Treatment ·························································································· 78

3.4 DISCUSSION···················································································································· 82

CHAPTER 4: CHANGES IN CB1 RECEPTOR DENSITY AND AFFINITY FOLLOWING BILATERAL

VESTIBULAR DEAFFERENTATION IN RATS ················································································· 93

4.1 INTRODUCTION ·············································································································· 94


4.2.1 Western Blotting

Table of Contents xi

4.2.1.1 Animals ······················································································· 97

4.2.1.2 Tissue Collection ········································································· 97

4.2.1.3 Tissue Homogenisation ································································ 97

4.2.1.4 Bradford Assay ············································································ 97

4.2.1.5 Western Blot ················································································ 98

4.2.1.6 Data Acquisition ········································································ 100

4.2.1.7 Statistical Analysis ····································································· 101

4.2.2 Radioligand Binding Assay

4.2.2.1 Animals ····················································································· 101

4.2.2.2 Cannabinoid Ligands ································································ 101

4.2.2.3 Membrane Preparation ······························································ 102

4.2.2.4 [3H]-CP 55,940 Binding Assay ·················································· 103

4.2.2.5 Data Acquisition and Statistical Analysis ··································· 104

4.3 RESULTS······················································································································· 105


4.3.1.1 Optimum Sample Amount ·························································· 105

4.3.1.2 CB1 Receptor Antibody Specificity ············································· 106

4.3.1.3 Changes in CB1 Receptor Protein Density ································· 106


4.3.2.1 Optimisation of the Protein Amount and Radioligand Concentration

································································································· 108

4.3.2.2 Changes in the CB1 Receptor Binding Affinity ··························· 109

4.4 DISCUSSION···················································································································110

CHAPTER 5: DISCUSSION AND CONCLUSIONS ···········································································116

APPENDICES ································································································································ 123

APPENDIX 1: THE SPECIFICITY OF CANNABINOID CB1 RECEPTOR LABELLING IN THE RAT

HIPPOCAMPUS ····························································································································· 124

1.1 INTRODUCTION ············································································································ 124

1.2 MATERIALS AND METHODS·························································································· 127

1.2.1 Removal of Brain ························································································ 127

1.2.2 Tissue Sectioning························································································· 127

Table of Contents xii

1.2.3 Immunohistochemistry ················································································ 128

1.2.4 Double Label Immunofluorescence ····························································· 129

1.2.5 Data Acquisition ························································································· 130

1.3 RESULTS······················································································································· 131

1.3.1 Optimisation of the CB1 Receptor Primary Antibody ··································· 131

1.3.2 Optimisation of the Secondary Antibody ······················································ 133

1.3.3 Double Labelling Immunofluorescence························································ 135

1.4 DISCUSSION·················································································································· 137

APPENDIX 2: CHANGES IN CB1 RECEPTOR EXPRESSION FOLLOWING BILATERAL

VESTIBULAR DEAFFERENTATION IN RATS ··············································································· 139

2.1 INTRODUCTION ············································································································ 139


2.2.1 Animals ······································································································· 142



2.2.4 Statistical Analysis ······················································································ 142

2.3 RESULTS······················································································································· 143

2.3.1 Spatial Distribution of the CB1 Receptors in the Hippocampus ···················· 143

2.4 DISCUSSION·················································································································· 146

APPENDIX 3: THE SPECIFICITY OF CANNABINOID CB2 RECEPTOR LABELLING IN THE RAT

HIPPOCAMPUS ····························································································································· 147

3.1 INTRODUCTION ············································································································ 147


3.2.1 Animals ······································································································· 147

3.2.2 Western Blotting ·························································································· 147


3.2.4 Double Label Immunofluorescence ····························································· 152


Table of Contents xiii

3.3 RESULTS······················································································································· 154

3.3.1 Western Blotting ·························································································· 154

3.3.2 Immunohistochemical Analysis of the CB2-Specific Antibody······················· 154

3.3.3 Double Labelling Immunofluorescence························································ 158

3.4 DISCUSSION·················································································································· 161

REFERENCES ······························································································································· 163

The New Beginning···················································································································· 206

List of Figures and Tables xiv

LIST OF FIGURES AND TABLES

CHAPTER 1: INTRODUCTION

Figure 1.1 Venn diagram showing the possible association between the vestibular and

endocannabinoid systems, together with their discrete involvement in cognitive function and

synaptic plasticity ·················································································································· 2

Figure 1.2 A schematic representation of the structure of the vestibular labyrinth with its

innervating vestibular and cochlear nerves ············································································· 4

Figure 1.3 A schematic representation of the structure and organisation of different types of

hair cells in the vestibular sensory epithelium ········································································ 6

Figure 1.4 Simplified diagram representing the organisation of the key areas and pathways

for the production of VORs and VSRs ··················································································· 9

Figure 1.5 Some potential vestibulo-hippocampal connections ············································ 14

Table 1.1 Neurochemical changes in the hippocampus and in the neocortex following

vestibular damage ················································································································ 16

Figure 1.6 Schematic diagram of the synthesis, signalling, and degradation of

endocannabinoids AEA and 2-AG at the synaptic cleft ························································· 24

CHAPTER 2: THE EFFECT OF THE CANNABINOID RECEPTOR AGONIST, WIN 55212-2, ON

OBJECT RECOGNITION MEMORY IN RATS

Figure 2.1 The object recognition memory testing conditions for each day ·························· 38

Figure 2.2 Total exploration time (in seconds) for session 1 (before drug administration) and

session 2 (after drug administration) for the vehicle treated group and groups receiving 1, 3 or

5 mg/kg WIN ······················································································································· 39

Figure 2.3 Mean exploration time (in seconds) for familiar and novel objects for the vehicle-

treated group and the groups receiving 1, 3 or 5 mg/kg WIN ················································ 40

Figure 2.4 The discrimination indices for the vehicle-treated group and the groups receiving 1,

List of Figures and Tables xv

3 or 5 mg/kg WIN ················································································································ 41

CHAPTER 3: THE EFFECT OF CANNABINOIDS ON SPATIAL MEMORY FOLLOWING BILATERAL

VESTIBULAR DEAFFERENTATION IN RATS

Figure 3.1 The foraging task apparatus ················································································ 52

Figure 3.2 The display window of the custom-coded path tracing software on a PC············· 55

Figure 3.3 Schematic diagram of measuring the initial heading angle ·································· 55

Figure 3.4 Number of pre-training sessions required for the sham and BVD animals to reach

the foraging task criterion ···································································································· 58

Figure 3.5 Searching and homing time of the sham and BVD animals in the last 6 days of the

pre-training ·························································································································· 59

Figure 3.6 First and second home choices of the light probe trial ········································· 60

Figure 3.7 Foraging task parameters displayed by sham-vehicle, sham-WIN, BVD-vehicle,

and BVD-WIN animals in the light probe trial ····································································· 61

Figure 3.8 The velocity comparison between searching and homing in the light probe trial · 62

Figure 3.9 The average number of visits to the old home, and the average number of errors

made before reaching the correct home by sham-vehicle, sham-WIN, BVD-vehicle, and

BVD-WIN animals in the light probe trial ············································································ 63

Figure 3.10 Linear graph of the searching distance (cm) for different treatment groups during

the dark training and the AUC of the searching distance for the sham and BVD animals to

show the effect of surgery, dose and the interactions····························································· 64

Figure 3.11 Linear graph of the searching time (sec) for different treatment groups during the

dark training and the AUC of the searching time showing the effect of surgery, treatment, and

dose ····································································································································· 65

Figure 3.12 Linear graph of the searching velocity (cm/sec) for different treatment groups

during the dark training and the AUC of the searching velocity showing the effect of surgery,

treatment, and dose ·············································································································· 66

List of Figures and Tables xvi

Figure 3.13 An example of the homeward path taken by a sham and a BVD rat in the dark

training ································································································································ 67

Figure 3.14 Linear graph of the homing distance (cm) for different treatment groups during

the dark training and the AUC of the homing distance showing the effect of surgery, treatment,

and dose ······························································································································· 68

Figure 3.15 Linear graph of the homing time (sec) for different treatment groups during the

dark training and the AUC of the homing time showing the effect of surgery, treatment, and

dose ····································································································································· 69

Figure 3.16 Linear graph of the homing velocity (cm/sec) for different treatment groups

during the dark training and the AUC of the homing velocity showing the effect of surgery,

treatment, and dose ·············································································································· 70

Figure 3.17 The velocity comparison between searching and homing during the dark training..

············································································································································ 71

Figure 3.18 Rose diagram showing the initial heading angles of the sham and BVD animals

for the dark training ············································································································· 72

Figure 3.19 Initial heading angles of animals in different treatment groups for 1 and 2 mg/kg

WIN treatment ····················································································································· 73

Figure 3.20 First and second home choices of the sham and BVD animals with 1 and 2 mg/kg

WIN treatment in the dark training ······················································································· 74

Figure 3.21 Linear graph of the number of errors made before reaching the correct home by

animals in different treatment groups and the AUC of the number of errors for the sham and

BVD animals to show the effect of surgery, drug treatment, dose, and their interactions ······· 75

Figure 3.22 The number of animals that completed the task during the dark training ··········· 76

Figure 3.23 Simple regression analysis performed to predict the number of errors made by

vehicle-treated sham and BVD animals or by all of the sham and the BVD animals from their

searching velocities ·············································································································· 77

Figure 3.24 Foraging task parameters displayed by sham-vehicle, sham-WIN, BVD-vehicle,

and BVD-WIN animals with AM251 treatment on day 21 of the dark training ····················· 79

List of Figures and Tables xvii

Figure 3.25 The velocity comparison between searching and homing with AM251 treatment

on day 21 of the dark training ······························································································· 78

Figure 3.26 First and second home choices of the BVD and sham animals on day 21 of the

dark training with the treatment of AM251 prior to vehicle or WIN administration ·············· 80

Figure 3.27 Initial heading angles of vehicle- or WIN-treated animals on day 21 of the dark

training with the treatment of AM251 prior to vehicle or WIN administration ······················ 81

Figure 3.28 The graph showing the average number of errors made before reaching the

correct home by the sham-vehicle, sham-WIN, BVD-vehicle, and BVD-WIN animals on day

21 of the dark training ·········································································································· 81

CHAPTER 4: CHANGES IN CB1 RECEPTOR DENSITY AND AFFINITY FOLLOWING BILATERAL


Figure 4.1 The CB1 receptor band intensity increased as the amount of sample protein

increased. This change can be visualised in the western blots for the CB1 receptor and its

corresponding actin ············································································································ 105

Figure 4.2 Western blot images showing the concerning lack of specificity of the CB1

receptor antibody for CB1 receptors ··················································································· 106

Figure 4.3 The levels of CB1 receptor protein in the CA1, CA3, DG of the hippocampus at 1,

3, and 7 days following BVD compared to the sham controls ············································· 107

Figure 4.4 Saturation binding curve with increasing amount of hippocampal membrane

preparation for different concentrations of [3H]-CP 55,940. Specific binding of [

3H]-CP

55,940 was calculated from the difference between total and non-specific binding ············· 108

Figure 4.5 Displacement curves of the [3H]-CP 55,940 binding with WIN as a competitive

inhibitor and the IC50 values of WIN at 1 day, 3 days, and 7 days following sham or BVD

surgery ······························································································································· 109

APPENDIX 1: THE SPECIFICITY OF CB1 RECEPTOR LABELLING IN THE RAT HIPPOCAMPUS

Table 1.1 Various combinations of CB1 receptor primary and subsequent secondary antibody

List of Figures and Tables xviii

titres trialled and their incubation durations ········································································ 129

Figure 1.1 CB1 receptor labelling with different primary antibody incubation durations ···· 131

Figure 1.2 CB1 receptor labelling with different primary antibody concentrations·············· 132

Figure 1.3 CB1 receptor labelling with different secondary antibody incubation durations · 133

Figure 1.4 CB1 receptor labelling with different secondary antibody concentrations ·········· 134

Figure 1.5 Double labelling immunofluorescence of the CB1 receptor antibody with an

antibody for NeuN, in the hippocampus ············································································· 135

Figure 1.6 Immunohistochemical labelling of the CB1 receptor in axons and dendrites in the

stratum oriens and DG regions of the hippocampus ···························································· 136

APPENDIX 2: CHANGES IN CB1 RECEPTOR EXPRESSION FOLLOWING BILATERAL


Figure 2.1 Representative immunohistochemical sections of the CB1 receptor labelling in the

hippocampus of a sham and a BVD animal ········································································ 143

Figure 2.2 The spatial distribution of the CB1 receptors across the CA1, CA3, and DG of the

hippocampus at 1, 3, and 7 days following BVD compared to the sham controls ················ 144

Figure 2.3 The AUC of the spatial distribution of CB1 receptors in the dorsal and ventral

hippocampus at 1, 3, and 7 days following BVD compared to the sham controls ················ 145

APPENDIX 3: THE SPECIFICITY OF CB2 RECEPTOR LABELLING IN THE RAT HIPPOCAMPUS

Figure 3.1 A CB2 receptor diagram showing the locations of different commercial and

privately made CB2 receptor primary antibodies targeting different epitopes of the CB2

receptor protein ·················································································································· 150

Table 3.1 Various combinations of CB2 receptor primary (1o) and subsequent secondary (2

o)

antibody titres trialled and their incubation durations ························································· 152

Figure 3.2 Western blots of hippocampal tissue (Hp1 and Hp2) and spleen (sp, as a positive

List of Figures and Tables xix

control), using the Santa Cruz and Abcam CB2 receptor antibodies ···································· 154

Figure 3.3 Immunohistochemical comparison of CB2 receptor labelling with different

primary antibody concentrations in the dentate gyrus (DG) of the rat hippocampus ············ 155


primary antibody concentrations in the DG of the rat hippocampus ···································· 156


secondary antibody concentrations in the DG of the rat hippocampus································· 156


antibody incubation durations in the DG of the rat hippocampus ········································ 157

Figure 3.7 Immunohistochemical labelling of CB2 receptors in the hippocampus ·············· 158

Figure 3.8 Double labelling immunofluorescence of the CB2 receptor antibody with a NeuN

antibody, a neuronal marker in various regions of the hippocampus ··································· 159

Figure 3.9 Immunofluorescent labelling showing the distribution of the CB2 receptors in the

CA3 region of the hippocampus using antibodies from the Abcam (rabbit polyclonal) and

Santa Cruz (goat polyclonal) ······························································································ 160

List of Abbreviations xx

LIST OF ABBREVIATIONS

Abbreviation Explanation

2-AG 2-arachidonyl glycerol

ADN Anterodorsal nucleus

AEA Anandamide

AMPA -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

ANOVA Analysis of variance

APS Ammonium persulfate

AUC Area under the curve

BSA Bovine serum albumin

BVD Bilateral vestibular deafferentation

CA1 Cornu Ammonis area 1

CA3 Cornu Ammonis area 3

cAMP Cyclic adenosinemonophosphate

CB1 receptor Cannabinoid 1 receptor

CB2 receptor Cannabinoid 2 receptor

cDNA Complimentary deoxyribonucleic acid

CNS Central nervous system

CT Computed tomography

DAB Diaminobenzidine

DAG Diacylglycerol

DAGL Diacylglycerol lipase

DG Dentate gyrus

dH2O Distilled water

DMS Delayed matching to sample

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DNMS Delayed nonmatch to sample

DSE Depolarisation-induced suppression of excitation

DSI Depolarisation-induced suppression of inhibition

DTN Dorsal tegmental nucleus

EC Entorhinal cortex

List of Abbreviations xxi

EEG Electroencephalography

EMT Endocannabinoid membrane transporter

FAAH Fatty acid amide hydrolase

GABA -aminobutyric acid

GLM General linear model

GPCR G-protein coupled receptor

HRP Horse radish peroxidase

IAC Internal auditory canal

IC50 Half maximal inhibitory concentration

IPSC Inhibitory postsynaptic current

LMM Linear mixed model

LMN Lateral mammillary nucleus

LTD Long-term depression

LTP Long-term potentiation

MAGL Monoacylglycerol lipase

mGluR Metabotropic glutamate receptor

MRI Magnetic resonance imaging

NADA N-arachidonoyl-dopamine

NAPE-PLD N-acyl phosphatidyl ethanolamine phospholipase D

NArPE N-arachidonoyl-phosphtidyl ethanolamine

NF-L Neurofilament-L

NMDA N-methyl-D-aspartate

NOS Nitric oxide synthase

PLC Phospholipase C

PPTN Pedunculopontine tegmental nucleus

PS Post-subiculum

RNA Ribonucleic acid

SDS Sodium dodecylsulfate

SNAP-25 Synaptosome-associated protein of 25 kDa

SOR Spontaneous object recognition

SUM Supramammillary nucleus

TEMED N,N,N‟,N‟-tetremethylethylenediamine

UVD Unilateral vestibular deafferentation

VCR Vestibulocollic reflex

VNC Vestibular nucleus complex

List of Abbreviations xxii

VOR Vestibulo-ocular reflex

VSR Vestibulospinal reflex

WIN WIN 55212-2

o C Degrees Celsius

9-THC Delta-9-tetrahydrocannabinol

1

Chapter 1

Introduction

Chapter 1: Introduction 2

The vestibular system in the inner ear is the sensory system primarily responsible for

sensing self-motion and for stabilising the eyes and the body. It has been reported that damage

to the vestibular system leads to debilitating psychological and emotional problems as well as

cognitive impairments in humans and animals. Furthermore, significant synaptic changes

occur following vestibular damage in the brain areas that are involved in processing vestibular

information. As it has been demonstrated that the endocannabinoid system is critically

involved in synaptic plasticity and in cognitive function, this thesis examines the role of the

endocannabinoid system in the brain following vestibular damage, and investigates neural

mechanisms that may underlie the debilitating effects of vestibular damage (Figure 1.1).

Figure 1.1

Venn diagram showing the possible association between the vestibular and endocannabinoid

systems, together with their discrete involvement in cognitive function and synaptic plasticity.

Synaptic Plasticity

&

Cognitive Function

Endocannabinoid

System

Vestibular

System

?


1.1 The Vestibular System

Unlike other sensory systems, the functions and sensations of the vestibular system are

not prominent, readily recognisable, or localisable in our consciousness. However, the

influence of the vestibular system is crucial for almost every aspect of human daily activities

and „normal‟ behaviour. The physiological significance of this “hidden” sensory system was

uncovered by early animal experimental pioneers such as Flourens, Goltz, Stefani, Breuer,

Mach, von Cyon, Ewald, Magnus, Ménière, and Bárány. They demonstrated that surgical

ablation and mechanical stimulation of the vestibular apparatus led to a dramatic and often

peculiar disorganisation of posture and movement (Choi et al., 2007; Heskin-Sweezie et al.,

2010; Parietti-Winkler et al., 2010; see Horak, 2009 for review).

Stimulation of the vestibular system can be achieved by linear or angular accelerations

of the whole body. The vestibular system receives information about the movement and the

position of the head in space, in order to maintain head and body posture (Peterson and

Richmond 1988) and to stabilise gaze during walking and running (Grossman et al., 1988,

1989). Head movements in space are often a complex combination of angular rotation and

linear translation. Nevertheless, the vestibular system has evolved to sense changes in angular

and linear velocity in all three dimensions, and allows the brain to receive exact information

about head movement and position even without visual input in most organisms, including in

humans (see Angelaki and Cullen, 2008; Angelaki et al., 2009 for reviews).

1.1.1 Anatomical Structure and Function

In order to identify anomalies under conditions of vestibular dysfunction, it is necessary

to understand the normal structure and function of the vestibular system. The vestibular

system is composed of three components: a peripheral sensory apparatus, a central processor,

and a mechanism for motor output. The peripheral apparatus consists of a set of acceleration

sensors that together are known as the vestibular labyrinth, which sends information to the

central nervous system (CNS) about head angular and linear acceleration, and orientation of

the head with respect to gravity. The major role of the peripheral vestibular apparatus is to

signal the brain about the acceleration of the head or changes in gravitational forces, so that

related muscle groups are activated to produce movements of the body and eyes to allow the

organism to adapt to maintain appropriate posture and balance (see Cohen and Keshner, 1989;

Keshner and Cohen, 1989 for reviews).


The vestibular labyrinth is located lateral and posterior to the cochlea in the inner ear,

and is contained in the petrous portion of the temporal bone. The peripheral vestibular system

has two different types of detectors: the semicircular canals and the otoliths (Figure 1.2). The

three semicircular canals (the horizontal, anterior, and posterior canals), detect primarily

angular acceleration of the head in space and the two otolith organs (the utricle and the

saccule), have a unique role in registering changes in gravitational force or linear acceleration

by gravity (see Kondrachuk, 2001 for review).

Figure 1.2

A schematic representation of the structure of the vestibular labyrinth (purple) with its

innervating vestibular and cochlear nerves (orange). The illustration has been modified from

Glover (2009), with permission from Elsevier.

It is often assumed that the three semicircular canals lie at right angles to one another

and that the left and right sets of canals represent true mirror images of each other on either

side of the sagittal plane. However, a series of anatomical studies by Markham and colleagues

has shown that neither of these assumptions is true (Blanks et al., 1972, 1975; Curthoys et al.,

1975, 1977). Furthermore, with the advances in high-resolution computed tomography (CT)

and magnetic resonance imaging (MRI), it has become possible to obtain accurate knowledge

of the absolute orientation and position of the semicircular canals (Della Santina et al., 2005;

Lane et al., 2008; Bradshaw et al., 2010). When the horizontal canal in humans is positioned

so that it is parallel to the earth‟s horizontal, the anterior canal is positioned at an angle of

approximately 110 degrees with respect to the horizontal canal while the posterior canal is at

an angle of approximately 95 degrees. In addition, the angles between semicircular canals


vary between individuals and species (see Wilson and Melvill Jones, 1979).

Each semicircular canal is comprised of a bony and membranous duct. Within the bony

labyrinth, membranous ducts and sacs are separated from the bony part by a fluid called

perilymph. In all three canals, the membranous duct opens into a sac known as the utricle, and

each canal together with the utricle forms a closed ring in which the second fluid, called

endolymph, circulates. Each semicircular canal is widened at one end to form an ampulla,

where the hair cells and their supporting cells are located in a crista. Angular acceleration in

the plane of the canal creates inertial force that acts on the endolymph circulation around the

canal, which in turn induces physical displacement of the stereocilia of the hair cells in the

same direction, stimulating the hair cells. As the membranous labyrinths are anchored within

the otic capsule, the displacement of the hair cells accurately corresponds to the acceleration

of the skull at all times (see Wilson and Melvill Jones, 1979).

The macula of the two otolith organs is a structure analogous to the crista in the

semicircular canals. The utricular macula is horizontally oriented along the base of the utricle,

and is primarily responsible for transduction of earth-horizontal linear acceleration in any

direction, and registers static tilt with respect to gravity. Beneath the utricle lies the saccule,

containing the saccular macula. This component has a similar role to that of the utricular

macula, but is arranged in an approximately orthogonal plane on the medial wall of the

saccule to detect acceleration in the vertical plane (Buttner-Ennever, 1999; Curthoys et al.,

2009).

Hair cells in the otolith organs are organised in a similar way to the ampulla except for

the presence of calcium carbonate crystals, the otoconia, above the otolithic membrane. In the

utricle and saccule, the otolithic membrane covers the stereocilia of the hair cells. The relative

displacement of the otoconia, by linear acceleration or the gravity-induced inertial force

acting in the plane of the macula, causes the stereocilia to bend. This mechanical deflection of

the cilia stimulates the sensory hair cells, which in turn generates trains of action potentials in

the primary afferent nerve fibres (see Wilson and Melvill Jones, 1979).

There are two morphologically different types of hair cells in the vestibular sensory

epithelium (Figure 1.3). The type I cells are flask-shaped and are surrounded by a single large

primary afferent nerve terminal in the form of a nerve chalice. The type II cells are more

cylindrically shaped, and are innervated by a series of bouton-type nerve terminals at their


base. These sensory cells are called hair cells due to the stereocilia that project from the apical

surface of the cell. They are found in the cristae at the ampullary end of the semicircular

canals, and in the maculae of the utricle and saccule (see Zajonc and Roland, 2005). The

bundle of stereocilia found on the top of these hair cells is arranged in a highly systematic

manner. The stereocilia are arranged in rows of increasing height, where the tallest

stereocilium lies next to a single cilium of a different kind, the kinocilium (Figure 1.3). The

circulation of the endolymph in the semicircular canals, and the otoconial movement in the

case of the maculae, causes the mechanical bending of the stereocilia of the hair cells, which

in turn is translated into a graded electrical potential. These graded electrical potentials in the

hair cells are then translated into action potentials that propagate down the afferent nerves

towards the vestibular nuclei (see Wilson and Melvill Jones, 1979).

Figure 1.3

A schematic representation of the structure and organisation of different types of hair cells in

the vestibular sensory epithelium. The illustration has been adapted from Meyers et al. (2009),

with permission from Elsevier.

The direction of this mechanical deflection, either towards or away from the kinocilium,

is associated with excitation or suppression of primary afferent neural activity, respectively.

Deflection of the stereocilia towards the kinocilium causes the hair cells to depolarise, which

in turn increases the rate of firing in the vestibular afferent fibres. In contrast, if the stereocilia


are bent away from the kinocilium, the hair cell becomes hyperpolarised, subsequently

reducing the afferent firing rate (Loewenstein, 1974; Goldberg and Hudspeth, 2000). Given

that the opposing physiologic responses arise from mechanical deflections of the stereocilia,

the hair cells in the vestibular organs are arranged in a specific orientation in order to signal

the precise direction of the angular or linear acceleration. In the semicircular canals, the

ampullary hair cells of the complementary pairs of canals are arranged in such a way that the

depolarisation of the hair cells occurs in opposite directions in response to any component

rotational acceleration in the plane of those canals. Therefore, the three semicircular canals,

lying approximately orthogonal to one another, are able to respond accurately to any

rotational movement made in three-dimensional space. In the two otolith organs, each of the

utricular and saccular maculae is divided into two areas by curved lines, the striolae. The hair

cells are polarised in opposite directions on either side of the striolae, which effectively

separate the receptors into two morphologically opposed groups. Due to the curvature of the

striolae, the otolithic hair cells are able to respond sensitively to head positions and linear

accelerations in all directions (Wilson and Melvill Jones, 1979).

1.1.2 Vestibular Pathways

The peripheral vestibular nerve fibres that arise from the receptor hair cells in the

semicircular canals and the otolith organs converge as they course toward Scarpa‟s (vestibular)

ganglion, which contains the bipolar ganglion cell bodies of the afferent nerve fibres within

the bony internal auditory canal (IAC). In addition to the vestibular nerve, the IAC contains

the cochlear nerve, the facial nerve, the nervus intermedius (a branch of the facial nerve), and

the labyrinthine artery (see Wilson and Melvill Jones, 1979; Figure 1.2). Inputs from the

primary vestibular afferents reach two main areas in the brain: the vestibular nucleus complex

(VNC) and the cerebellum (Carleton and Carpenter, 1984). The VNC is located along the

lateral wall of the fourth ventricle between the roof of the brainstem and the floor of the

cerebellum. The VNC is divided into four “major” nuclear groups; superior, medial, lateral,

and descending (inferior), as well as some smaller cell groups (Brodal and Pompeiano, 1957;

Brodal, 1974). However, the boundaries of the specific vestibular nuclei are difficult to

distinguish based on their cytological characteristics (see Barmack, 2003; Highstein and

Holstein, 2006 for reviews). Once head motion signals are received by the VNC, various

subnuclei (the superior, medial, lateral, descending vestibular nuclei and the prepositus

hypoglossi nucleus), process specific types of angular and linear velocity information. This

process is highly ordered. Both of the superior and medial vestibular nuclei are responsible for


the vestibulo-ocular reflexes (VORs), and the lateral vestibular nucleus is the nucleus

primarily responsible for the vestibulo-spinal reflexes (VSRs). However, the medial vestibular

nucleus is also involved in the VSRs. The descending nucleus communicates with all of the

other nuclei, yet does not have a primary output of its own. Primary vestibular fibres do not

cross the midline of the brainstem, yet the labyrinth on one side greatly influences the activity

of the VNC of the other. This marked effect is achieved through the commissural fibres that

interconnect the VNC of the two sides (Hain and Helminski, 2007). In the VNC, processing of

vestibular sensory inputs occurs simultaneously with the processing of other sensory

information such as proprioceptive, visual, tactile, and auditory inputs (see Smith et al., 2005a

for review).

The secondary vestibular fibres from the VNC provide an ascending input to cranial

motor nuclei III, IV and VI, controlling the reciprocal contractions of extraocular muscles to

make reflexive eye movements (i.e. VORs) to compensate for head movement (Figure 1.4).

Outputs of the VNC not only evoke reflexes mediated by extraocular muscles, they also evoke

skeletal muscle reflexes. Descending projections of the VNC to spinal motor centres originate

from the lateral, medial, and descending vestibular nuclei (Brodal, 1981) to form lateral and

medial vestibulospinal tracts. Some of the vestibular information is also relayed by

reticulospinal tracts, which originate in the pontomedullary reticular formation (Wilson and

Melville Jones, 1979). These projections allow for the generation of a variety of reflexes of

the body and limb musculature to control body posture and maintain balance (i.e. VSR)

during head motion and against gravity (Figure 1.4). Some of the vestibular information from

the VNC is also relayed onto the cerebellum (see Highstein and Holstein, 2006 for review;

Figure 1.4). Although the cerebellum is not required for vestibular reflexes, vestibular reflexes

cannot be regulated or function effectively when this structure is absent (Beraneck et al., 2008;

Cullen et al., 2009; see Mackay and Murphy, 1979 for review). Conscious awareness of self-

motion in darkness relies on ascending pathways from the VNC to the thalamus, from which

vestibular information is projected into many areas of the neorcortex and limbic system (see

Smith et al., 2005a for review).


Vestibular input

Non-vestibular

sensory input

VNC

Cranial motor nuclei

Cerebellum

Spinal motor centre

VOR

VSR

Figure 1.4

Simplified diagram representing the organisation of the key areas and pathways for the

production of the VORs and VSRs. Abbreviations: VNC, vestibular nucleus complex; VOR,

vestibulo-ocular reflex; VSR, vestibulo-spinal reflex.

1.1.3 Peripheral Vestibular Deafferentation

As the brain processes vestibular signals by comparing those received from both ears, it

is critical that both vestibular labyrinths are intact in order to achieve normal vestibular

reflexes. However, should a loss of vestibular function occur, vestibular reflex activity is

disturbed, resulting in a syndrome of ocular motor and postural disorders due to the

interruption of central vestibulo-ocular and vestibulo-spinal pathways.

Much of our understanding of vestibular function is due to accumulated clinical and

experimental observations in humans and animals with unilateral and bilateral vestibular

lesions. The effects of unilateral or bilateral vestibular deafferentation (UVD and BVD,

respectively) have been studied extensively in animals (Smith et al., 1986; Smith and

Curthoys, 1988a, b; Zennou-Azogui et al., 1993; Hamann et al., 1998; Newlands et al., 2005;

Goddard et al., 2008a; Zheng et al., 2004, 2006, 2007, 2009b) and in humans (Honrubia et al,

1984, 1985; Fetter et al., 1991; Brandt et al., 2005; Hüfner et al., 2007; Lopez et al., 2007;

Mbongo et al., 2009). The symptoms that occur following UVD can be categorised into two

groups: static or dynamic. Static symptoms include spontaneous ocular nystagmus (quick

phases towards the intact side), yaw head tilt and, roll head tilt, and dynamic symptoms

include abnormal amplitude (gain) and timing (phase) of the VORs and VSRs in response to

head movement (see Darlington and Smith, 2000 for review).

Following UVD, the spontaneous resting activity of neurons in the VNC ipsilateral to

the lesion decreases, while it increases on the contralateral side (see Smith and Curthoys,

1989 for review). As a result of this imbalance in spontaneous resting activity between

neurons in the ipsilateral and contralateral VNCs, asymmetrical activation of the vestibular


reflexes causes a dramatic series of symptoms such as spontaneous nystagmus, postural

instability, and inadequate compensatory responses to head movement (see Smith and

Curthoys, 1989 for review).

In humans, a sudden unilateral loss of labyrinthine function can result from accidents,

ototoxicity, diseases, or from surgical treatment for diseases such as Ménière‟s disease. The

consequence of a sudden loss of unilateral labyrinthine function is dramatic, and patients

suffer from severe dizziness, nausea and vomiting (Baloh, 1979). Although UVD patients

show “normal” upright stance, provided that at least one accurate sensory input (visual or

somatosensory) is present, severe postural disturbances are observed when UVD patients are

forced to rely solely on vestibular afferent input (Nashner et al., 1982; Horak et al., 1990;

Fetter et al., 1991; Maurer et al., 2000; Mergner et al., 2009). The severe ocular and postural

deficits due to UVD, however, are temporary and are partially reduced by a recovery process

known as „vestibular compensation‟ in both animals (Norre et al., 1984; Sirkin et al., 1984;

Smith et al., 1986; Newlands and Perachio, 1990; Gilchrist et al., 1998; Heskin-Sweezie et al.,

2010) and humans (Fetter et al., 1991; Choi et al., 2007; Parietti-Winkler et al., 2010; see

Horak, 2009; Saman et al. 2009 for review). The vestibular compensation process is thought

to involve synaptic plasticity within the VNC (zu Eulenburg et al., 2010; see Dieringer, 1995;

Darlington et al., 2002; Olabi et al., 2009 for reviews). During vestibular compensation,

altered resting activity in each of the two VNCs returns to near normal, and as it does so,

some of the symptoms of UVD decrease (Yamanaka et al., 2000; Him and Dutia, 2001;

Bergquist et al., 2008; Helmchen et al., 2009; Heskin-Sweezie et al., 2010; see Lacour and

Tighilet, 2010 for review). However, the compensation process is never complete. Although

many of the static symptoms due to UVD diminish within a few days to a week, the

symptoms related to the deficits in dynamic function (i.e. gain and phase) of the vestibular

reflexes remain (Fetter et al., 1996; Hamann et al., 1998; Gilchrist et al., 1998; Lopez et al.,

2007; Heskin-Sweezie et al., 2010; Parietti-Winkler et al., 2010; see Dutia, 2010 for review).

The effects of BVD are different to UVD. The static symptoms of UVD do not develop

following BVD due to an equal reduction in the resting activity in both VNCs (Ris and

Godaux, 1998; see Smith and Curthoys, 1989 for review). However, since BVD results in

complete loss of the VORs and VSRs, severe oscillopsia (a blurring of the vision during head

movement) and ataxia (uncoordinated muscle movements) occur as a result (see Brandt,

1996). Following BVD, despite the regeneration of spontaneous resting activity in the VNC

neurons, the dynamic sensitivity to head movement never recovers (Ryu and McCabe, 1976;


Waespe et al,. 1992; Yakushin et al., 2005). Therefore, there is no recovery in VOR function

following BVD, although there is some evidence to suggest that some compensation may

occur by substituting smooth pursuit eye movements for the absent VORs (Bense et al., 2004;

Bockisch et al., 2004; Dieterich et al., 2007; Herdman et al., 2007). While ataxia gradually

decreases over time (Igarashi and Guitiierrez, 1983; Igarashi et al., 1988), some residual VSR

symptoms such as high-acceleration postural perturbation are observed (Allum and Pfaltz,

1985; Horak et al., 1990).

Interestingly, BVD results in hyperactivity in rats while reducing exploratory behaviour

and thigmotaxis (bodily wall contact during movement) (Goddard et al., 2008a). Furthermore,

BVD has been shown to cause emotional changes not only in humans (Furman and Jacob,

2001; Staab and Ruckenstein, 2003; Sang et al., 2006; Jáuregui-Renaud et al., 2008; Kalueff

et al., 2008), but also in animals (Goddard et al., 2008a; Zheng et al., 2008). It is clear that

following UVD or BVD, the processing of visual, auditory, and proprioceptive sensory

information is disrupted. These disturbances cause significant changes in neural activity in the

VNC, which may result in changes further upstream in the processing networks of the brain.

Furthermore, vestibular damage also results in cognitive deficits in animals (Wallace et al.,

2002b; Russell et al., 2003a; Zheng et al., 2004, 2006, 2007, 2009b) and in humans

(Schautzer et al., 2003; Brandt et al., 2005), which are thought to be related to vestibular

input to the hippocampal region of the brain.

1.1.4 Vestibular-Hippocampal Connections

A great deal of research on learning and memory has focused on the hippocampus, a

brain structure implicated in these processes (see Olton and Papas, 1979; Squire, 1992; Rolls,

1996; Burgess et al., 2002; Squire et al., 2004 for reviews). Furthermore, a considerable

amount of data suggests the importance of the hippocampus for the construction and retrieval

of spatial memory maps and the ability to navigate through environments (Morris et al., 1982;

Jarrard 1993; Burgess et al., 2002; Yim et al., 2008; de Oliveira Alvares et al., 2008), with a

number of studies showing impairments in spatial tasks following hippocampal damage for

both animals (Whishaw and Maaswinkel, 1998; Whishaw and Gorny, 1999; Gilbert and

Kesner, 2002; Broadbent et al., 2004; Talpos et al., 2008; Faraji et al., 2008; Iordanova et al.,

2009) and humans (Altemus and Almli, 1997; Kessels et al., 2001; Spiers et al., 2001; Astur

et al., 2002).


Interest in the possibility of a connection between the vestibular system and the

hippocampus has been provided by previous studies in both experimental animals and human

patients showing that the vestibular system contributes to the development of spatial memory

(Etienne, 1980; Matthews et al., 1989; Israel et al., 1996; Cuthbert et al., 2000; Russell et al.,

2003a, 2006; McGauran et al., 2005; Allen et al., 2007; Nardini et al., 2008).

Spatial memory deficits in BVD animals have been demonstrated in numerous

behavioural tasks such as the radial arm maze (Matthews et al., 1989; Ossenkopp and

Hargreaves, 1993; Russell et al., 2003a), the spatial forced-alternation T-maze (Zheng et al.,

2007), and the foraging task (Wallace et al., 2002c; Zheng et al., 2006, 2009b). In a more

recent study by Zheng et al. (2009b), it was shown that although rats with BVD have been

found to be able to use visual cues to navigate themselves home under light conditions, their

ability to perform in the foraging task was severely impaired in darkness when compared with

sham rats (Zheng et al., 2009b). Although UVD animals have been found to show spatial

memory deficits at 3 months post-operation in performing the foraging task, recovery from

these deficits has been observed by 6 months post-operation (Zheng et al., 2006).

In parallel with animal studies, impairments in the spatial memory and navigational

abilities of humans with vestibular dysfunction have been shown to endure for up to 10 years

following the loss of vestibular function (Grimm et al., 1989; Risey and Briner, 1990; Peruch

et al., 1999; Schautzer et al., 2003; Redfern et al., 2004; Brandt et al., 2005; see Smith et al.,

2005b; Hanes and McCollum, 2006 for reviews). A landmark study was published by Brandt

and colleagues in 2005 in which the volume of the hippocampus and performance in spatial

memory tests in patients with acquired chronic bilateral vestibular loss were compared with

age-, sex-, and education-matched controls (Brandt et al., 2005). Intriguingly, patients with

bilateral vestibular loss exhibited significant impairment in performing in spatial memory and

navigation tasks when tested with a virtual Morris water maze. Moreover, MRI scans of these

patients revealed a selective and bilateral atrophy of the hippocampus, suggesting that spatial

memory and navigation rely on vestibular input (Brandt et al., 2005). Interestingly, Hüfner et

al., (2007) showed that while patients with left vestibular loss showed no significant deficits

in spatial memory and navigation, patients with right vestibular loss showed a greater heading

error compared to left UVD patients or to control subjects in the probe trial in a virtual Morris

water maze. Furthermore, hippocampal atrophy was not observed in patients with unilateral

vestibular loss whether the vestibular loss was due to acoustic neurinoma (Hüfner et al., 2007)

or vestibular schwannoma removal (Hüfner et al., 2009). However, more recently, zu


Eulenberg and colleagues demonstrated that patients with unilateral vestibular loss due to

vestibular neuritis exhibited an atrophy of the left posterior hippocampus (zu Eulenberg et al.,

2010). The authors postulated that this atrophy may be due to a reduced inflow of vestibular

input after an incident of vestibular neuritis (zu Eulenberg et al., 2010). Interestingly,

structural and functional plasticity of the hippocampus in healthy humans has also been

demonstrated. For example, London taxi drivers (Maguire et al., 2000), professional dancers

and slackliners (Hüfner et al., 2010) have all shown a decrease in anterior hippocampal

volume with an increase in posterior hippocampal volume compared to age- and sex-matched

controls. Taken together, these results emphasise the role of the vestibular system in spatial

memory function, and outline the importance of vestibular input to the hippocampus for

maintaining its integrity.

A number of anatomical connections between the VNC and the hippocampus have been

suggested. Some vestibular information reaching the hippocampus passes through the

thalamus, the parietal cortex, and the entorhinal or perirhinal cortex and then to the

hippocampus (Figure 1.5). However, more direct connections between the VNC and the

hippocampus are also possible. These include a theta-generating pathway leading from the

pedunculopontine tegmental nucleus via the supramammillary nucleus and medial septum to

the hippocampus, or the head direction system passing through the dorsal tegmental nucleus,

lateral mammillary nucleus, anterodorsal thalamic nucleus, and the post-subiculum to the

hippocampus (see Smith, 1997; Smith et al., 2005a for reviews; Figure 1.5).


Vestibular Nucleus

Thalamus

Parietal cortex

Entorhinal/Perihinal

cortices

Hippocampus

PPTN

SUM

Medial

septum

DTN

LMN

ADN

PS

Figure 1.5

Some potential vestibulo-hippocampal connections. Colour codes for the pathways are:

thalamo-cortical route; theta-generating route; and head-direction route. Abbreviations: DTN,

dorsal tegmental nucleus; PPTN, pedunculopontine tegmental nucleus; SUM, supramammillary

nucleus; LMN, lateral mammillary nucleus; ADN, anterodorsal nucleus; PS, post-subiculum.

Many studies have reported evidence for vestibular-hippocampal connections through

the use of neurochemical, electrophysiological, and imaging methods. The first neurochemical

evidence to support the link between the vestibular system and the hippocampus was provided

by Horii et al. (1994). In this study, electrical stimulation of the vestibular nerve caused a

large increase in acetylcholine release in the hippocampus, which was inhibited by the

administration of a non-NMDA (N-methyl-D-aspartate) receptor antagonist into the VNC

(Horii et al., 1994). Although the authors postulated that an increase in histamine release in

the medial septum following the electrical stimulation of vestibular nerve (Mochizuki et al.,

1994) might affect acetylcholine release in the hippocampus, depletion of neuronal histamine

did not prevent the increased release of acetylcholine in the hippocampus (Horii et al., 1995,

1996). Since then, numerous studies have demonstrated neurochemical changes in the

hippocampus following peripheral vestibular damage, providing compelling evidence for

neural connections between the vestibular system and the hippocampus (Table 1.1). In

particular, in a study by Ashton et al. (2004b), although there was no change in cannabinoid

CB1 receptor expression in the hippocampus following UVD in rats, this was the only study


that investigated possible changes in the endocannabinoid system following vestibular

damage. However, it must be remembered that the Ashton et al. (2004b) study was carried out

in UVD animals and given that UVD and BVD result in quite different symptoms, no change

in the CB1 receptors following UVD does not necessarily imply that there would be no

change following BVD.

Evidence for a connection between the vestibular system and the hippocampus has been

further provided by studies showing that vestibular stimulation leads to activation of the

hippocampus in animals (O‟Mara et al., 1994; Gavrilov et al., 1995, 1996; Sharp et al., 1995;

Cuthbert et al., 2000; Hicks et al., 2004; Horii et al., 2004) and in humans (Vitte et al., 1996;

Suzuki et al., 2001; Dieterich et al., 2003). In particular, Cuthbert et al. (2000) demonstrated

that high frequency electrical stimulation in one vestibular labyrinth of guinea pigs evoked

field potentials in the hippocampus bilaterally with a latency of approximately 40

milliseconds. The electrical stimulation used in this study was below the threshold to produce

eye movements, suggesting that the hippocampal responses were not confounded by feedback

from the eye muscles or from efferent copy signals (Cuthbert et al., 2000). Similar findings

were reported by Hicks and others who also showed that these hippocampal responses can be

elicited from multiple areas of the guinea pig vestibular labyrinth including the canal

ampullae, utricle and saccule (Hicks et al., 2004).


Table 1.1

Neurochemical changes in the hippocampus and in the neocortex following vestibular damage.

Neural Substrate Changes in Level of Expression Reference

UVD

eNOS

↓ in the contralateral CA2/3

↑ in the contralateral DG Liu et al., 2003c

↑ in the contralateral EC and PRC Liu et al., 2004

nNOS ↓ in the ipsilateral DG

Zheng et al., 2001;

Liu et al., 2003c

↓ in the contralateral EC and PRC Liu et al., 2004

Arginase I ↑ in the contralateral PRC

↓ in the ipsilateral PRC Liu et al., 2004

Arginase II No change in the hippocampus Liu et al., 2003c

↑ in the ipsilateral EC Liu et al., 2004

NMDA receptor s

subunits

↓ in the ipsilateral CA2/3 (NR1)

Liu et al., 2003b ↓ in the ipsi- and contralateral CA2/3

(NR2A)

↑ in the ipsilateral CA1 (NR2A)

CB1 receptors No change in the hippocampus Ashton et al., 2004b

Glucocorticoid

rereceptors No change in the hippocampus Lindsay et al., 2005

BVD

SNAP-25 ↑ in the DG

Goddard et al., 2008c Synaptophysin

No change in the hippocampus, EC,

PRC, FC Drebrin

NF-L

Serotonin

tratransporter ↓ in the FL and CA1

Goddard et al., 2008b

Tryptophan

hyhydroxylase ↑ in the FL, CA2/3, and DG

Dopamine--

hyhydroxylase


PRC, FL

Dopamine

tratransporter


PRC, FL

Tyrosine

hyhydroxylase


PRC, FL

Abbreviations: DG, dentate gyrus; EC, entorhinal cortex; eNOS, endothelial nitric oxide synthase; FL,

frontal lobes; nNOS, neuronal nitric oxide synthase; NF-L, neurofilament-L; NMDA, N-methyl-D-aspartate;

PRC, perirhinal cortex; SNAP-25, synaptosome-associated protein of 25 kDa.


Under normal conditions, hippocampal place cells fire when an animal moves through a

specific location in an environment (O‟Keefe and Dostrovsky, 1971), and enable animals to

construct their location within the environment (O‟Keefe and Nadel, 1978). The spatial-

specific firing pattern of the hippocampal place cells is maintained even in the absence of

visual input (Hill and Best, 1981; Quirk et al., 1990; Save et al., 1998), and these

hippocampal place cells are thought to have an essential role in spatial navigation (Knierim et

al., 1995; Wiener et al., 1995; see O‟Keefe, 1979 for review). Interestingly, the activity of

these place cells has been shown to be influenced by electrical stimulation of the VNC. For

example, Horii et al. (2004) showed that the firing rates of hippocampal CA1 neurons were

altered following the electrical stimulation of the medial vestibular nucleus in rats using single

unit recording. The firing rates of the place cells in the hippocampus (i.e. complex spike cells)

were increased in a current intensity-dependent manner, while only some of the theta cells (i.e.

non-complex spiking cells) responded to vestibular stimulation (Horii et al., 2004).

Following bilateral inactivation of the vestibular system, whether the damage is

temporary (e.g. transtympanic injection of tetrodotoxin; Stackman et al., 2002) or permanent

(surgical lesions of the labyrinth; Russell et al., 2003b, 2006), the response of place cells and

theta activity in the CA1 region of the hippocampus has been shown to be disrupted.

Specifically, Stackman and colleagues showed that tetrodotoxin-induced vestibular

inactivation disrupted the location-specific firing of hippocampal place cells and disintegrated

the place fields. Furthermore, place cell spatial coherence and spatial information content

were significantly decreased, while place cell peak or average firing rate was unaffected

(Stackman et al., 2002). Spatial coherence is a measure of the orderliness of discharge within

and outside the place fields (Kubie et al., 1990) and spatial information content is a

quantitative measure of the amount of information conveyed by each spike (Skaggs et al.,

1993). Russell et al. (2003b) reported that the firing fields of place cells in BVD animals were

larger compared to sham animals, indicating that the location-specific firing of place cells in

BVD animals was disturbed. The place cells of BVD animals had lower spatial coherence and

information content, while the average firing rate, but not the peak firing rate, was higher

(Russell et al., 2003b). In addition, a larger field size and average firing rate in theta cells was

observed in BVD animals (Russell et al., 2003b), but Stackman et al. (2002) did not find any

changes in theta activity. Nevertheless, a decrease in the rhythmicity of theta activity in the

hippocampus has been observed in BVD rats (Russell et al., 2006).

In an attempt to further clarify the mechanism underlying the dysfunction of


hippocampal place cells following vestibular damage, Zheng and colleagues carried out in

vitro electrophysiological studies involving hippocampal brain slices from UVD rats. In this

study, there was a significant reduction in field potential responses to electrical stimulation in

the Schaffer collaterals in the CA1 area at 5-6 months following the lesion, indicating a

significant loss of normal electrical excitability (Zheng et al., 2003). Although more profound

effects on hippocampal electrical excitability were predicted following the complete loss of

vestibular function, recently Zheng et al. (2010) reported that this was not the case, at least in

vivo. Neither the baseline field potential, population spike height in the dentate gyrus, field

potential in the CA1 area, nor the induction or maintenance of long-term potentiation (LTP) in

vivo were affected by BVD in rats at 6 weeks or 7 months following surgery (Zheng et al.,

2010). The authors postulated that in spite of place cell dysfunction and interruption in theta

activity following BVD (Stackman et al., 2002; Russell et al., 2003b, 2006), the BVD-

induced spatial memory impairments may not be due to inability of the hippocampus to

generate LTP (Zheng et al., 2010).

Overall, these findings suggest that vestibular signals may exert an important influence

on spatial information processing in the hippocampus, which may explain, at least in part, the

navigational cognitive deficits of animals and humans with vestibular dysfunction. However,

knowledge of how this influence occurs is far from complete, and the findings outlined here

highlight a need for further research into the connection between the vestibular system and

hippocampal function.


1.2 The Endocannabinoid System

Marijuana (Cannabis sativa), or Cannabis, is one of the oldest drugs of abuse in the

world, but is also the drug with the longest recorded history of medicinal value. The major

psychoactive constituent of marijuana, 9-tetrahydrocannabinol (

9-THC), was identified by

Ganoi and Mechoulam (1964), who subsequently revealed its chemical structure and activity

(Mechoulam and Gaoni, 1967; Mechoulam, 1970; Mechoulam et al., 1970). Furthermore,

studies of the biological effects of 9-THC and its synthetic analogues have shown a precise

structural selectivity (Hollister, 1974) as well as stereo-selectivity (Jones et al., 1974),

foreshadowing the existence of a drug-receptor interaction. Besides marijuana‟s known

euphoric effect, its use also results in changes in learning and memory, anxiety, analgesia,

hypothermia, stimulation of food intake, anti-emetic effects, and vasorelaxation in humans

(see Jones, 1977; Maykut, 1985; Taylor, 1998; Hall and Degenhardt, 2009 for reviews). In

rodents, Cannabis and its related compounds have been shown to produce a characteristic

combination of up to four of the following symptoms: hypothermia, analgesia, hypoactivity

and catalepsy (Grunfeld and Edery, 1969; Compton et al., 1993; see Adams and Martin, 1996;

Chaperon and Thiébot, 1999 for review).

The discovery of 9-THC and the chemical synthesis of its analogues provided the

foundation for Cannabis research, and since then a growing body of evidence has emerged

focusing on the role of the endocannabinoid system in numerous diseases and disorders. The

endocannabinoid system is comprised of: 1) cannabinoid receptors, 2) endogenous

cannabinoids, also known as endocannabinoids, and 3) enzymes responsible for the

production, transport, and degradation of these cannabinoids and their receptors (see Elphick

and Egertova, 2001 for review).

1.2.1 Cannabinoid Receptors and Endocannabinoids

CB1 receptors

The first report that 9-THC might exert its effects by interacting with a specific

receptor protein in the brain was by Howlett (1984), when it was demonstrated that

cannabinoids decrease cyclic adenosine monophosphate (cAMP) levels in neuroblastoma cell

cultures, suggesting a Gi/o-coupled receptor-mediated action (Howlett and Fleming, 1984;

Howlett, 1985; Howlett et al., 1986). Further characterisation of this putative cannabinoid

receptor was carried out by Devane et al. (1988), who demonstrated the high-affinity,


saturable, stereospecific binding of a synthetic cannabinoid agonist, [1,2-(R)-5]-5-(1,1-

dimethyleptyl)-2-[5-hydroxy-2-(3-hydroxy propyl) cyclohexyl] phenol (CP-55,940) in rat

brain homogenates. Two years later, Herkenham and others performed an autoradiographic

study using tritiated CP-55,940 ([3H]-CP-55,940) to show the distribution of cannabinoid

binding sites in the brain (Herkenham et al., 1990, 1991). Following this, Matsuda and others

reported the cloning of a complimentary deoxyribonucleic acid (cDNA) sequence, encoding a

cannabinoid 1 receptor (CB1) from a rat brain cDNA library (Matsuda et al., 1990). Two

splice variants of the CB1 receptor have also been discovered: CB1A, which has an altered

amino-terminal sequence (Shire et al., 1995), and CB1B, which has an in-frame deletion of 33

amino acids at the amino-terminus (Ryberg et al., 2005).

The CB1 receptor is a seven transmembrane-domain receptor, coupled to the pertussis

toxin-sensitive G protein, Gi/o. As a member of the G protein-coupled receptor (GPCR)

superfamily, the actions of CB1 receptors are transduced via the activation of these G proteins

(Prather et al., 2000). CB1 receptor activation of G-proteins results in the inhibition of

adenylyl cyclase (Howlett, 1984), inhibition of calcium (Ca2+

) conductances (Mackie and

Hille, 1992; Caulfield and Brown, 1993; Mackie et al., 1995), stimulation of potassium (K+)

conductances (Deadwyler et al., 1995; Mackie et al., 1995), and stimulation of the mitogen-

activated protein kinase pathway (Bouaboula et al., 1995; see Demuth and Molleman, 2006

for review). The CB1 receptors are among the most abundant GPCRs in the rat and human

CNS (Herkenham et al., 1991; Mailleux and Vanderhaeghen, 1992; Glass et al., 1997), and

the density of CB1 receptors in the brain rivals that of ionotropic receptors for glutamate and

-aminobutyric acid (GABA) (see Herkenham, 1995 for review). Among the various brain

regions, the CB1 receptors are highly expressed in the basal ganglia, the molecular layer of the

cerebellum, the hippocampus, the cerebral cortex, the rostral ventromedial medulla, certain

nuclei of the thalamus and amygdala, the periaqueductal grey and dorsal primary afferent

spinal cord regions (Herkenham et al., 1990, 1991; Tsou et al., 1998; Ashton et al., 2004a;

Suarez et al., 2008). In the hippocampus, CB1 receptors have been found to be mainly

expressed in GABAergic inhibitory interneurons, most of which originate from

cholecystokinin-positive basket cells (Katona et al., 1999; Tsou et al., 1999; Egertova and

Elphick, 2000; Nyiri et al., 2005; Neu et al., 2007). Recently, the presence of presynaptic CB1

receptors has also been reported in hippocampal glutamatergic axon terminals, suggesting

their role in the inhibition of glutamate release (Katona et al., 2006; Kawamura et al., 2006;

Takahashi and Castillo, 2006). However, it is important to note that the expression of CB1

receptors in these excitatory synapses is at least 20 times lower than that at inhibitory


interneuron sites (Kawamura et al., 2006). CB1 receptors are also present, although at much

lower levels, in a variety of peripheral organs and tissues, among them being immune cells,

the spleen, the adrenal and pituitary glands, sympathetic ganglia, the heart, the lungs, and in

parts of the reproductive, urinary and gastrointestinal tracts (Galiègue et al., 1995; see

Pertwee, 1997 for review).

Following the initial anatomical evidence for the localisation of CB1 receptors in the

brain (Herkenham et al., 1990, 1991), subsequent light microscopy studies have revealed the

expression of numerous CB1 receptor-positive fibres throughout the brain (Egertova et al.,

1998; Tsou et al., 1998; Egertova and Elphick, 2000; Hájos et al., 2000; Katona et al., 1999,

2000, 2001). These fibres were tentatively identified as axons based on their distinctive

morphology (thin and rich in varicosities). This identification of presynaptic localisation was

then confirmed from work conducted in the rat hippocampus using electron microscopy,

where silver-impregnated gold particles represented the CB1-immunopositive sites (Katona et

al., 1999). The varicosities observed using light microscopy corresponded to axon terminals,

which were found to be densely covered with CB1 receptors and packed with synaptic

vesicles (Katona et al., 1999, 2000).

It has been shown that activation of the CB1 receptor causes the inhibition of the release

of the associated neurotransmitter, which may vary depending on the location of the CB1

receptor. One of the first studies of a cannabinoid receptor causing inhibition of

neurotransmitter release was carried out by Gill et al. in 1970. In this study, an electrically-

evoked twitch response of the guinea-pig ileum (which involves the release of acetylcholine)

was depressed by the application of 9-THC (Gill et al., 1970). Further evidence that the

activation of presynaptic CB1 receptors decreases the release of various neurotransmitters has

been demonstrated in in vitro assays as well as in various in vivo experimental animal models

and in humans (Gifford and Ashby, 1996; Gessa et al., 1997; Kathmann et al., 1999; Katona

et al., 1999; Gifford et al., 2000; Hájos et al., 200l; Szabo et al., 2002; Wang, 2003; Foldy et

al., 2006; Kawamura et al., 2006; Lee et al., 2010; see Schlicker and Kathmann, 2001 for

review).

CB2 receptors

Following the identification of the CB1 receptor, a second cannabinoid receptor subtype,

CB2, was isolated and cloned from the human promyelocytic cell line HL60 (Munro et al.,

1993). The CB1 and CB2 receptors share 44% overall identity in their primary protein


structure, showing the highest degree of homology in the transmembrane domains (see

Childers and Breivogel, 1998 for review). In contrast to presynaptic localisation of CB1

receptors in the brain, immunohistochemical studies suggest postsynaptic localisation of CB2

receptors (Gong et al., 2006; Onaivi et al., 2006), which has recently been confirmed by the

use of electron microscopy (Brusco et al., 2008b).

CB2 receptors are mainly expressed in cells of the immune and haematopoietic systems,

among them leukocytes, and cells in the spleen and the tonsils (Munro et al., 1993). Due to

the expression of CB2 receptors in the immune system, CB2 receptor activation elicits

immunomodulatory responses, such as a decrease in antigen presenting cell activity and a

down regulation of cytokine (IFN- and TNF-) production during inflammatory responses

(Klein et al., 2001; Lombard et al., 2007). However, there have also been recent

investigations showing that CB2 receptors are found in the CNS in response to injury. The

presence of CB2 receptors has been reported in glial cells associated with neuritic plaques in

Alzheimer‟s disease (Benito et al., 2003; Ramirez et al., 2005) and Down‟s syndrome brains

(Núñez et al., 2008), in the CNS of animals with experimental autoimmune encephalomyelitis

(an animal model of human multiple sclerosis) (Maresz et al., 2005), in plaque cell subtypes

in human multiple sclerosis (Benito et al., 2007), in brain tumours (Ellert-Miklaszewska et al.,

2007), in macrophages following middle cerebral artery occlusion in rats (Ashton et al., 2007),

and in activated microglia in an animal model of Parkinson‟s disease (Price et al., 2009).

From these results, it seems possible that CB2 receptor expression may be induced in discrete

brain regions following neuropathological insults. Furthermore, the expression of CB2

receptors has also been found in the brains of healthy subjects (Van Sickle et al., 2005; Ashton

et al., 2006; Gong et al., 2006; Onaivi, et al., 2006; Baek et al., 2008). Consequently, the

original concept that CB1 receptors play an exclusive role in the brain, and CB2 receptors in

the immune system, has evolved into the idea that both cannabinoid receptor types can be

involved in controlling both central and peripheral functions. Moreover, functional studies

have suggested that the activation of CB2 receptors by the administration of exogenous CB2

receptor ligands may open novel therapeutic avenues for a number of pathological CNS

conditions such as Alzheimer‟s disease, stroke, and neuropathic pain (Ramirez et al., 2005;

Zhang et al., 2007, 2008; Jhaveri et al., 2008; see Rivers and Ashton, 2010 for review).

Endocannabinoids

The discovery of the cannabinoid receptors opened the way for the identification of

their endogenous ligands, known as „endocannabinoids‟. Endocannabinoids represent a class


of lipophilic compounds based on the general structure of modified arachidonic acid

derivatives. Arachidonyl ethanolamide or anandamide (AEA) and 2-arachidonyl glycerol (2-

AG) are the two best characterised endocannabinoids in the mammalian CNS (see Piomelli,

2003 for review). AEA was isolated in 1992 by Devane et al., and it acts as a partial agonist at

both CB1 and CB2 receptors (Sugiura et al., 2002). The other major endocannabinoid, 2-AG,

was initially isolated from the canine gut (Mechoulam et al., 1995) and the rat brain (Sugiura

et al., 1995). The concentration of 2-AG in the mammalian brain is much higher than that of

AEA, and it activates both CB1 and CB2 receptors with greater efficacy than does AEA (see

Sugiura et al., 2006; Sugiura, 2009 for reviews). Other endocannabinoids have also been

isolated during the last decade, including 2-arachidonyl-glycerol ether (noladin ether) (Hanus

et al., 2001), N-arachidonoyl-dopamine (NADA) (Bisogno et al., 2000; Huang et al., 2002)

and virodhamine (Porter et al., 2002). However, the pharmacological and physiological

activities of these endocannabinoids have not yet been fully characterised.

Due to the lipophilic nature of AEA and 2-AG, they are not stored in the aqueous

interior of synaptic vesicles, but are synthesised and released from postsynaptic neurons on

demand, and target presynaptic cannabinoid receptors (Di Marzo et al., 1994; Stella et al.,

1997; see Kano et al., 2009 for review; Figure 1.6). There have been two major pathways

described for the activation of endocannabinoid synthesis from postsynaptic neurons. These

are the phospholipase C (PLC)-dependent pathway and the Ca2+

-dependent pathway (see

Heifets and Castillo, 2009 for review; Figure 1.6). The PLC-dependent pathway is triggered

by the activation of Gq-coupled receptors including group 1 metabotropic glutamate receptors

(mGluRs) (Maejima et al., 2001; Varma et al., 2001; Ohno-Shosaku et al., 2002) and M1/M3

muscarinic receptors (Kim et al., 2002; Ohno-Shosaku et al., 2003; Fukudome et al., 2004).

The other described pathway, the Ca2+

-dependent pathway, is driven by an increase in

intracellular Ca2+

concentration via activation of voltage-gated Ca2+

channels (Brenowitz and

Regehr, 2003; Adermark and Lovinger, 2007). AEA is synthesised from a phospholipid

precursor, N-arachidonoyl-phosphtidyl ethanolamine (NArPE), which is catalysed by N-acyl

phosphatidyl ethanolamine phospholipase D (NAPE-PLD; Figure 1.6). The synthesis of 2-AG

occurs through the formation from phospholipids of a diacylglycerol (DAG) precursor, which

is catalysed by PLC, followed by the hydrolysis of DAG lipase (DAGL; Figure 1.6).


Figure 1.6

Schematic diagram of the synthesis, signalling, and degradation of endocannabinoids AEA and

2-AG at the synaptic cleft. Abbreviations are: AEA, anandamide; 2-AG, 2-arachidonoyl glycerol;

DAGL, diacylglycerol lipase; EC, endocannabinoid; EMT, endocannabinoid membrane

transporter; FAAH, fatty acid amide hydrolase; MAGL, monoacylglycerol lipase; mGluR,

metabotropic glutamate receptor; M1/M3, muscarinic receptor types M1 and M3; NAPE-PLD, N-

acyl phosphatidyl ethanolamine phospholipase D; NArPE, N-arachidonoyl-phosphtidyl

ethanolamine; PLC, phopholipase C. Figure reproduced from Lutz and Marsicano (2009),

with copyright permission from Elsevier.

Endocannabinoids are removed from synaptic clefts by a two-step mechanism: re-

uptake and subsequent intracellular enzymatic degradation (Hillard and Jarrahian, 2000;

Fowler and Jacobsson, 2002; McFarland and Barker, 2004). There are at least three models

proposed for AEA uptake by cells. The first model is that AEA is transported via the

endocannabinoid membrane transporter (EMT), which transports AEA from one side of the

membrane to the other (Fegley et al., 2004; Ligresti et al., 2004). The second model is that

AEA passes through the membrane by simple diffusion, which is facilitated by the


concentration gradient resulting from intracellular degradation (Glaser et al., 2003). The third

model is that AEA undergoes endocytosis through a caveolae-related uptake process

(McFarland et al., 2004). Contrary to extensive investigation of the AEA re-uptake

mechanism, there is relatively little known about 2-AG uptake. However, there are several

studies suggesting that the 2-AG uptake mechanism is similar to that of AEA (Piomelli et al.,

1999; Beltramo and Piomelli, 2000; Bisogno et al., 2001). Once the endocannabinoids are

taken into a cell, AEA is catalysed by fatty acid amide hydrolase (FAAH) and 2-AG by

monoacylglycerol lipase (MAGL) in addition to FAAH (Goparaju et al., 1998; Deutsch et al.,

2001; Glaser et al., 2003; Day et al., 2001; Figure 1.6).

Overall, the postsynaptic release of endocannabinoids, together with the presence of

presynaptic CB1 receptors on both the inhibitory and excitatory synapses, suggests that

endocannabinoids play an important role in modulating synaptic transmission in the nervous

system.

1.2.2 Neuromodulatory Role of the Endocannabinoid System

The role of endocannabinoids as retrograde messengers mediating short-term (< 1

minute) inhibition of neurotransmitter release was first postulated in studies by Llano et al.

(1991) and Alger (Pitler and Alger, 1992). In these studies, a brief depolarisation of principal

neurons (Purkinje cells of the cerebellum and pyramidal cells of the hippocampus) triggered a

transient suppression of GABAergic synaptic input in the cerebellum (Llano et al., 1991) and

in the hippocampus (Pitler and Alger, 1992). This phenomenon was termed depolarisation-

induced suppression of inhibition (DSI). Over the next decade, numerous studies were

published confirming that endocannabinoids were the “mysterious” retrograde messengers

that mediated cerebellar (Kreitzer and Regehr, 2001a; Diana et al., 2002; Yoshida et al., 2002)

and hippocampal (Ohno-Shosaku et al., 2001; Wilson and Nicoll, 2001) DSI. Furthermore,

Kreitzer and Regehr (2001b) discovered an analogous phenomenon, termed depolarisation-

induced suppression of excitation (DSE) in the cerebellum, which was also shown to be

mediated by endocannabinoids acting upon glutamatergic synapses. More recently, DSE has

also been demonstrated in the dentate gyrus region of the hippocampus (Chiu and Castillo,

2008). Taken together, these results demonstrate that endocannabinoids are important

mediators of short-term plasticity in the brain.

It has been shown that endocannabinoids have the capacity for inducing not only short-


term, but also long-term synaptic plasticity (minutes to hours) in the brain, which has

indicated the potential long-term effect of endocannabinoids on brain function (see Gerdeman

and Lovinger, 2003; Chevaleyre et al., 2006 for reviews). Endocannabinoid-mediated long-

term plasticity takes the form of depression, in which the activation of presynaptic CB1

receptors by endocannabinoids causes a long-lasting reduction of neurotransmitter release.

This endocannabinoid-mediated long-term depression (LTD) has been shown to be a

widespread phenomenon in the brain, that has been observed at both excitatory (glutamatergic)

(Gerdeman et al., 2002; Robbe et al., 2002; Sjostrom et al., 2003; Mato et al., 2008; Haj-

Dahmane and Shen, 2010) and inhibitory (GABAergic) (Chevaleyre and Castillo, 2003;

Chevaleyre et al., 2007; Adermark et al., 2009) synapses in various brain structures including

the dorsal striatum (Gerdeman et al., 2002; Adermark et al., 2009), the nucleus accumbens

(Robbe et al., 2002; Mato et al., 2008), the amygdala (Chevaleyre et al., 2007), the

hippocampus (Chevaleyre and Castillo, 2003; Chevaleyre et al., 2007; see Lafourcade, 2009

for review), the neocortex (Sjostrom et al., 2003), and the ventral tegmental area (Szabo et al.,

2002; Haj-Dahmane and Shen, 2010; see Heifets and Castillo, 2009 for review). Interestingly,

persistent CB1 receptor activation is not required to maintain endocannabinoid-mediated LTD.

This phenomenon was demonstrated in studies which showed that, once established,

endocannabinoid-mediated LTD was not reversed by the administration of CB1 receptor

inverse agonists, such as N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-

methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716A) and N-(piperidin-1-yl)-5-(4-

iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride

(AM251) (Robbe et al., 2002; Sjostrom et al., 2003; Chevaleyre and Castillo, 2003; Ronesi et

al., 2004; Adermark et al., 2009).

Reflecting the widespread distribution of CB1 receptors in the brain (Herkenham et al.,

1990; Tsou et al., 1998), administration of exogenous agonists for the CB1 receptors also

reliably suppresses synaptic transmission in various regions of the brain. These regions

include the hippocampus (Shen et al., 1996; Hoffman and Lupica, 2000; Chiu and Castillo,

2008; Bajo et al., 2009; Serpa et al., 2009), the substantia nigra pars compacta (Chan et al.,

1998; Szabo et al., 2000; Wallmichrath and Szabo, 2002), the cerebellum (Levenes et al.,

1998; Daniel et al., 2004; Kawamura et al., 2006), and the nucleus accumbens (Pistis et al.,

2002; Robbe et al., 2003). This suppression of synaptic transmission by exogenous CB1

receptor agonists has been shown to occur at both excitatory (Kawamura et al., 2006; Bajo et

al., 2009; Fan et al., 2010) and inhibitory (Foldy et al., 2006; Inada et al., 2010; Lee et al.,

2010) synapses. Administration of CB1 receptor inverse agonists prevented or reversed


cannabinoid-induced suppression of synaptic transmission, which confirmed that exogenous

cannabinoids act via the CB1 receptors (Wallmichrath and Szabo, 2002; Bajo et al., 2009;

Serpa et al., 2009; Inada et al., 2010; Lee et al., 2010). Furthermore, administration of

cannabinoid receptor agonists in CB1 receptor knock-out mice has been observed not to

produce any suppression of synaptic transmission, which is evidence further demonstrating

the involvement of CB1 receptors in the modulation of synaptic transmission (Kawamura et

al., 2006; Fan et al., 2010). Aside from synaptic changes, CB1 receptor activation has also

been shown to modulate synaptic plasticity by influencing neurogenesis in the hippocampus,

as the activation of CB1 receptors has been found to promote neurogenesis in the

hippocampus in animals (Jin et al., 2004; Marchalant et al., 2009; Wolf et al., 2010).

Overall, cannabinoid-mediated synaptic plasticity via CB1 receptors is a widespread

phenomenon in the brain, and it reflects the role of the endocannabinoid system in brain

function.

1.2.3 The Role of the Endocannabinoid System in Cognitive Function

In the years since the identification of cannabinoid receptors and the development of

synthetic cannabinoids, there has been significant progress in assessing the effects of

cannabinoids on cognitive processes. Impairments in learning and memory are among the best

known behavioural effects of cannabinoids. In humans, it has long been recognised that

thoughts become fragmented and short term memory is impaired following Cannabis intake

(Bromberg, 1934; Ames, 1958; Abel, 1971; Miller and Branconnier, 1983; Heishman et al.,

1997; Nestor et al., 2008; Weinstein et al., 2008; see Ranganathan and D'Souza, 2006 for

review). Similarly, the disruptive effects of cannabinoid receptor agonists on learning and/or

on the performance of diverse memory tasks have also been thoroughly documented in non-

human primates (Zimmerberg et al., 1971; Aigner, 1988; Winsauer et al., 1999; Nakamura-

Palacios et al., 2000) and in rodents (Carlini et al., 1970; Litchman et al., 1995; Ferrari et al.,

1999; Hampson and Deadwyler, 2000; Cha et al., 2006; Barna et al., 2007; Pamplona et al.,

2008; Suenaga and Ichitani, 2008).

The hippocampus is a brain structure that has been strongly implicated in learning and

memory in both animals and in humans (Altemus and Almli, 1997; Whishaw et al., 2001;

Brandt et al., 2005; Iordanova et al., 2009; Goodrich-Hunsaker et al., 2010). There is

evidence to suggest that the effects of cannabinoids on learning and memory may be due, at


least in part, to effects of cannabinoids on the hippocampus. Not only are CB1 receptors

highly expressed in the hippocampus (Herkenham et al., 1990; see Mackie, 2005 for review),

but also a great deal of evidence demonstrates that the activation of these receptors results in

impairment in various memory tasks that are known to be sensitive to disruption of

hippocampal functions in animals. For example, systemic or intrahippocampal administration

of several CB1 receptor agonists led to impaired performance by rats in the Morris water maze

(Robinson et al., 2003, 2007; Yim et al., 2008), the radial arm maze (Lichtman et al., 1995;

Egashira et al., 2002; Suenaga et al., 2008; Wegener et al., 2008; Wise et al., 2009), and an

object recognition task (Schneider and Koch, 2002; Barna et al., 2007; Clarke et al., 2008;

Schneider et al., 2008; Suenaga and Ichitani, 2008; Baek et al., 2009). The memory-impairing

effects of CB1 receptor agonists have been shown to be mediated at least in part via the CB1

receptors, as these agonist-induced deficits are reversed by the administration of CB1 receptor

inverse agonists such as SR141716A and AM251 (Lichtman and Martin, 1996; Brodkin and

Moerschbaechier, 1997; Chaperon et al., 1998; Mallet and Beninger, 1998; Da Silva and

Takahashi, 2002; Suenaga et al., 2008; Wise et al., 2009). Furthermore, these cannabinoid-

induced memory impairments resemble the effects of hippocampal lesions on learning and

memory in humans (Kessels et al., 2001; Spiers et al., 2001; Astur et al., 2002; Goodrich-

Hunsaker et al., 2010) and in rats (Altemus and Almli, 1997; Gilbert et al., 2002; Broadbent

et al., 2004; Talpos et al., 2008; Faraji et al., 2008; Iordanova et al., 2009; Brady et al., 2010).

For these reasons, the detrimental effects of CB1 receptor agonists on learning and memory

have been attributed primarily to their effect on hippocampal function.

As treatment with CB1 receptor agonists produces memory impairment, it has been

hypothesised that blocking CB1 receptors might enhance or improve memory. However, the

effects of CB1 receptor inverse agonists in rats have been inconsistent. While in some studies

the CB1 receptor inverse agonists such as SR141716A and AM251, seemed to enhance

memory (Terranova et al., 1996; Takahashi et al., 2005; Lichtman, 2000; Wolff and Leander,

2003; de Oliveira Alvares et al., 2008), other studies have shown no effect (Brodkin and

Moerschbaecher, 1997; Da and Takahashi, 2002; Suenaga et al., 2008) or even impairment of

memory (de Oliveira Alvares et al., 2005, 2006; Arenos et al., 2006). Furthermore, studies

with mice lacking CB1 receptors showed that these animals have a prolonged aversive

memory and suggested that the endocannabinoid system may have a role in facilitating

extinction and/or forgetting processes (Reibaud et al., 1999; Varvel and Lichtman, 2002;

Marsicano et al., 2002; Varvel et al., 2005; see Valverde et al., 2005 for review).


Modifications of the strength of synaptic connections between neurons have been

shown to underlie the mechanism of memory formation and storage in the mammalian brain

(see Maren and Baudry, 1995 for review). LTP and LTD are forms of synaptic plasticity in the

hippocampus which have been suggested to have a role in learning and memory processes

(see Martin et al., 2000 for review). Interestingly, previous studies have shown that the

activation of CB1 receptors in hippocampal slices, either by synthetic cannabinoids or by

endocannabinoids, impairs LTP (Collins et al., 1994, 1995; Terranova et al., 1995; Stella et al.,

1997; Lees and Dougalis, 2004; Hoffman et al., 2007; Bajo et al., 2009) and LTD (Misner and

Sullivan, 1999; Mato et al., 2004; see Chevaleyre et al., 2006 for review), indicating the

involvement of the hippocampal CB1 receptors in learning and memory processes.

Furthermore, in vivo studies have demonstrated the disruption of hippocampal neuronal

function and responsiveness following the administration of CB1 receptor agonists (Hampson

and Deadwyler, 2000; Robbe et al., 2006; Robinson et al., 2007; Abush and Akirav, 2009;

Robbe and Buzsaki, 2009; Goonawardena et al., 2010). For example, hippocampal

multineuron recordings revealed that the administration of 9-THC or (R)-(+)-[2,3-dihydro-5-

methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin -6-yl]-1-

naphthalenylmethanone (WIN 55212-2; a synthetic cannabinoid CB1/CB2 receptor agonist)

dose-dependently reduced the firing rate of hippocampal principal cells during the encoding

(sample) phase of a delayed nonmatch to sample (DNMS) task (Hampson and Deadwyler,

2000). This result suggests that the encoding of information by hippocampal neurons is

important in the performance of the DNMS task (Hampson and Deadwyler, 2000). Similarly,

Robinson and others showed that the administration of the synthetic cannabinoid agonist

(6aR,10aR)-9-(Hydroxymethyl)-6,6-dimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10,a-

tetrahydrobenzo [c]chromen-1-ol (HU-210), resulted in reductions in firing frequency, and in

the bursting of CA1 and CA3 principal neurons of the hippocampus, which were proposed as

underlying mechanisms for the spatial memory deficits observed with the same drug

(Robinson et al., 2007). Overall, these results show that the endocannabinoid system plays an

important role in cognitive function.


1.3 The Association Between the Vestibular and Endocannabinoid

Systems

As previously explained (see sections 1.1.4 Vestibular-Hippocampal Connection and

1.2.3 The Role of the Endocannabinoid System in Cognitive Function), it has been suggested

that both the vestibular and endocannabinoid systems play a significant role in cognitive

function in animals and humans. Furthermore, numerous human (Scoville and Milner, 1957;

Frisk and Milner, 1990; Burgess et al., 2002; Maguire et al., 2006; Goodrich-Hunsaker et al.,

2010) and animal (Olton and Werz, 1978; Altemus and Almli, 1997; Forwood et al., 2005;

Talpos et al., 2008; Faraji et al., 2008; Iordanova et al., 2009) studies demonstrate that

hippocampal integrity is important for normal learning and memory functions which require

ongoing neuroplastic modifications, such as the formation of new synapses or a

reorganization of existing synpases (Bailey and Kandel, 1993; Moser, 1999; Rubino et al.,

2009b). Interestingly, cognitive deficits and alterations in hippocampal synaptic plasticity

following either cannabinoid receptor agonist treatment or BVD show similarities in both

experimental animal models and in human patients. A number of behavioural studies have

shown that both cannabinoid receptor agonist administration (Lichtman et al., 1995; Egashira

et al., 2002; Robinson et al., 2007; Wegener et al., 2008; Wise et al., 2009) and BVD

(Wallace et al., 2002c; Russell et al., 2003a; Schautzer et al., 2003; Brandt et al., 2005; Zheng

et al., 2007, 2009b) cause spatial memory impairment in hippocampus-dependent tasks in

both animals and humans. Such results suggest that the spatial memory deficits may, at least

in part, be due to detrimental effects of cannabinoids and BVD on the hippocampus.

Furthermore, as it has been demonstrated that many hippocampal synaptic changes are seen in

the brain after vestibular damage (Stackman et al., 2002; Russell et al., 2003b, 2006; Zheng et

al., 2010) and that the endocannabinoid system is critically involved in synaptic plasticity in

the hippocampus (see Mackie, 2008 for review), it is possible that the endocannabinoid

system may be contributing significantly to the hippocampal plasticity observed following

lesions of the vestibular system.


1.4 Research Goals

Although extensive research has been carried out on the vestibular system, the focus in

the past has been largely on the role of the vestibular system in the ocular motor and postural

reflexes, as these reflexive movements are directly associated with vestibular function

(Honrubia et al, 1984, 1985; Zennou-Azogui et al., 1993; Hamann et al., 1998; Mbongo et al.,

2009). However, there has been a persistent accumulation of evidence showing detrimental

effects of vestibular damage on cognitive functions, including attention and, learning and

memory in both animals and humans (Wallace et al., 2002c; Russell et al., 2003a; Brandt et

al., 2005; Zheng et al., 2009a, b). This has led to more quantitative investigations, and

eventually to studies of the effects of peripheral vestibular damage on the hippocampus.

While both the vestibular and endocannabinoid systems have been investigated in great

detail, no previous study has investigated the possible involvement of the endocannabinoid

system in the cognitive impairments that result from vestibular dysfunction. Therefore, the

aim of this research was to determine whether the hippocampal endocannabinoid system plays

a role in the functional changes seen after vestibular damage. By better understanding the

mechanism(s) of this phenomenon, it may be possible to minimise this pathological process,

and also to develop potential treatments which can restore cognitive function.

Therefore, the goals of this research were to:

1) Determine the optimum memory-impairing dose of the cannabinoid CB1/CB2 receptor

agonist, WIN 55212-2 („WIN‟), in an object recognition task, prior to investigating its

effects on the cognitive deficits in BVD rats (Chapter 2),

2) Determine whether BVD-induced spatial deficits would persist at 14 months following

BVD and whether any spatial deficits apparent at 14 months post-BVD could be

modulated by the administration of WIN, or the CB1 receptor inverse agonist, AM251

(Chapter 3),

3) Determine whether the density and the affinity of CB1 receptors in the hippocampus

changes following BVD (Chapter 4).

32

Chapter 2

The Effect of the Cannabinoid Receptor Agonist, WIN

55212-2, on Object Recognition Memory in Rats

Chapter 2: Cannabinoids and Object Recognition Memory in Rats 33

2.1 Introduction

It was necessary to determine the optimum dose of the cannabinoid CB1/CB2 receptor

agonist, WIN 55212-2 (WIN), prior to investigating its effect on the cognitive deficits in rats

induced by bilateral vestibular deafferentation (BVD). The object recognition memory task

was chosen to achieve this goal as this particular behavioural paradigm does not require any

pre-training, and therefore the effects of WIN in rats could be determined in a relatively short

period of time.

Learning and memory impairments are among the most renowned effects of

cannabinoids in both humans and animals (see Adams and Martin, 1996; Ameri, 1999; Diana

and Marty, 2004 for reviews). In humans, the cannabinoid receptor agonist 9-THC produces

a variety of “intoxicating” psychoactive and motor effects, including a marked impairment

and disruption of short-term memory as well as several other perceptual and disorientation

effects (Miller and Branconnier, 1983; Murray, 1986; Chait and Perry, 1992). In addition,

chronic use of marijuana also leads to a lasting impairment in cognition (Solowij et al., 1995;

Pope and Yurgelun-Todd, 1996; Pope et al., 2001), which may be due to changes in

hippocampal morphology (Lawston et al., 2000; Tagliaferro et al., 2006). Numerous

behavioural studies in animals have shown that systemic or intrahippocampal administration

of natural cannabinoid receptor agonists like 9-THC or anandamide, or synthetic agonists,

such as WIN, CP-55,940 or HU-210, impairs performance in hippocampus-dependent

memory tasks, such as the radial arm maze (Nakamura et al., 1991; Lichtman et al., 1995;

Suenaga et al., 2008; Wise et al., 2009), the water maze (Robinson et al., 2003; Marchalant et

al., 2008), and the delayed alternation task (Heyser et al., 1993;Nava et al., 2000, 2001;

Suenaga et al., 2008).

Recognition memory, although it can be defined in many ways, is generally regarded as

the ability to discriminate the familiarity of items or events that have previously been

encountered and is considered to be a critical component of declarative memory (Mumby,

2001). Declarative memory is characterised as the conscious memory for facts and events and

is further divided into episodic memory (memory for personal events) and semantic memory

(memory for general information; Squire and Zola, 1996; Winters et al., 2008). On the other

hand, procedural memory for habits or skills is defined as a non-declarative memory, and

requires an extensive acquisition phase (Winters et al., 2008). Declarative memory can be

acquired with relatively fewer exposures to the material to be learned, compared to procedural


memory.

Object recognition memory is commonly impaired in amnesic patients who lose their

ability to recognise people and objects they have come across since the onset of amnesia. For

the past 20 years, a variety of behavioural methods for studying object-recognition memory in

animals have been developed, which include the delayed nonmatching-to-sample (DNMS)

task, delayed matching-to-sample (DMS) task, and spontaneous object recognition (SOR) task

(see Mumby, 2001; Winters et al., 2008 for reviews).

The SOR task is a variation of the DNMS paradigm to assess object recognition

memory. It is referred to as spontaneous because it exploits the innate tendency of rats to

explore novel stimuli in preference to familiar stimuli (Ennaceur and Delacour, 1988).

Rodents readily approach objects and investigate them physically by touching and sniffing the

objects, rearing upon and trying to manipulate them with their forepaws (Aggleton, 1985).

The similarity between the DNMS and SOR tasks is that the behaviour of normal animals in

the test or choice phase is driven by a single exposure to sample objects and the subsequent

recognition (Winters et al., 2008). In the standard version of the SOR task, a rat is placed in

an open field arena and allowed to explore the sample objects in the arena for a fixed duration,

usually few minutes (5-10 minutes). This period is called the sample phase. The rat is then

taken away from the arena for a retention delay, which lasts several minutes to hours, and then

returned to the arena with one of the sample objects changed to a novel one. This is called the

test phase. By nature, rats should spend more time exploring the novel object than previously

encountered objects (Dere et al., 2007). Therefore, when there is a bias in time towards

exploring the novel objects compared to sample objects, it is suggested that the rat remembers

the sample objects and therefore this can be taken as an index of recognition of the familiar

sample object. The major advantage of this task over food-rewarding maze learning tasks and

classical DNMS or DMS tasks, is that it does not require spatial learning, food or water

deprivation, the application of any explicit reinforcement (food or electric shock delivery), the

learning of test rules or of response-reward correlations. Therefore, the study can be done

with little or no training and it is also less stressful for the animals than the negative

reinforcement-based tasks, such as the hidden platform version of the water maze, inhibitory

and active avoidance, or fear conditioning tasks, which have been extensively utilised to study

the neurobiology of learning and memory in rodents (see Winters et al., 2008 for review).

Many experimental paradigms, such as Morris water maze, radial arm maze, and


instrumental discrimination tasks, have been employed to investigate the role of cannabinoids

in learning and memory. However, the majority of these paradigms are based on conditioning,

yet recognition memory is a non-conditioned behaviour. Therefore, the evaluation of the

influence of cannabinoids on recognition memory can further provide evidence regarding the

involvement of the endocannabinoid system in learning and memory processes. Recognition

memory impairments induced by the systemic administration of cannabinoid receptor agonists

have been reported previously (Ciccocioppo et al., 2002; Schneider and Koch, 2002; Kosiorek

et al., 2003; Schneider et al., 2008). Furthermore, there are many studies involving direct

administration of cannabinoids into the hippocampus that have investigated performance in

the SOR task (Barna et al., 2007; Clarke et al., 2008; Suenaga and Ichitani, 2008). Although

some studies have reported object recognition deficits in adult rats (Schneider and Koch, 2002;

O‟Shea et al., 2006; Barna et al., 2007), most have reported that pubertal or pre-pubertal

exposure to CB1 receptor agonists is more detrimental (Schneider et al., 2005, 2007; O‟Shea

et al., 2006; Quinn et al., 2008). Furthermore, these studies support the notion that exposure

to Cannabis sativa during adolescence may predispose humans to cognitive impairment and

possibly even psychosis, later in life (Schneider and Koch, 2003; Schneider et al., 2008).

The aim of this study was to further investigate the issue of whether exposure to a CB1

receptor agonist affects rats during adulthood, by examining the dose-dependent effects of

WIN in an object recognition task in adult rats. Since most studies have employed multiple

injections (O‟Shea et al., 2004, 2006; Schneider and Koch, 2003, 2005, 2007; Quinn et al.,

2008; Schneider et al., 2008), the second issue of interest was whether a single injection of

the drug would interfere with object recognition. Furthermore, the results from the present

study would reveal the optimum dose of WIN for rats, to then be used to treat BVD animals

during performance in the foraging task (Chapter 3).


2.2 Materials and Methods

All experiments described throughout this thesis were approved by the University of

Otago Animal Ethics Commiittee (07/06). The animals were treated with respect and every

effort was made to ensure their wellbeing. In order to minimise the number of animals

involved in the study, efforts were made to maximise the data yield from each animal. The

animals were maintained on a twelve-hour light-dark cycle at 22o C and housed four per cage.

All experimental procedures throughout this thesis were conducted during the light period of

the light-dark cycle.

2.2.1 Animals

Twenty-nine naïve adult male Wistar rats (7 months old), weighing 500-600 g, were

used for this study. These animals were taken from another project in which the animals were

not exposed to any drug. Animals were randomly divided into four groups: (1) vehicle control

(n = 7); (2) 1 mg/kg WIN (n = 7); (3) 3 mg/kg WIN (n = 7); (4) 5 mg/kg WIN (n = 8).

2.2.2 Drugs

The cannabinoid agonist, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-

morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin -6-yl]-1-naphthalenylmethanone (WIN

55212-2, WIN) was purchased from Tocris Bioscience (Bristol, UK). WIN was dissolved in

dimethyl sulfoxide (DMSO, Merck) and diluted in saline (0.9% sodium chloride), at a final

concentration of 1:1 ratio (DMSO:Saline). The drug was administered subcutaneously (s.c.) at

doses of 1, 3, or 5 mg/kg. The doses utilised were determined based on previous studies

showing the effects of cannabinoids on learning and memory (Lichtman et al., 1995;

Schneider, M. & Koch, M. 2002; Robinson et al., 2003; Chambers et al., 2004; Bhatti et al.,

2009; Shoaib, 2008). The vehicle solution was prepared in a similar manner, with the

exception that the drug was omitted (50% DMSO in saline). Each animal was weighed on the

day of injection and received a single dose of WIN or vehicle, 30 minutes prior to the testing

session.

2.2.3 Object Recognition Memory Test

The test was modified from Zheng et al. (2004). The duration of testing was three days.


The test was carried out in a 60 x 60 x 35 cm grey-painted wooden box, in which rats were

tested individually (Figure 2.1d). A video camera was mounted on the ceiling directly above

the testing apparatus to record animals‟ behaviour for future analysis. On day 1, each animal

was placed in the box for 10 minutes for habituation. No objects were placed in the arena

(Figure 2.1a). On day 2, each animal had two identical 10 minute sessions exploring four

identical plastic objects (4 x 6 cm) placed in the box (Figure 2.1b). The inter-trial interval was

30 minutes. The location of each object was kept constant for each rat and counter-balanced

across the rats and groups. All objects and the test arena were cleaned with diluted

disinfectant and thoroughly dried before each session and between each rat. On day 3, animals

were tested for their reaction to novelty. In the first session of day 3, animals were placed in

the arena with identical conditions to the previous day (Figure 2.1b). After the first session, a

subcutaneous injection of WIN or vehicle was given to each rat. Thirty minutes later, the

second session was run, in which one of the sample objects was replaced with a novel one

(Figure 2.1c). The location of the novel object was carefully randomised across the groups in

order to prevent location effects for particular groups. In another words, different groups

received equal exposure to the novel object in the different spatial position. The exploration

was defined as the duration of time the animal spent directing its nose at a distance within 2

cm of the object and/or touching the object. The total exploration time and time spent

exploring each object was recorded and analysed. Following Winters et al. (2004), a

discrimination index, defined as the difference between the time spent exploring the novel

versus the familiar objects, divided by the total time spent exploring objects, was also

calculated in order to control for the general effects of WIN on object exploration. A decrease

in the discrimination index would indicate a decline in an animal‟s ability to discriminate the

novel object from the familiar ones.

2.2.4 Statistical Analysis

Prior to any analysis, the data were tested for the parametric statistical assumptions of

normality and homogeneity of variance. The data were found to violate these assumptions and

therefore were square root transformed. Two-way analyses of variance (ANOVAs) with

repeated measures on time were then performed followed by Tukey‟s post-hoc comparisons

(Winer et al., 1991). The discrimination index data were subjected to a one-way ANOVA

followed by Tukey‟s post-hoc comparisons (Winer et al., 1991). P < 0.05 was considered

significant in all cases.


a) Day 1 b) Day 2 & 1st Session of Day 3

c) 2nd

Session of Day 3 d) Test Apparatus

Figure 2.1

The object recognition memory testing conditions for each day. (a) Habituation in the testing

arena without any objects on day 1. (b) Sampling phase with 4 identical objects (red circles) on

day 2 and on first session of day 3. (c) Reaction to novelty on day 3, where one of the sample

objects is changed to a novel one. (d) Photograph of object recognition memory task testing

arena with a novel and sample objects.


2.3 Results

2.3.1 Total Exploration Time

The total exploration time was significantly reduced with the administration of WIN in

a dose dependent manner (F(3,50) = 3.09, P = 0.035; Figure 2.2). When the novel object was

introduced in session 2, there was an apparent increase in the exploration time in the vehicle-

treated group. However, this increase was not significant as there was no significant session

effect or any significant interaction between the drug and the session. Post hoc comparisons

revealed that only 5 mg/kg WIN-treated animals spent significantly less time overall

exploring objects (both familiar and novel) compared to the vehicle treated animals (P =

0.033; Figure 2.2).

Figure 2.2

Total exploration time (in seconds) for session 1 (before drug administration) and session 2

(after drug administration) for the vehicle treated group and groups receiving 1, 3 or 5 mg/kg

WIN. Data are represented as means + SEM. There was a significant difference between the

vehicle and the 5 mg/kg WIN group only (* P = 0.033).

0

10

20

30

40

50

60

Vehicle 1 mg/kg WIN 3 mg/kg WIN 5 mg/kg WIN

Tota

l E

xp

lora

tion

Tim

e (

s)

Session 1

Session 2*


2.3.2 Exploration Time by Objects

When the exploration time towards each object was analysed, WIN-treated animals

spent significantly less time exploring the novel object compared to vehicle-treated animals

(F(3,50) = 7.06, P = 0.000; Figure 2.3). However, animals in all groups spent a longer time

exploring the novel object than the familiar object (F(1,50) = 53.55, P = 0.000) and there was a

significant interaction between the drug and the object (F(3,50) = 6.02, P = 0.001; Figure 2.3).

This suggests that the increase in the exploration time for the novel object was smaller for the

WIN-treated animals. Post hoc comparisons indicated that the time spent exploring the novel

object for the 5 mg/kg WIN-treated group was significantly lower than for the vehicle- (P =

0.0005) and 1 mg/kg WIN-treated groups (P = 0.008).

Figure 2.3

Mean exploration time (in seconds) for familiar and novel objects for the vehicle-treated group

and the groups receiving 1, 3 or 5 mg/kg WIN. Data are represented as means + SEM. Post hoc

comparisons indicated that the time spent exploring the novel object for the vehicle group was

significantly greater than for the 5 mg/kg WIN group (* P = 0.0005), and exploration time of the

novel object for the 1 mg/kg WIN group was significantly greater than for the 5 mg/kg WIN

group (* P = 0.008).

0

10

20

30

40

50


Exp

lora

tion

Tim

e b

y O

bje

cts

(s) Novel

Familiar

*

*


2.3.3 Discrimination Index

The discrimination index was calculated and analysed in order to control for the general

effect of WIN on object exploration. Vehicle-treated animals showed a significantly higher

discrimination index compared to the three WIN groups (F(3,28) = 3.51, P = 0.03; Figure 2.4).

Post hoc comparisons showed that this was due to the 1 mg/kg WIN-treated group having a

significantly lower discrimination index than the vehicle group (P = 0.028).

Figure 2.4

The discrimination indices for the vehicle-treated group and the groups receiving 1, 3 or 5

mg/kg WIN. Data are represented as means + SEM. The discrimination index was significantly

lower in the 1 mg/kg WIN-treated group compared to the vehicle group (* P = 0.028), while there

were no differences between the discrimination index of the vehicle group and animals treated

with the higher doses of WIN.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8


Dis

crim

inati

on

In

dex

*


2.4 Discussion

In this study, activation of cannabinoid receptors dose-dependently decreased the

exploration time towards objects, whether novel or familiar, in adult rats. However, this was

significant only in the 5 mg/kg WIN-treated animals compared to vehicle. When a novel

object was introduced, vehicle-treated animals spent significantly more time exploring the

novel object, while WIN-treated animals did not. The discrimination index set a distinction

between the measures of exploration, motor activity, and novelty-preference as this index

controls for the general effects of WIN on the exploration of familiar as well as novel objects.

As shown in Figure 2.4, only the lowest dose of 1 mg/kg WIN specifically reduced the

discrimination index, indicating that the impairment in object recognition was only at the

lowest dose. Moreover, no significant changes in the total exploration time in the vehicle- and

1, 3 mg/kg WIN-treated groups further indicates that novelty-seeking behaviour and motor

activity are differentially altered by administration of WIN. Therefore, an apparent deficit in

object recognition at a high dose of WIN (i.e. 5 mg/kg) may be compromised as a result of a

general decrease in exploration that affects familiar as well as novel objects. The results of the

present study are consistent with those reported by others in which the effective cannabinoid

drug dose for producing object recognition memory deficits ranges from 0.3 to 1.2 mg/kg

(Schneider and Koch, 2003; O‟Shea et al., 2006; Schneider et al., 2008).

The results of the present study confirm that the adverse effects of CB1 receptor agonists

on object recognition are not restricted to juveniles, and are supported by similar results

reported by others in adult rats (Schneider and Koch, 2002; O‟Shea et al., 2005; Schneider et

al., 2008). Thus the present study adds to a growing body of evidence that object recognition

in adult rats is significantly affected by WIN, despite well-established findings that

behavioural and cognitive deficits seen following CB1 receptor agonist treatment are more

pronounced in pubertal than in adult rats (Schneider and Koch, 2003; O‟Shea et al., 2004;

Cha et al., 2006; Quinn et al., 2008).

Another confirmational finding of the current study is that a single WIN treatment is

sufficiently potent to induce deficits in object recognition in adult rats. Although numerous

studies have used chronic injections of cannabinoid receptor agonists such as 9-THC (Quinn

et al., 2008), WIN (Schneider and Koch, 2003, 2005, 2007; Schneider et al., 2008), or CP-

55,940 (O‟Shea et al., 2004, 2006), only a small number of studies have used acute treatment

with cannabinoid receptor agonists to determine their effect on object recognition memory in


rats (Schneider and Koch, 2002; Schneider et al., 2008; Fox et al., 2009). Furthermore, a

study by Schneider et al. showed that repeated administration of WIN in adult rats did not

induce any permanent alterations in the discrimination index, object recognition and object

discrimination, whereas acute WIN treatment showed a significant reduction (Schneider et al.,

2008). Since animals have shown the ability to develop tolerance to cannabinoid drugs upon

repeated exposure (see Gonzalez et al., 2005 for review), it is advantageous that single WIN

treatment can induce recognition memory impairments in adult rats.

Object recognition memory is often attributed to the perirhinal cortex and CB1 receptors

are known to be expressed in high densities in this brain region (Liu et al., 2003a). However,

numerous studies have shown that direct administration of cannabinoids into the hippocampus

impaired object recognition memory (Barna et al., 2007; Clarke et al., 2008; Suenaga and

Ichitani, 2008). Furthermore, hippocampal lesion studies have also shown impairment in

object recognition memory, suggesting that the hippocampus also plays a role in the object

recognition memory (Clark et al., 2000; Zola et al., 2000; Broadbent et al., 2004, 2009).

Overall, the results from this investigation suggest that 1 mg/kg WIN is sufficient to produce

cognitive deficits in healthy adult rats. Therefore, whether or not the administration of this

drug will influence spatial memory deficits in BVD rats can now be investigated, and is

described in the next Chapter of this thesis.

44

Chapter 3

The Effect of Cannabinoids on Spatial Memory Following

Bilateral Vestibular Deafferentation in Rats

Chapter 3: Cannabinoids and Spatial Memory in BVD Rats 45

3.1 Introduction

In the previous Chapter, it was revealed that 1 mg/kg WIN is sufficient to produce

cognitive deficits in healthy adult rats. Therefore, the effect of this dose of WIN on the

performance of BVD rats in a spatial memory task was investigated in the present study. The

administration of cannabinoid drugs to the BVD animals will address the issue of whether or

not the endocannabinoid system is involved in the pathophysiology of the cognitive

impairments that are observed following bilateral vestibular damage.

Rodents, like many other small animals, must be able to forage for food in their home

ranges while minimising the risk of predation. For successful foraging, animals must

remember their home location and/or keep track of their movements and position in relation

to the home base. The ability to navigate by using external (allothetic) cues, such as visual,

auditory, and magnetic cues, is referred to as piloting. This ability has been shown to exist in

mammals (Sutherland and Dyck, 1984; Collett, 1987; Schenk, 1987), birds (Vander Wall,

1982; Cheng, 1988; 1989), and insects (Collett and Land 1975; Cartwright and Collett, 1982,

1983; Gould, 1987). Although the use of external cues allows animals to navigate accurately,

a single strategy is insufficient in the unpredictable nature of environments. In other words, if

the animal is exposed to a novel or unfamiliar environment where external cues are either

absent or in conflict with previous experience, then the animal has to rely on self-movement

cues, to return to the point where movement originated.

It is a long-standing concept that the vestibular apparatus of vertebrates might serve as

the prime sensing element for animal navigation. In the early 1960s, Schmidt-Koenig, (1965)

defined navigation as an animal‟s ability to maintain or establish reference to a goal other than

through recognition of known landmarks. Indeed navigation requires the information of

present position, direction and magnitude of motion with respect to other points of reference

(Fernandez and Macomber, 1962). Furthermore, Maaswinkel and Whishaw (1999) suggested

that there is a hierarchy in the use of sensory cues during spatial navigation, at least in rats.

Visual cues are used primarily, and then olfactory cues, then other self-movement cues. Other

cues that provide information about self-motion are referred to as idiothetic cues, which

include vestibular information, proprioceptive information, sensory flow, and efferent copies

of motor commands.

The navigational strategy that uses idiothetic cues is known as „dead reckoning‟ or „path


integration‟ and is colloquially called „sense of direction‟. Particularly important in dead

reckoning is the information from the vestibular system, as it provides the concepts of

horizontality and verticality and allows the detection of changes in acceleration of the body.

The linear and angular acceleration signals from the vestibular system are critical self-

movement cues as they allow the computation of current location and direction in space

relative to a starting location. This is hugely advantageous for an animal, because it means

that the animal can return directly to its home base, even following a complex outward trip.

Directional estimation based on self-movement cues during a foraging task has been shown to

be impaired when the vestibular system is damaged (Wallace et al., 2002c; Zheng et al., 2006,

2009b). While it has been suggested that rats appear to preferentially use allothetic cues,

(primarily visual), over idiothetic cues, many cues can be used flexibly and concurrently

(Maaswinkel and Whishaw, 1999).

Numerous electrophysiological studies provide evidence for the contribution of the

vestibular system to spatial guidance (Sharp et al., 1995; Stackman and Taube, 1997;

Stackman et al., 2002; Russell et al., 2003b, 2006; van der Meer et al., 2007; Bassett et al,

2007; see Smith et al., 2005a, b for reviews). Dead reckoning was first suggested by Darwin

(1873) as a form of navigation and it has been demonstrated in many controlled laboratory

conditions by removing allothetic cues from a testing situation (Etienne et al., 1986;

Maaswinkel et al., 1999; Wallace et al., 2002b; Hines and Whishaw, 2005; Zheng et al.,

2009b). Moreover, a number of animal behavioural studies have demonstrated that vestibular-

lesioned rats are highly dependent upon allothetic cues when performing in a spatial

navigation task, and their ability to utilise dead reckoning is impaired (Stackman and Herbert,

2002; Wallace et al., 2002c; Zheng et al., 2006, 2009b). This reliance on allothetic cues,

especially visual cues, has also been shown in vestibular-deficient patients, where only minor

navigational deficits were observed in the light condition, but marked impairments in spatial

navigation were observed in the dark or when patients were blind-folded (Heimbrand et al.,

1991; Pozzo et al., 1991; Brookes et al., 1993).

In order to effectively assess the role of vestibular information in spatial navigation, it is

important to use a task in which the dead reckoning calculation is made immediately

following the completion of a circuitous outward trip, where each trial is different. Rats have

a natural tendency to collect food and carry it back to a refuge for consumption. This unique

characteristic has been shown to be a robust means by which to dissociate the use of allothetic

and idiothetic cues when foraging in a visually rich environment. First developed by Whishaw


and Tomie (1997), the foraging task has been used widely to assess animals‟ exploratory

behaviour and their navigational strategies. The task involves a rat leaving a home base and

foraging for a hidden food pellet on a large open circular table. The location of the food pellet

changes between trials, therefore, the circuitous outward searching paths made by a rat will

also change in length, duration, and complexity, depending upon how quickly the food is

found. Rats display different food-handling behaviour toward different sizes of food pellet

(Whishaw et al., 1990). Therefore, the size of the food pellet has to be sufficiently large so

that the rat will carry it back home to eat it, rather than eating it on the table (Whishaw et al.,

1990, 1995). The home base is positioned at the periphery of the table and is hidden

underneath the surface so that it is indistinguishable from seven other holes. Therefore, each

trip is unique with respect to direction and distance. For an accurate return to the home base, a

rat must know the location of the home base in relation to visual cues learned from previous

trials, or else calculate the home direction from its own self movement cues, and thus generate

a direct homeward route. Starting the animals from novel locations and performing the task in

darkness are the versatile features of the foraging task that allow the assessment of which

method is preferentially used when navigating. If a rat returns to an old home location, it is an

indication that it used allothetic cues, mainly visual cues, to guide itself home. However, if a

rat chooses the novel location, then that is an indication that it disregarded the previous

experience and responded to idiothetic cues derived from its just-completed trip.

As previously described, both the vestibular and endocannabinoid systems play a role in

spatial memory functions in animals and humans (see sections 1.1.4 Vestibular-Hippocampal

Connection and 1.2.3 The Role of the Endocannabinoid System in Cognitive Function in

Chapter 1). While Zheng and others have shown that BVD animals have spatial memory

impairment at 5-7 months following BVD surgery in a foraging task (Zheng et al., 2009b),

whether this effect of BVD on spatial navigation is permanent or not has never been

investigated. Furthermore, although the impairment in spatial learning and memory following

cannabinoid receptor agonist treatment has been thoroughly analysed using the classic spatial

memory tasks such as the radial arm maze and the Morris water maze, these tasks cannot

separate the use of self-movement cues from the allothetic cues used by animals. Given that

both the administration of cannabinoid receptor agonists (Lichtman et al., 1995; Robinson et

al., 2003, 2007; Wegener et al., 2008; Yim et al., 2008; Wise et al., 2009) and damage to the

vestibular system (Russell et al., 2003a; Zheng et al., 2006, 2009b) impair animals‟ spatial

memory, the present study aimed to investigate whether BVD-induced impairment in spatial

navigation may be influenced by the administration of a CB1/CB2 receptor agonist, WIN


55212-2 (WIN) and/or a CB1 receptor inverse agonist, AM251.



3.2.1 Animals

Thirty-two adult male Wistar rats were used, each weighing 250 – 300 g at the time of

surgery. All animals received either the sham or BVD surgery 14 months prior to the

behavioural testing. However, 4 animals were lost due to aging before the behavioural testing

commenced. The animals were randomly divided into four treatment groups: (1) sham surgery

with vehicle control (n = 8); (2) sham with WIN (n = 7); (3) BVD with vehicle control (n = 6);

(4) BVD with WIN (n = 7). The animals were food deprived to 85% of their normal feeding

weight before and during the behavioural testing. The animals‟ body weight was measured

and recorded every day. Large oat and wheat honey cereal loops were used as food rewards

during behavioural testing. The size of the food reward used in the current study was

validated in previous studies (Zheng et al., 2006, 2009b). After each day of testing, the

animals were given supplementary laboratory rodent food in their home cage in order to

maintain body weight. The necks of rats were spray-painted black (Donaghys Industries Ltd,

Christchurch, New Zealand) to enable tracking and analysis of their movement using the

custom-made software (RatTracker, designed and programmed by the Departmental

Technician, Mr. Kevin Markham).

3.2.2 Peripheral Vestibular Lesion Surgery

In the rat, the surgical lesion of the peripheral vestibular apparatus is a complicated

procedure due to its anatomical location in the inner ear. Moreover, the stapedial artery across

the oval window beneath the stapes, sets further restrictions on the procedure. Because the

vestibular apparatus is accessed via the cochlea, lesions of the cochlea are unavoidable. As a

result, auditory function is completely lost in BVD surgery, in addition to vestibular function.

The surgery was conducted under a general anaesthetic of ketamine hydrochloride (760

g/kg, s.c.), medetomidine hydrochloride (300g/kg, s.c.) and atropine sulfate (80g/kg,

s.c.). The wound margin was anaesthesised locally with xylocaine (5 mg/wound margin, s.c.).

Under microscopic control, the tympanic membrane was exposed using a retro-auricular

approach and the tympanic membrane, malleus, and incus were removed. The stapedial artery

was cauterised and the horizontal and the anterior semicircular canal ampullae drilled open.

The contents of the canal ampullae and the utricle and saccule were then aspirated, and the

temporal bone was sealed with dental cement. Carprofen (5 mg/kg, s.c.) was used for post-


operative analgesia and atipamezole hydrochloride (5 mg/kg, s.c.) was used to reverse the

effect of medetomidine hydrochloride. Using temporal bone histology, our previous studies

have shown that the BVD surgical procedure produces a complete and permanent lesion of

the vestibular labyrinth with no damage beyond the temporal bone (Zheng et al., 2006).

Sham surgery consisted of exposing the temporal bone and removing the tympanic

membrane without producing a vestibular lesion. This procedure provided a partial auditory

control that involved damage to the tympanic membrane only, with no other surgical trauma.

All other procedures such as anaesthesia and recovery were the same for the sham as for the

lesioned animals.

3.2.3 Drugs

The cannabinoid CB1/CB2 receptor agonist, WIN 55212-2 („WIN‟), and the cannabinoid

CB1 receptor inverse agonist, AM251, were both purchased from Tocris Bioscience (Bristol,

UK). AM251 is chemically similar to SR141617A except that it has a 4-iodophenyl group

instead of 4-chlorphenyl in SR141617A, with a similar potency to SR141716A in tissue

homogenate binding assays (Gatley et al., 1998). The half-lives of WIN and AM251 in rat are

approximately 3 and 22 hrs, respectively (McLaughlin et al., 2003; Agu et al., 2006). WIN

was dissolved in dimethyl sulfoxide (DMSO, Merck) and diluted with saline (0.9% sodium

chloride) in a 1:1 ratio (DMSO : saline) yielding a final concentration of 1 mg/mL, whereas

AM251 was dissolved in a 75% DMSO solution in saline at a concentration of 1 mg/mL. The

drugs were administered s.c. at doses of 1 and 2 mg/kg for WIN and 3 mg/kg for AM251. The

doses utilised were determined based on the previous studies (for WIN, see Chapter 2; Baek

et al., 2009; for AM251, Rodgers et al., 2005; Arenos et al., 2006; Chambers et al., 2006;

Nawata et al., 2010). WIN or vehicle was administered once daily for 21 days. Even though

there may be differences in the effects of WIN under conditions of single versus multiple

injections, the initial effect of WIN on day 1 would essentially be the same as giving a single

injection. The higher 2 mg/kg dose was used when 1 mg/kg did not appear to have a

substantial effect on performance in the foraging task. The vehicle solution was prepared in a

similar manner, with the exception that the drug was omitted.

3.2.4 Foraging Task Apparatus

The apparatus was similar to those used by other researchers and in previous studies in


our research group (Whishaw and Tomie, 1997; Maaswinkel et al., 1999; Wallace et al.,

2002c; Zheng et al., 2006, 2008b). The apparatus consisted of a 140 cm diameter circular

wooden white table that was elevated 105 cm above the floor (Figure 3.1). Eight 10 cm

diameter holes were located at equal distances around the perimeter of the table, centered 10.5

cm from the table‟s edge, which served as potential home bases for the rats. The table was

mounted on a central bearing such that the table could be rotated between animals. A smaller

circular table (32 cm in diameter) was fixed in the centre of the large table so that it remained

stable when the large table was rotated. This setup was to ensure that the potential odour cues

left on the table could be displaced. These characteristics of the apparatus allowed the home

cage location to be either fixed or variable in relation to the room. A home cage was placed

beneath one of the eight holes from which a rat could climb out onto the table. A metal sheet

was inserted at the bottom of the other seven holes to prevent accidental falls by BVD rats.

Black fabric was placed on top of the metal sheet in order to prevent reflection from the lights

directly above the table, or from the infrared light in the dark condition. The 8 holes had an

identical appearance when viewed from the table surface. A 5.5 cm high transparent perspex

edging was also fitted around the table to prevent falls. Twenty-three food cups (4 cm in

diameter and 1 cm in height) were evenly distributed on the surface of the table.

The apparatus was located in a test room. In this room, many visual cues were available

which included pictures on the wall, the door, and furniture such as cabinets and the computer.

A disc (195 cm in diameter) with a centre bearing was mounted on the ceiling, 130 cm above

the table. An opaque curtain was hung on this disc so that the table could be enclosed from all

visible light during dark conditions. The room lights remained off for both light and dark

conditions. In the light conditions, the apparatus and the room were illuminated by three disc

lights, which were positioned on the disc, 120o apart from each other. In the dark conditions,

the disc lights were turned off and an infrared light source, located inside the curtain, was

used for visualisation of a rat‟s movement through the camera. Rats are unable to see infrared

light (Neitz and Jacobs, 1986). An infrared camera was placed above the centre of the table to

record the movements of the animals under both light and dark conditions. A speaker playing

constant white noise was also mounted above the centre of the table. This was used to

eliminate any possible polarised auditory cues which may affect the performance of the

animals (Rossier et al., 2000; Watanabe and Yoshida, 2007).


Rotatable

table

32 cm

Fixed

platform

10.5 cm 10 cm

Food cup

Hole

Transparent

edging

Ceiling

disc

130 cm

195 cm

10 cm

10 cm

Lights

Home cage

140 cm

105 cm

Ceiling

Floor

Figure 3.1

The foraging task apparatus. (a) A side view of the apparatus. The home cage was hidden

beneath the surface of the circular table. The lights and white noise generator were located on

the ceiling disc above the table. (b) Dorsal view of the table with 8 potential exit holes around

the periphery. The smaller circular table (dashed line) was fixed in the centre of the larger table,

allowing the larger table to be rotated. The positions of 23 food cups are indicated by open

circles (not drawn to scale).

a)

b)


3.2.5 Foraging Task Procedure

Pre-Training

Before the commencement of the formal tests, the rats were trained until they met the

criterion, which was to retrieve 4 food pellets per 10 minute session. All of the food cups were

baited for the first 2-3 days of the pre-training period. The number of baited cups was

gradually decreased on subsequent days until only 1 food cup was baited per trial. A trial was

defined as an exit from the home cage and a return to the cage with a food pellet. On some

occasions, well-trained rats failed to return to the home cage with a food pellet. Such

responses were classified as a failed trial. The home cage was located beneath the same hole

and at the same location in relation to the room throughout the pre-training. During the pre-

training, the rats learned to climb out of the home cage, search for the food pellet on the table,

and carry it back to the cage. After the rat retrieved a food pellet and returned to the home

cage, a new food pellet was placed in one of the food cups while the rat ate. Some of the rats

initially attempted to eat the food on the table surface rather than carrying it back to the home

cage. They were discouraged from doing so by taking away the pellet or poking them gently

every time they began to eat. All rats quickly learned to discontinue this behaviour and carried

the pellet to the home cage. Pre-training was completed when the rat‟s performance was

stabilised to the 4 search and retrieval trials in succession. The room lights were turned off,

the ceiling frame lights were turned on, and the curtain surrounding the table was drawn back.

Light Probe Trial

In principle, when rats navigate, they can use allothetic cues, idiothetic cues, a

combination of both types, or interchange between cues (Maaswinkel and Whishaw, 1999).

Therefore, in order to determine which cues rats primarily use in light, a probe trial was given

after the completion of pre-training. Throughout the pre-training, the animals were trained

from a single starting location. However, in the light probe trial, the home location was

changed to a novel position, which was located 90o from that in the pre-training. All other

room settings remained exactly the same as in the pre-training phase. Thirty minutes prior to

the trial, each rat was given either the vehicle control or 1 mg/kg WIN (s.c.). Before each rat

began the trial, the foraging table was cleaned with disinfectant to remove any surface cues.

Dark Training

Following the light probe trial, the rats were given 1 day (4 trials) of „normal‟ training in

light where the home cage was positioned at the same location as during the pre-training trials.

The rats did not receive any drug treatment on the „normal‟ training day. After a day of


„normal‟ training, the rats were then tested in the dark with the curtain surrounding the table.

The training lasted for 21 days. Each rat was subjected to 1 trial per day, in which the position

of the home cage was changed and a different food cup was baited each day. The table was

cleaned with disinfectant between animals. Each animal was administered either vehicle

control or WIN, 30 minutes prior to each trial throughout dark training. One mg/kg WIN (s.c.)

was administered from day 1 to 10, and then the dose was increased to 2 mg/kg from day 11

to 20, due to the apparent lack of effect of the 1 mg/kg dose (described in Results section). On

day 21, all of the animals received AM251 (3 mg/kg, s.c.) 15 minutes before the vehicle

control or WIN injection to determine whether the effect of WIN could be blocked by a single

injection of the cannabinoid receptor inverse agonist.

3.2.6 Data Acquisition

Animals were tested in the foraging task 14 months following BVD surgery. The

number of sessions to reach the foraging task criterion (the retrieval of 4 food pellets per

session per day; Rubino et al., 2009a, b) was recorded in the pre-training phase. Each rat‟s

foraging trip was divided into two segments: searching and homing. The path length (cm), trip

duration (sec) and velocity (cm/sec) for both the searching and homing trips were obtained. In

addition, the first home choice, the second home choice, and the number of choices made

before reaching home (number of errors), and the initial heading angle (o; the direction that

the animal turned immediately after finding food to return home, see detailed description

below), were also obtained from the homeward trip segment. During the light probe trial, the

number of visits made to the old home was also recorded. The number of animals that

completed the task on days 1, 10, 11, and 20 of the dark training was also recorded (Rubino et

al., 2009a, b). These days were chosen as they marked either the first or the last day of WIN

administration at 1 mg/kg or 2 mg/kg. Each exploratory trip was analysed using DVD

playback displayed on a PC and using custom-coded path-tracing software (Figure 3.2). The

sampling rate of the path tracing software was 20 frames/sec.

To measure the initial heading angle, the image constructed by the custom tracking

software was transferred to Adobe Photoshop and enlarged. From this image, the initial

heading angle was measured by using a protractor, where 0o was defined as the most direct

angle possible toward the correct home from the baited food-cup. The angle was measured in

a counter-clockwise direction from a point where the animal had travelled 15 cm from the

baited food cup on the foraging table (Figure 3.3).


Figure 3.2

The display window of the custom-coded path tracing software on a PC. (a) A window showing

the current camera view of the foraging table. (b) A window that played the DVD recordings of

the animals’ movement for path tracking. (c) Control panel for setting the frame capture rate

and also to start and end the path tracking. (d) An example of the tracing image constructed for

each animal. The initial heading angle was measured from this image.

Figure 3.3

Schematic diagram of measuring the initial heading angle, derived from measuring the angle

between the line of the shortest return route to home (green line) and the line constructed from

the food cup to the heading point where the animal had travelled 15 cm along the homeward

path (red line) on the foraging table.

a) b)

c)

d)

Homeward path

Food

Heading angle

Home 15 cm



Statistical analyses were performed using various statistical packages including SPSS

17, Oriana 2, Prism 5, and Minitab 15. Anderson-Darling and Levene‟s tests were used to

ensure that the parametric assumptions of normality and homogeneity of variance,

respectively, were not violated. Where assumptions were found to be violated, a natural log

(ln) or square-root data transformation was applied and the data were retested for normality

and equal variance (Rice, 2007). The number of animals in each group varied slightly

between analyses as animals that did not complete the task could not be included in the

analysis.

The number of sessions needed to reach the foraging task criterion was analysed using a

non-parametric Mann-Whitney U test, as the data showed a non-normal distribution which

could not be resolved by data transformation. An area under the curve (AUC) calculation was

used to extract information that was contained in repeated measurements over time (Pruessner

et al., 2003). The AUC values of the pre-training searching and homing time were used to

perform a one-way ANOVA. All of the light probe trial data were analysed using two-way

ANOVAs, except the home choice data which were analysed using circular statistics.

The dark training data analyses for the searching and homing distance, time, velocity,

and the number of errors were performed using a linear mixed model (LMM) analysis using

SPSS 17. The LMM was chosen as an alternative to the general linear model (GLM) with

multi-level ANOVAs, as the data were measured repeatedly, were heavily correlated, and had

unequal samples sizes (Rice, 2007). LMM was performed using a restricted maximum

likelihood procedure, and the most appropriate covariance matrix structure was chosen based

on the smallest Schwarz‟s Bayesian Criterion (Kutner et al., 2005). The AUC calculation was

used to extract information that was contained in repeated measurements over time (Pruessner

et al., 2003). The AUC calculation was essentially an integration of the repeatedly measured

data. After calculating AUC values in Prism 5, three-way ANOVAs were carried out using

SPSS 17 with surgery, treatment, and dose as the factors for each of the dependent variables:

searching and homing distance, time, velocity, and number of errors. For the dose effect, the

AUC values were blocked into 1 and 2 mg/kg WIN treatment.

The analysis of the initial heading angle was conducted using circular statistics and the

Oriana 2 statistics program. A significant Rayleigh‟s test (P < 0.05) suggested that the data

were not uniformly distributed and were clustered around a mean direction (Zar, 1999). The


percentage of animals that headed to a mean direction was also calculated and was presented

as tendency to a mean direction. The data for the number of sessions to reach criterion were

cube-root transformed to fulfill the assumption of normality and were analysed using a two

sample t-test (SPSS 17). Chi-square analysis was employed to analyse the number of animals

that completed the task (SPSS 17).

Simple and multiple stepwise regression analyses were carried out using Minitab 15 to

determine whether or not the memory impairment could be predicted from the motor activity

of animals, especially in the BVD animals. For the simple regression analysis, searching

velocity AUC was chosen as a predictor variable because it was a measure of hyperactivity,

yet independent from any measure of velocity on homing that might be related to memory.

The AUC for the number of errors was chosen as an index of animals‟ memory and served as

a dependent variable. For the multiple regression analyses, the independent variables were

searching and homing distance, time, and velocity. AUC values were used in these analyses,

as the AUC calculation standardised the units, making the data comparable. An R2 or adjusted

R2 value, (for simple and multiple regression, respectively), indicated how well the regression

explained the data. The closer the R2 value to 1, the better the model predicted the number of

errors from the independent variables.

The data from the light probe trial and from AM251 treatment (day 21), were analysed

using the GLM two-way ANOVAs using SPSS 17.


3.3 Results

3.3.1 General Behavioural Observations

In general, both the sham and the BVD rats quickly learned to forage for food and

return to the home base. The rat usually poked its head out of the hole a few times before

making its exit. It exited by pulling itself up with its forepaws and pushing with the hind paws.

Once on the table, both the sham and the BVD rats showed a random search for food on the

table. However, the BVD animals were more hyperactive than the sham animals and showed

typical characteristics of BVD animals, such as head weaving, circling, and some of them,

walking backwards. Once a rat found the food pellet, it grasped it in its mouth and set off for

home. The same number of sham animals as BVD animals tried to eat on the table, but they

all quickly learned not to do so. On their return, some of the BVD animals accidentally fell

into the refuge rather than entering stably as all of the sham animals did. Individual rats took

20-30 sec to eat the food before making another foraging trip. More BVD animals exited

home and returned without finding a food pellet (a failed trial) than sham animals. However,

in general, the BVD animals were as motivated as the sham animals to search for food and

were able to carry the food back home to eat.

3.3.2 Pre-Training

Number of Sessions to Reach the Foraging Task Criterion

Although the BVD animals had to be trained for a longer period of time to reach the

foraging task criterion, it was not significantly different (P = 0.290) from the sham animals

(Figure 3.4).

Figure 3.4

Number of pre-training sessions required for the sham (n = 15) and BVD (n = 13) animals to

reach the foraging task criterion (retrieval of 4 food pellets per 10 min session per day). The

data are represented as mean + SEM.


Searching and Homing Time

The BVD animals had a significantly longer searching time compared to the sham

animals until session 15 (F(1,19) = 6.18; P = 0.022; Figure 3.5b). However, on the last two days

of the pre-training, there was no significant difference between the two groups in their

searching time (F(1,20) = 0.036; P = 0.851; Figure 3.5c), suggesting that the BVD animals

could search for the food as effectively as the sham animals. Regression analysis indicated

that there was no significant change in homing time across the last six days of the pre-training

sessions, which indicated that the animals had a stable performance (R2 = 0.056; Figure 3.5d).

However, the BVD animals showed a significantly longer homing time compared to sham

controls (F(1,20) = 61.60; P = 0.000; Figure 3.5e).

SEARCHING

HOMING

Figure 3.5

Searching (a) and homing (d) time of the sham and BVD animals in the last 6 days of the pre-

training. (b) and (c) The AUC of the searching time in sessions 12-15 and 16-17, respectively

and (e) The AUC of the homing time. Data are represented as mean + SEM.

a) b)

c)

d) e)


3.3.3 Light Probe Trial

First and Second Home Choices

Upon finding the food, most of the sham animals went directly to the old home location

where they had been trained during the pre-training phase, but the BVD animals did not.

Circular statistical analysis on the first home choice demonstrated that sham animals had a

significant preference for the old home location, whereas BVD animals did not (Rayleigh test;

r = 0.76; P = 3.31 x 10-4

for sham; r = 0.098; P = 0.930 for BVD). However, once they

discovered that the home cage was no longer at the old home location, the animals carried on

searching for home. The second home choice was randomly distributed for both sham and

BVD animals (r = 0.45; P = 0.113 for sham; r = 0.51; P = 0.287 for BVD; Figure 3.6). There

was no significant difference between the WIN- and vehicle-treated animals for the first or

second home choice.

Figure 3.6

First and second home choices for the light probe trial. The large circle represents the foraging

table and eight circles on the periphery of the table represent the potential home bases. The

hatched circle represents the old home location, and the closed one represents the novel home

location. The coloured circles outside the table represent the individual animals in various

groups. The direction and the length of the arrow in the middle of the table represents the

mean direction and tendency for the first and second home choices for sham (green) and BVD

(purple) animals. The black dotted line represents the direction for the correct home (that is, 0o

or 360o), and it serves as a reference to show animals’ deviation on their home choices.

1st Choice

Old home New home

2nd

Choice

SHAM-Veh SHAM-WIN BVD-Veh BVD-WIN


Searching and Homing Distance, Time, Velocity

The effects of surgery and drug treatment were not statistically significant and there

were no significant interactions (Figure 3.7).

Figure 3.7

Foraging task parameters displayed by sham-vehicle, sham-WIN, BVD-vehicle, and BVD-WIN

animals in the light probe trial. (a), (b), (c) Searching distance, time, velocity, respectively; (d),

(e), (f) homing distance, time, velocity, respectively. The data are represented as mean + SEM.

SEARCHING HOMING

a) d)

b)

c)

e)

f)


b)

Searching and Homing Velocity Comparison

Both the sham and BVD animals had a significantly higher homing velocity than

searching velocity during the light probe trial (three-way ANOVA; F(1,31) = 24.36; P = 0.000;

Figure 3.8a). There were no statistically significant effects for surgery (F(1,31) = 0.64; P =

0.429), drug treatment (F(1,31) = 2.10; P = 0.157; Figure 3.8b), or of any of the interactions

between factors.

Figure 3.8

The velocity comparison between searching and homing in the light probe trial. (a) The

difference in searching and homing velocities between the sham and the BVD animals; (b) the

effect of vehicle and WIN treatment on the sham and the BVD animals’ velocity. Data are

represented as mean + SEM.

d)

a)


Number of Visits to Old Home

Before reaching the correct home, sham animals made significantly more visits to the

old home compared to the BVD animals (two-way ANOVA; F(1,16) = 6.81; P = 0.019).

However, there was neither a significant drug treatment effect (F(1,16) = 0.47; P = 0.501) nor a

significant interaction between the surgery and the drug treatment (F(1,16) = 0.57; P = 0.463;

Figure 3.9a).

Number of Errors

There was no significant difference between the sham and the BVD animals in the

number of errors made before reaching the correct home (two-way ANOVA; F(1,15) = 0.94; P

= 0.348). Furthermore, there was no significant drug treatment effect or interaction (F(1,15) =

0.006; P = 0.939 for drug treatment; F(1,15) = 0.082; P = 0.778 for surgery x drug treatment

interaction; Figure 3.9b).

Figure 3.9

(a) The average number of visits to the old home, and (b) the average number of errors made

before reaching the correct home by sham-vehicle, sham-WIN, BVD-vehicle, and BVD-WIN

animals in the light probe trial. The data are represented as mean + SEM.

a) b)


3.3.4 Dark Training

Searching Distance

BVD animals travelled a significantly longer distance in searching for food compared to

the sham animals (linear mixed model (LMM) analysis; P = 0.000). However, there was no

significant drug effect (P = 0.628; Figure 3.10a). The three-way ANOVA on the AUC values

further confirmed the above results (F(1,78) = 24.59; P = 0.000 for surgery; F(1,78) = 2.30; P =

0.134 for drug treatment; Figure 3.10b) and showed a significant dose effect, where both the

sham and BVD animals had a significantly shorter searching distance in the 2 mg/kg WIN

dose than in the 1 mg/kg (F(1,78) = 3.99; P = 0.049; Figure 3.10b). There was also a significant

interaction between surgery, treatment and dose (F(1,78) = 5.06; P = 0.027; Figures 3.10c, d),

but no other significant interactions.

Figure 3.10

(a) Linear graph of the searching distance (cm) for different treatment groups during the dark

training with the data reflecting a block of two sessions. (b), (c), and (d) The AUC of the

searching distance for the sham and BVD animals to show the effect of surgery, dose and the

interactions between surgery, treatment, and dose. Data are represented as mean + SEM.

a)

b) c) d)


Searching Time

Neither surgery nor drug treatment had any significant effect on searching time in the

dark training (LMM analysis; P = 0.202 for surgery; P = 0.365 for drug treatment; Figure

3.11a). The three-way ANOVA on the AUC values further confirmed these results (F(1,78) =

1.10; P = 0.298 for surgery; F(1,78) = 2.93 ; P = 0.091 for drug treatment; Figure 3.11b, c).

There was no significant dose effect (F(1,78) = 0.91; P = 0.343) or any significant interactions

between factors.

Figure 3.11

(a) Linear graph of the searching time (sec) for different treatment groups during the dark

training with the data reflecting a block of two sessions. (b) and (c) The AUC of the searching

time showing the effect of surgery, treatment, and dose. Data are represented as mean + SEM.

a)

b) c)


Searching Velocity

BVD animals had a significantly higher searching velocity compared to sham animals

(LMM analysis; P = 0.000). However, there was no significant drug effect (P = 0.082; Figure

3.12a). The three-way ANOVA on the AUC values confirmed the above results (F(1,78) = 8.33;

P = 0.005 for surgery; Figure 3.12b; F(1,78) = 2.84; P = 0.096 for drug treatment; Figure 3.12c).

There was a significant dose effect, where animals‟ searching velocity was significantly

decreased with 2 mg/kg WIN treatment compared to 1 mg/kg (F(1,78) = 10.35; P = 0.002).

There were no significant interactions between surgery, drug treatment, and dose.

Figure 3.12

(a) Linear graph of the searching velocity (cm/sec) for different treatment groups during the

dark training with the data reflecting a block of two sessions. (b) and (c) The AUC of the

searching velocity showing the effect of surgery, treatment, and dose. Data are represented as

mean + SEM.

a)

b) c)


Homeward Path

The homeward path taken by the sham and the BVD animals in the dark training clearly

showed that the sham animals took the most direct route home (Figure 3.13a), whereas the

BVD animals did not (Figure 3.13b). The BVD animals made directionless movements across

the entire foraging table; some visited every other hole until they serendipitously found the

home.

Figure 3.13

An example of the homeward path taken by a (a) sham and a (b) BVD rat in the dark training.

The large circle represents the foraging table. The small black circle in the middle of the table

represents the location of the food pellet, and the other black circle near the periphery

represents the home location.

a) Sham b) BVD


Homing Distance

The BVD animals travelled a significantly longer distance to return home compared to

sham animals (LMM analysis; P = 0.000). However, there was no significant drug effect (P =

0.188; Figure 3.14a). The three-way ANOVA on the AUC values further confirmed these

results (F(1,78) = 50.03; P = 0.000 for surgery; Figure 3.14b; F(1,78) = 0.88; P = 0.353 for drug

treatment; Figure 3.14c) and showed a significant dose effect, where animals had a

significantly shorter homing distance for the higher WIN dose (F(1,78) = 28.37; P = 0.000;

Figure 3.14c). Furthermore, there was a significant interaction between surgery and dose,

indicating that the BVD animals had a shorter homing distance for 2 mg/kg WIN, while the

sham animals did not show any change (F(1,78) = 18.74; P = 0.000; Figure 3.14b). There were

no other significant interactions.

Figure 3.14

(a) Linear graph of the homing distance (cm) for different treatment groups during the dark

training with the data reflecting a block of two sessions. (b) and (c) The AUC of the homing

distance showing the effect of surgery, treatment, and dose. Data are represented as mean +

SEM.

a)

b) c)


Homing Time

The BVD animals had a significantly longer homing time compared to the sham

animals (LMM analysis; P = 0.000). However, there was no significant drug effect (P = 0.991;

Figure 3.15a). The three-way ANOVA on the AUC values further confirmed these results

(F(1,78) = 17.18; P = 0.000 for surgery; Figure 3.15b; F(1,78) = 2.32; P = 0.132 for drug

treatment; Figure 3.15c) and showed a significant dose effect (F(1,78) = 20.42; P = 0.000;

Figure 3.15c). Furthermore, there was a significant interaction between surgery and dose,

indicating that the BVD animals had a shorter homing time for 2 mg/kg WIN, while the sham

animals did not show any change (F(1,78) = 10.33; P = 0.002; Figure 3.15b). There were no

other significant interactions.

Figure 3.15

(a) Linear graph of the homing time (sec) for different treatment groups during the dark training

with the data reflecting a block of two sessions. (b) and (c) The AUC of the homing time

showing the effect of surgery, treatment, and dose. Data are represented as mean + SEM.

b) c)

a)

b) c)


Homing Velocity

The BVD animals had a significantly higher homing velocity compared to sham

animals (LMM analysis; P = 0.000), but there was no significant drug effect (P = 0.680;

Figure 3.16a). However, the three-way ANOVA on the AUC values showed no significant

surgery or dose effect (F(1,78) = 3.71; P = 0.058 for surgery; F(1,78) = 2.82; P = 0.097 for dose;

Figure 3.16b). Furthermore, WIN treatment significantly decreased animals‟ homing velocity

(F(1,78) = 5.25; P = 0.025; Figure 3.16c) There were no significant interactions between the

surgery, treatment, and dose.

Figure 3.16

(a) Linear graph of the homing velocity (cm/sec) for different treatment groups during the dark

training with the data reflecting a block of two sessions. (b) and (c) The AUC of the homing

velocity showing the effect of surgery, treatment, and dose. Data are represented as mean +

SEM.

a)

b) c)




searching velocity in the dark training (3-way ANOVA; F(1,20) = 34.05; P = 0.000; Figure

3.17a). In addition, the BVD animals had a significantly higher overall velocity compared to

the sham animals (F(1,20) = 7.20; P = 0.009). However, WIN treatment significantly reduced

animals‟ velocity (F(1,20) = 8.37; P = 0.005; Figure 3.17b). However, there were no significant

interactions between factors.

Figure 3.17

The velocity comparison between searching and homing during the dark training. (a) The

difference in searching and homing velocities between the sham and the BVD animals; (b) The

effect of vehicle and WIN treatment on the sham and the BVD animals’ velocity. Data are

represented as mean + SEM.

a) b)


Heading angle - Sham0

90

180

270 50 50

50

50

40 40

40

40

30 30

30

30

20 20

20

20

10 10

10

10

Heading angle - BVD0

90

180

270 15 15

15

15

12.5 12.5

12.5

12.5

10 10

10

10

7.5 7.5

7.5

7.5

5 5

5

5

2.5 2.5

2.5

2.5

Initial Heading Angles

When they found the food, the sham animals made a clear head turn toward the correct

home and went home in almost a straight path. A Rayleigh test on the initial heading angle

confirmed that there was a clear orientation towards home for the sham animals (r = 0.60; P <

1 x 10-12

; Figure 3.18). However, the BVD animals‟ heading angles were uniformly

distributed around 360o (r = 0.072; P = 0.498; Figure 3.18). Furthermore, the WIN treatment

did not affect sham animals‟ clear orientation toward home nor did it have any effect on BVD

animals (Figure 3.19).

SHAM

BVD

Figure 3.18

Rose diagram showing the initial heading angles of the sham and BVD animals for the dark

training. The mean vector is indicated by the black line and 95% confidence interval (C.I.) for

the mean is indicated by the line extending either side. The 95% C.I. values for the BVD animals

were unreliable due to low concentration of vectors, hence the red line. The inner circles

(dotted line) indicate the number of observations for the given vectors (blue triangles).


1 mg/kg WIN treatment

SH

AM

– V

ehic

le

SH

AM

– W

IN

BV

D –

Veh

icle

BV

D –

WIN


SH

AM

– V

ehic

le

SH

AM

– W

IN

BV

D –

Veh

icle

BV

D –

WIN

Figure 3.19

Initial heading angles of animals in different treatment groups for 1 and 2 mg/kg WIN treatment.

SHAM-Veh0

90

180

270 25 25

25

25

20 20

20

20

15 15

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15

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SHAM-WIN0

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BVD-Veh0

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2.5

BVD-WIN0

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270 6 6

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5 5

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4 4

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3 3

3

3

2 2

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1 1

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SHAM-Veh0

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270 25 25

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20 20

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SHAM-WIN0

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BVD-Veh0

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Most of the sham animals made their home choice the correct home even on the first

day in the dark (r = 0.73; P = 9.97 x 10-5

). The sham animals had a significant preference for

the correct home on their first and second home choice throughout the dark training, whereas

the BVD animals did not (Figure 3.20). Both the sham and BVD animals‟ home choices were

not affected by the WIN treatment. However, vehicle-treated BVD animals had a preference

for an incorrect hole on their first home choice in the 2 mg/kg WIN treatment period.

Figure 3.20

First and second home choices of the sham and BVD animals with 1 and 2 mg/kg WIN

treatment in the dark training. The large circle represents the foraging table and eight circles

on the periphery of the table represent the potential home bases. The closed circle represents

the correct home location. The direction and the length of the arrows in the middle of the table

represent the mean direction and tendency of the home choices for sham (green) and BVD

(purple) animals. The black dotted line represents the direction for the correct home (that is, 0o

or 360o), which served as a reference to show animals’ deviation on their home choices.

2n

d choice


1st ch

oice



Number of Errors

The BVD animals made significantly more errors before reaching the correct home

compared to the sham animals (LMM analysis; P = 0.000; Figure 3.21a; three-way ANOVA;

F(1,78) = 78.64; P = 0.000; Figure 3.21b). Although there was no significant drug effect overall

(LMM analysis; P = 0.267; Figure 3.21a; three-way ANOVA; F(1,78) = 1.59; P = 0.211; Figure

3.21c, d), WIN treatment significantly decreased the number of errors made by the BVD

animals, (F(1,78) = 6.37; P = 0.014; Figure 3.21c), particularly at the higher dose (F(1,78) = 8.02;

P = 0.006; Figure 3.21b). In fact, animals made significantly fewer errors overall with 2

mg/kg WIN treatment compared to 1 mg/kg (F(1,78) = 8.87; P = 0.004; Figure 3.21d). There

were no other significant interactions.

Figure 3.21

(a) Linear graph of the number of errors made before reaching the correct home by animals in

different treatment groups with the data reflecting a block of two sessions. (b), (c), and (d) The

AUC of the number of errors for the sham and BVD animals to show the effect of surgery, drug

treatment, dose, and their interactions. Data are represented as mean + SEM.

a)

b) d)

c)


Number of Animals that Completed the Task

There were no significant surgery or drug treatment effects for the number of animals

that completed the foraging task throughout the dark training (Figure 3.22).

Figure 3.22

The number of animals that completed the task during the dark training.

Simple Regression Analysis

A simple regression analysis was performed on the AUC for the number of errors made

(dependent variable) against the AUC for the searching velocity (predictor variable) of the

vehicle-treated sham and BVD animals. It showed that the number of errors made by the

animals could not be predicted from their searching velocities (R2 = 0.004 for sham, Figure

3.23a; R2 = 0.115 for BVD, Figure 3.23b). When WIN-treated animals were included in the

analysis, no statistically reliable prediction could be drawn (R2 = 0.104 for sham, Figure 3.23c;

R2 = 0.017 for BVD, Figure 3.23d).

Multiple Stepwise Regression Analysis

The multiple stepwise regression analysis was done to determine whether the AUC for

the number of errors made by sham or BVD animals could be predicted from the AUCs for

various predictor variables, such as searching and homing distance, time, or velocity.

Although the stepwise process revealed that the homing time and velocity predicted the

number of errors made, the adjusted R2 values were too low to make this prediction

statistically reliable (adjusted R2 = 0.453 for sham; adjusted R

2 = 0.435 for BVD).

Vehicle group only WIN group included

SH

AM

200190180170160150140130120

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14

12

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8

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AUC searching velocity

AU

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Figure 3.23

Simple regression analysis performed to predict the number of errors made by vehicle-treated (a) sham and (b) BVD animals or by (c) all of the sham and

(d) the BVD animals from their searching velocities. The AUC values were used to perform the regression analysis.

b)

a) c)

d)

R2 = 0.004

R2 = 0.115

R2 = 0.104

R2 = 0.017


3.3.5 AM251 Treatment

Searching and Homing Distance, Time, Velocity

When treated with AM251, the BVD animals had significantly higher searching and

homing velocities compared to the sham animals (two-way ANOVA; F(1,14) = 18.12; P = 0.001

for searching; F(1,14) = 5.25; P = 0.038 for homing; Figure 3.24). There were no statistically

significant differences in any other measurements taken.



searching velocity (three-way ANOVA; F(1,28) = 31.72; P = 0.000; Figure 3.25a). Furthermore,

the BVD animals had a significantly higher overall velocity than the sham animals (F(1,28) =

23.82; P = 0.000; Figure 3.25a, b). However, there was no significant drug treatment effect

(F(1,28) = 0.099; P = 0.756) or any interactions.

Figure 3.25

The velocity comparison between searching and homing with AM251 treatment on day 21 of

the dark training. (a) The difference in searching and homing velocities between the sham and

the BVD animals; (b) The effect of drug treatments on the sham and the BVD animals’ velocity.

Data are represented as mean + SEM.

a) b)


Figure 3.24

Foraging task parameters displayed by sham-vehicle, sham-WIN, BVD-vehicle, and BVD-WIN

animals with AM251 treatment on day 21 of the dark training. (a), (b), (c) Searching distance,

time, velocity, respectively; (d), (e), (f) homing distance, time, velocity, respectively. The data

are represented as mean + SEM.

SEARCHING

a)

b)

c)

d)

e)

f)

HOMING



AM251-WIN treated animals showed a significant preference for the correct home on

their first home choice (Rayleigh test; r = 0.68; P = 0.054), while AM251-vehicle treated

animals did not (r = 0.27; P = 0.425). However, AM251-vehicle treated animals showed a

significant preference for the correct home on their second choice (r = 0.64; P = 0.021). The

number of AM251-WIN treated animals that made the second choice was too small (n = 2) to

perform a statistical analysis. The sham animals had a significant preference for the correct

home base on their first home choice, whereas BVD animals did not (r = 0.63; P = 0.01 for

sham; r = 0.25; P = 0.653 for BVD; Figure 3.26). The second home choice was randomly

distributed for both sham and BVD animals (r = 0.57; P = 0.199 for sham; r = 0.48; P = 0.255

for BVD; Figure 3.26).

Figure 3.26

First and second home choices of the BVD and sham animals on day 21 of the dark training

with the treatment of AM251 prior to vehicle or WIN administration. The large circle represents

the foraging table and eight circles on the periphery of the table represent the potential home

bases. The closed circle represents the correct home location. The coloured circles outside the

table represent the individual animals in various groups. The direction and the length of the

arrows in the middle of the table represent the mean direction and tendency of the home

choices for sham (green) and BVD (purple) animals. The black dotted line represents the

direction for the correct home (that is, 0o or 360

o), which served as a reference to show animals’

deviation on their home choices.

1st Choice

SHAM-Veh SHAM-WIN BVD-Veh BVD-WIN

2nd

Choice


Initial Heading Angles

AM251-vehicle treated animals showed a clear direction (Rayleigh test; r = 0.61; P =

0.008), while AM251-WIN treated animals did not (r = 0.492; P = 0.243; Figure 3.27).

Furthermore, the sham animals had a clear direction towards home, while the BVD animals‟

heading angles were uniformly distributed around 360o, indicating that the BVD animals had

no sense of direction when they turned to head back home (r = 0.73; P = 0.001 for sham; r =

0.419; P = 0.304 for BVD).

Figure 3.27

Initial heading angles of (a) vehicle- or (b) WIN-treated animals on day 21 of the dark training

with the treatment of AM251 prior to vehicle or WIN administration.

Number of Errors

The BVD animals made significantly more errors before reaching the correct home

compared to the sham animals (two-way ANOVA; F(1,14) = 4.69; P = 0.048). However, there

was no significant drug treatment effect (F(1,14) = 0.758; P = 0.399) or surgery x drug

treatment interaction (F(1,14) = 0.132; P = 0.722; Figure 3.28).

Figure 3.28

The graph showing the average number of errors made before reaching the correct home by

the sham-vehicle, sham-WIN, BVD-vehicle, and BVD-WIN animals on day 21 of the dark training.

The data are represented as mean + SEM.

a) AM251-Vehicle b) AM251-WIN


3.4 Discussion

This study had two major aims. The first was to investigate the effects of BVD on

spatial memory using the foraging task in animals that were at a time point long after the

surgical lesions – 14 months. The second was to determine whether a cannabinoid receptor

agonist, WIN, could exacerbate the spatial memory deficits caused by BVD, and whether an

inverse agonist, AM251, could reduce this effect. Aside from determining whether the

memory deficits could be modulated by cannabinoid drugs, such results would suggest

whether cannabinoid receptors, in the hippocampus and elsewhere, might be involved in the

effects of BVD on spatial memory.

This study provided strong evidence that the cognitive impairment due to the vestibular

damage is highly likely to be permanent. More specifically, at 14 months post-operation,

BVD animals were unable to use allothetic cues during their navigation to return home and

their spatial memory impairment was more severe in the dark than in the light condition.

Overall, these impairments appear to be more severe than at 5-7 months post-operation

(Zheng et al., 2009b) and administration of a cannabinoid receptor agonist showed a

complicated effect on these deficits.

There was no indication that the BVD animals were restricted in their movement or

showed any impairment in their ability to acquire the task. It has been reported that BVD

animals are more reluctant and hesitant to leave the home base compared to control animals in

the foraging task (Wallace et al., 2002c; Zheng et al., 2006, 2009b). One might argue that this

reluctance is due to the motor impairments that follow the vestibular lesions. However, this

could not have been the case because previous studies of BVD rats reported that not only are

they not limited in movement, but they are hyperactive (Russell et al., 2003a; Goddard et al.,

2008a; Zheng et al., 2006, 2007, 2008, 2009b), and also any vestibulo-spinal reflex deficits

due to BVD that might restrict animals‟ movement would have compensated to some extent

by the time of their performance (Smith and Curthoys, 1989).

Furthermore, in the pre-training, there was also no significant difference between the

sham and BVD animals in the searching distance. Indeed, BVD animals in the present study

showed hyperactivity, which was indicated by a higher locomotor velocity compared to sham

animals. Moreover, there was no significant difference in the searching distance between the

sham and BVD animals in the light probe trial. These results confirm that the BVD animals


were capable of generating the necessary motor activity to perform food searching. Further

evidence that BVD surgery left animals‟ ability to learn the task intact was provided in the

pre-training sessions where there was no significant difference between BVD and sham

animals in the number of trials to reach the criterion or in the searching time on the last 2 days

of training. This was consistent with a previous study where rats with bilateral sodium

arsanilate-induced vestibular lesions reached the criterion in an equivalent number of trials in

a spatial navigation task (Stackman and Herbert, 2002). Although BVD animals were capable

of homing during the pre-training, they did so with a significantly longer time. This was in

agreement with the previous study when the animals were tested at 5-7 months post-operation

(Zheng et al., 2009b). A question raised from this result was whether the BVD animals are

still able to use allothetic cues to guide them home as they did in the previous study.

Therefore, a light probe trial was given during which the home location was changed to a

novel position.

Effects of BVD on Spatial Navigation In Light

In the light probe trial, when the home choice and the number of visits to the old home

were analysed, the performance of the sham and the BVD animals was distinctly different.

The sham animals showed a significant preference for the old home location on their first

home choice, whereas the BVD animals did not. The sham animals‟ preference for the old

home location on their first choice was in agreement with previous studies (Maaswinkel and

Whishaw, 1999; Whishaw et al., 2001; Wallace et al., 2002c; Zheng et al., 2006, 2009b), and

suggests that visual cues were used during their piloting.

The BVD animals‟ random first home choice was a novel finding from the present study.

Both the Wallace et al. (2002c) and Zheng et al. (2009b) studies reported that BVD animals

showed a preference for the old home on their first choice. There are two possible

explanations for the lack of old home preference in the light probe trial by the BVD rats in the

current study. First, the extent of vestibular damage in the animals in the Wallace et al. (2002c)

study was variable as BVD was achieved by intratympanic injection of sodium arsanilate.

This magnitude of variation was indicated by the numerical scores given from 0 (no vestibular

damage) to 9 (complete vestibular damage). Although chemical ablation of the peripheral

vestibular labyrinth is often used to create a vestibular lesion in a rat (Ossenkopp and

Hargreaves, 1993; Stackman et al., 2002), this procedure has been shown to be unreliable, as

it often results in incomplete lesions (Jensen, 1983; Saxon et al., 2001). Therefore, it is

possible that the extent of vestibular damage may have accounted for the difference in the


homing choice. However, Zheng et al. (2009b) performed surgical ablation, which would

have caused complete vestibular damage without much variation. As the same surgical

technique was used in the present study, the absence of old home preference in the 14 months

post-BVD animals suggests that it was not just the degree of the vestibular damage that

contributed to the spatial deficits in the BVD animals in light, but also the length of post-

operation time. In other words, it is possible that the BVD animals‟ spatial deficits became

worse over time. In the Wallace et al. (2002c) study, BVD rats were tested only 2 weeks

following the surgery, while Zheng et al. (2009b) tested their animals at 5-7 months after the

surgery. In the present study, the animals were tested 14 months following the surgery.

Therefore, BVD animals‟ inability to use available allothetic cues and execute piloting as their

navigating strategy in the present study might be an indication that the BVD animals‟

cognitive deficits became worse between 7 and 14 months post-BVD.

The second home choice in the light probe trial was randomly distributed for both the

sham and the BVD animals. This result was consistent with the study by Zheng et al. (2009b).

However, the sham animals‟ random second home choice was contradictory to studies by

Wallace et al. and Whishaw et al. (Wallace et al. 2002c; Whishaw and Tomie, 1997; Whishaw

and Maaswinkel, 1998; Maaswinkel et al., 1999). In their studies, almost all of the control rats

proceeded directly to the new location after finding that the home was no longer present at the

old location. However, Zheng et al. (2009b) showed that when allothetic cues are available, it

is not common for the animals to switch to idiothetic cues even if they might have been

collected during navigation. This inflexibility in switching between the allothetic and

idiothetic cues is further evidenced by sham animals‟ persistent visits to the old home, since

the animals should have made their second home choice the new home location if idiothetic

cues had been used.

One possible explanation for the lack of ability in using allothetic cues in BVD animals

in light is that animals had poor vision and/or the lighting set up around the foraging table

may have been insufficient for them to utilise the available allothetic cues to navigate

themselves back to the previously correct home. However, sham animals‟ obvious preference

for the old home on their first home choice and persistent visits to the old home suggests that

the sham animals at least had adequate vision and the lighting around the foraging table was

sufficient. For the BVD animals, it is possible that they were still experiencing oscillopsia due

to permanent loss of their vestibulo-ocular reflexes (Smith and Curthoys, 1989), which may

have contributed to their poor performance in light. Nevertheless, it is curious that at 5-7


months post-BVD, the only deficit observed in light was a significantly longer homing time

(Zheng et al., 2009b). At 14 months post-BVD, it might be expected that the animals would

be better adapted to oscillopsia than at 5-7 months, and hence use visual cues more effectively.

Again, this suggests that BVD animals are more impaired in the foraging task at 14 months

than at 5-7 months post-operation and that their spatial memory deteriorates over time.

The random first home choice and the small number of visits to the old home by the

BVD animals in the light probe trial suggests that the BVD animals were more susceptible to

spatial reference memory impairment than the sham animals. This deficit might have been

due to the natural aging of the animals. Indeed, age-related deficits in spatial memory have

been previously observed in rats (Caprioli et al., 1990, 1991; Frick et al., 1995; Markowska,

1999; see Barnes, 2004 for review). In these studies, spatial reference memory deficits were

observed at the age of 17-18 months, but spatial working memory was not impaired until 24-

28 months of age. The animals in the present study were 16 months old when they were tested

in the foraging task. However, if the spatial reference memory deficit was due to aging, the

sham animals should have demonstrated the deficits as well. But this was not the case – sham

animals had a clear preference for the old home on their first home choice and they visited the

old home several times to ensure that the home was no longer there. Only the BVD animals

showed the impairment in their home choice and in the number of visits, which were

indications of the spatial reference memory deficits.

Another possible explanation for BVD animals‟ spatial impairment even in light is that

BVD animals might not have actually learnt the home location during pre-training, but

reached home by chance alone. Not only the probability of finding the home by chance would

have been equal between the sham and the BVD animals, the comparison of searching and

homing velocity in the light probe trial demonstrated that BVD animals were actively homing.

It has been shown that animals return to the home base with a greater velocity than in their

outward trip (Eilam and Golani, 1989; Drai et al., 2000; Whishaw et al., 2001; Wallace et al.,

2002b; Wallace and Whishaw, 2003). In the present study, both the sham and BVD animals

showed a significantly higher homing velocity than searching velocity, which suggested that

the BVD rats were actively homing, but possibly were unaware of the precise location of their

home base.

BVD animals‟ impaired spatial memory in light suggests that they were unable to

collect the information about the home location from vision alone. The next dominant sensory


cue in the rat is the olfactory cue (Maaswinkel and Whishaw, 1999). Since the BVD animals

would not have had any self movement cues from the vestibular system, it is possible that

they relied on olfactory cues more substantially than the sham animals. There has been

considerable debate concerning the use of olfactory cues by animals in maze tasks (Means et

al., 1971, 1992; Wallace et al., 2002a), and the contribution of olfactory cues in path

integration has previously been shown to be negligible (Maaswinkel and Whishaw, 1999;

Whishaw and Gorny, 1999; Whishaw et al., 2001; Wallace and Whishaw, 2003). Furthermore,

the results from the present study suggest that it is unlikely that homing was guided by

olfactory cues in the light probe trial. There are several reasons for this. First, the rats‟

foraging excursions were circuitous and therefore, it seemed improbable that there was a

direct track that guided animals‟ homeward movement. Second, if the animals used olfactory

cues, (for example, the odour of the cage that they had just left) or followed their own odour

trail, they would have returned to the new home location and not to the old location. However,

there was a clear preference for the old home by the sham animals. Not only did the BVD

animals did not show any preference for the old home in the light probe trial, they also did not

make their first home choice the new home, suggesting that the BVD animals did not use

olfactory cues as their dominant cue. This leads to the third reason. It has been shown that rats

following odour trails or a scented track travel much more slowly (Wallace and Whishaw,

2003). Comparison of searching and homing velocity between BVD and sham animals

demonstrated that homing velocity was higher than searching velocity not only in the sham

animals but also in the BVD animals, which suggests that the BVD animals were not

following any odour trails. Fourth, in a previous study in which animals were trained to

follow a scented string that leads to a food pellet in the dark, after the retrieval, the animals

took a relatively direct route back home without following the scented string which would

also have led them home (Whishaw and Gorny, 1999; Whishaw et al., 2001). These results

suggest that in the absence of visual cues, the self-motion cue is pre-eminent over the

olfactory cues. Finally, tracking rats have a distinct posture in which the nose is down and the

back is arched (Whishaw and Gorny, 1999; Wallace et al., 2002a), and this posture was not

observed by the homing sham or BVD rats in the current study. For these reasons, it can be

concluded that it is unlikely that the animals used olfactory cues for guidance in the foraging

task.

As chemical or surgical lesions of the vestibular labyrinth usually involve damage to the

cochlea as well, it is possible that any cognitive effects of the lesions are partly due to hearing

loss. For example, auditory stimulation, including noise trauma, has been reported to affect


place cell function (Sakurai, 1990, 1994; Goble et al., 2009). For this reason, sham animals

were included in the present study as partial auditory controls. Although some sound will still

be transmitted to the cochlea in the sham animals, these animals have consistently performed

significantly better in cognitive tasks than animals with vestibular lesions (Zheng et al., 2006,

2007, 2008, 2009a, b). This suggests that hearing loss is not the major cause of the spatial

memory deficits in animals subjected to bilateral vestibular lesions. Furthermore, animal

studies, using different kinds of aminoglycosides (i.e. streptomycin and neomycin) with

different toxicities for the auditory and vestibular hair cells, have shown that the effects of

auditory and vestibular lesions on learning and memory are different (Schaeppi et al., 1991).

In a radial arm maze task, rats treated with streptomycin, which lesioned the auditory and the

vestibular systems, exhibited impaired working memory; however, rats treated with neomycin,

which lesioned only the auditory system, did not (Schaeppi et al., 1991). Recently, it was

shown that rats subjected to acoustic trauma do not exhibit deficits in the Morris water maze

task, suggesting that their spatial memory is intact and that the auditory damage does not have

any profound effect on cogntion (unpublished observations).

Effects of BVD on Spatial Navigation In Darkness

The spatial navigation ability was most affected by the vestibular damage when the only

source of information available was idiothetic cues, that is, the vestibular and/or the

proprioceptive information. In darkness, the BVD animals had a significantly longer homing

distance and homing time compared to the sham animals. The BVD animals‟ circuitous

homing path and sham animals‟ direct path clearly indicated that the BVD animals had no

strategy for finding the correct home after retrieving the food, whereas the sham animals were

able to use self-movement cues to calculate the shortest route home. This was consistent with

previous publications reporting that vestibular information is required for path integration

(Wallace et al., 2002c; Zheng et al., 2006, 2009b). It could be argued that BVD rats‟ inability

to use a path integration strategy on their homeward trip in the dark training was due to their

longer searching distance compared to the sham rats, resulting in reduced homing accuracy. In

previous studies, similar searching distance, time, and velocity were observed for both sham

and BVD animals in the dark (Wallace et al., 2002c; Zheng et al., 2009b), suggesting that the

BVD animals were able to search for food effectively. However, this ability was impaired in

BVD animals at 14 months post-operation as evidenced by a significantly longer searching

distance. Nevertheless, such impairment seems to be compromised by the higher searching

velocity in BVD animals and as a result the searching time was not significantly different

between the sham and BVD rats, which suggested that both groups spent an equal amount of


time searching for food. Although the time spent searching for food could affect the homing

accuracy in the dark, searching distance is probably more important in terms of correctly

tracking the idiothetic cues on the searching path. This, together with the evidence that BVD

animals at 5-7 months were impaired in homing in the dark, but could still search for food

effectively, suggests that the BVD animals‟ longer searching distance observed in the present

study may account for their impairment in homing in the dark to some extent.

The impairment in BVD animals‟ ability in using self-movement cues to find their way

back home in darkness was further demonstrated by their initial heading angles and the home

choices. Throughout the dark training, the BVD animals‟ initial heading angles were

uniformly distributed around 360o, while the sham animals‟ initial heading angles were

clustered around the correct home base. Consistent with their initial heading angles, BVD

animals‟ first and second home choices were either random or clustered around an incorrect

location, whereas the sham animals showed a clear preference for the correct home location.

These results indicated that the BVD animals did not have any sense of direction. Similar

findings were reported for 5-7 months post-operative animals by Zheng et al. (2009b).

The impairment of homing in darkness was unlikely to be due to a lack of motivation in

the BVD animals. Although the BVD animals had significantly higher velocities in both

searching and homing, the comparison of the two types of velocities showed that the

hyperactivity of the BVD animals was not the same across the two trip segments; searching

and homing. The homing velocity was significantly higher than the searching velocity for

both the sham and BVD animals, which suggests that the BVD animals were actively homing

over and above their hyperactivity. This implies that any impairment in homing would not

have been due to BVD animals‟ hyperactivity or lack of motivation. Furthermore, although it

took the BVD animals longer to find home, they did poke their head into every potential

home location they passed by, inevitably resulting in a significantly higher number of errors

compared to the sham animals. This again suggests that the BVD animals were motivated to

return home without being aware of the exact home location. Similar results were observed in

the 5-7 months post-BVD animals in the Zheng et al. (2009b) study.

The hyperactivity observed in the BVD animals could potentially prevent them from

homing correctly by generating unnecessary movements or resulting in an attention deficit.

Indeed, BVD animals have been found to exhibit impairment in a 5-choice serial reaction time

task, which is used extensively to investigate animals‟ attention and such deficits could be part


of the explanation for the spatial memory deficits (Zheng, et al., 2009a). Nevertheless, the

simple regression analysis proved, for the first time, that the BVD animals‟ hyperactivity

could not predict their performance in the foraging task, as the number of errors (a measure of

animal‟s memory) could not be predicted from their searching velocity (a measure of

hyperactivity). This novel finding is of significance because BVD animals‟ hyperactivity has

been reported not only in surgically damaged BVD rats (Zheng et al., 2007, 2008, 2009b), but

also in sodium arsanilate-treated BVD animals (Ossenkopp et al., 1990; Porter et al., 1990;

Stackman and Herbert, 2002; Wallace 2002c). Moreover, as none of the measurements

(searching and homing distance, time, and velocity) could reliably predict the degree of

memory impairment in the animals, it seems that the BVD animals‟ motor activity is not

related to their performance in the spatial memory task. Taking these results together, it is

unlikely that hyperactivity accounted for the impairment in homing by BVD rats.

This study has shown, for the first time that rats with BVD do not recover from spatial

memory deficits even at very long time intervals following the surgery, such as 14 months.

From the estimated average life expectancies of rats (2.5 years, Baker et al., 1979) and

humans (85 years, Olshansky et al., 1990; Manton et al., 1991), 14 months post-BVD in rats

is calculated to be approximately 40 years for humans. Therefore, the results from the present

study suggest that that spatial memory deficits following vestibular damage may be

permanent.

Effects of Cannabinoid Drugs

The present study was the first to investigate the effect of cannabinoids using the

foraging task, and also was the first to demonstrate the effect of cannabinoids on spatial

learning and memory in BVD animals. It was expected that the administration of the

cannabinoid receptor agonist would exacerbate the observed spatial memory deficits in the

BVD animals as it has been shown to have disruptive effects on spatial learning and memory

in both animals and humans previously (Litchman et al., 1995; Cha et al., 2006; Barna et al.,

2007; Nestor et al., 2008; Pamplona et al., 2008; Suenaga and Ichitani, 2008; Weinstein et al.,

2008; Wise et al., 2009). However, the effect of WIN was more complicated than expected.

There have been concerns about cannabinoid receptor agonists affecting performance in

behavioural tasks by influencing motivation and reward-related behaviour. However, the

effect of cannabinoid receptor agonists on motivation has been inconsistent and contradictory,

where some studies have shown increased motivation (Higgs et al., 2005; Solinas and


Goldberg, 2005), while others have shown a reduction in motivation (Higgs et al., 2005;

Drews et al., 2005). Nevertheless, in the present study, WIN-treated animals showed no

significant differences in searching distances or searching times compared to the vehicle-

treated group either in light or in darkness, which suggested that WIN treatment did not

interfere with the animals‟ motivation to perform in the foraging task.

In the present study, WIN treatment significantly decreased animals‟ homing velocity

and their overall velocity, which raised a concern about whether the WIN treatment

contributed to the reduction in animals‟ motor activity. Indeed, cannabinoid receptor agonists

have been shown to decrease animals‟ motor activity (McGregor et al., 1996; Darmani, 2001;

Romero et al., 2002; Järbe et al., 2006; Fox et al., 2009). However, there are several reasons

why it is unlikely that the reduction in animals‟ velocity influenced their performance in the

foraging task. First, there was no significant difference between vehicle- and WIN-treated

animals in their searching time, which suggested that WIN-treated animals spent an equal

amount of time searching for food to the vehicle-treated animals. Second, if WIN treatment

reduced animals‟ motor activity, then the homing time should have been longer for WIN-

treated animals than vehicle-treated animals because WIN-treated animals would have moved

more slowly, and therefore, should have taken a longer time to return home. However, WIN

had no significant effect on homing time and in fact homing time was shorter for the higher

dose of WIN, which suggests that WIN treatment did not reduce animals‟ locomotor activity

at the doses used in the present study.

In the dark, the higher dose of WIN significantly reduced homing distance and time, and

the number of errors specifically in BVD animals, which suggested that WIN treatment might

have improved performance in the dark in BVD animals. However, WIN treatment was not

associated with any improvement in the initial heading angle or the first home choice. This

improvement was observed only in BVD animals, which suggested that BVD might have

resulted in changes in the endocannabinoid system in the brain (see Chapter 4). Moreover,

WIN might have led BVD animals to use different strategies other than path integration,

although at this stage it is not clear what these may be. However, given that the higher dose of

WIN significantly reduced homing velocity in animals, WIN treatment might have aided

BVD animals in effective homing by reducing their homing velocity.

There are some possible explanations for the general lack of effect of WIN on spatial

memory in the BVD and sham animals observed in the present study. One obvious


explanation is that the doses employed were too low. However, it needs to be noted that the

doses were chosen based on an object recognition study that was conducted in order to

determine doses that would impair memory without causing sedation (see Chapter 2; Baek et

al., 2009). Furthermore, based on previous studies (Wallace et al., 2002c; Zheng et al., 2009b),

it was predicted that because BVD rats were likely to exhibit spatial memory deficits, the

animals might be more sensitive to the adverse effects of WIN. For this reason, the animals

were first treated with the 1 mg/kg dose, and then the dose was increased to 2 mg/kg when it

became clear that the lower dose was having little effect, either on sham or BVD animals.

Although it is generally well accepted that cannabinoid receptor agonists impair spatial

memory (Suenaga and Ichitani, 2008; Robbe and Buzsaki, 2009; Wise et al., 2009), their

effects on memory are complex, dose-dependent (Abush and Akirav, 2009) and even memory

enhancement has been reported in some cases (Marchalant et al., 2008; Campolongo et al.,

2009). WIN did not seem to impair spatial memory even in the sham animals, as would be

expected. However, it is unlikely that this was due to the WIN doses being too low, because

the number of errors was actually reduced with the higher dose in darkness. It is conceivable

that the reason why WIN did not exacerbate spatial memory deficits in the BVD animals is

that they were already performing so poorly that the cannabinoid receptor agonist could not

impair this performance further.

To add to the complexity of the results, pre-treatment with AM251 abolished the effect

of WIN on homing time and the number of errors in the dark. Furthermore, animals that had

received AM251 treatment in combination with WIN showed a significant preference for the

correct home on their first home choice while AM251 treatment alone did not, suggesting that

AM251 treatment may impair path integration on its own, but does not have any effect when

given with WIN. Because inverse agonists such as SR141617A and AM251 have a tendency

to produce effects that are opposite to those produced by agonists for the CB1 receptors

(Landsman et al., 1997; Sim-Selly et al., 2001; see Pertwee, 2005a; Bergman et al., 2008 for

reviews), the behavioural consequences of their inverse agonist activity in combination with a

CB1 receptor agonist are difficult to interpret. However, recently, a CB1 receptor antagonist,

AM4113, has been developed (Sink et al., 2008). Although the effects of CB1 receptor

antagonists on animals‟ cognitive function are yet to be determined, future studies should be

carried out using these drugs to further clarify the function of the CB1 receptor and the

involvement of the endocannabinoid system in BVD animals.

An important issue to remember in the present study is that AM251 was given only once,


while WIN was administered for 20 days. While many studies have investigated the

consequences of the chronic administration of cannabinoid receptor agonists in animals,

studies involving cannabinoid receptor inverse agonists (AM251 and/or SR141617A) have

mainly focused on their effects in reversing the agonist-induced effects. Interestingly,

Hoffman et al. (2007) showed a significant enhancement of LTP in hippocampal slices taken

from animals that were treated with AM251 for 7 days (2 mg/kg/day, i.p.). However, an acute

exposure of hippocampal slices from drug-naïve rats to AM251 (1 M) did not have any

effect on LTP (Hoffman et al., 2007). The authors postulated that long-term treatment with

cannabinoid receptor inverse agonists may act to improve the mechanisms that support

memory (Hoffman et al., 2007). Based on these results, future studies should use long-term

treatment with AM251 as it might show a memory enhancement effect in BVD animals.

Furthermore, intrahippocampal administration of cannabinoids to the BVD animals should

also be considered for future investigation as it would provide evidence for the area specific

effects of cannabinoids in BVD animals and further clarify the involvement of the

endocannabinoid system in the cognitive deficits caused by bilateral loss of the vestibular

function.

Overall, although there was a lack of effect of cannabinoids, the present study was the

first study to demonstrate a possible connection between vestibular- and cannabinoid-

mediated memory impairment. Since the hippocampus has been shown to undergo plasticity

following BVD (Stackman et al., 2002; Russell et al., 2003b, 2006; Goddard et al., 2008c)

and CB1 receptors are highly expressed in the hippocampus, it is logical to examine CB1

receptor expression in the hippocampus following BVD. Furthermore, given that the density

of CB1 receptor expression is not necessarily indicative of the affinity or efficacy of the

receptor (Breivogel et al., 1997; Rubino et al., 2009a), it is also logical to investigate CB1

receptor binding affinity in BVD animals. These intriguing questions are investigated in the

next Chapter of this thesis.

93

Chapter 4

Changes in CB1 Receptor Density and Affinity Following

Bilateral Vestibular Deafferentation in Rats

Chapter 4: Changes in CB1 Receptor Density & Affinity Following BVD 94

4.1 Introduction

In the previous Chapter, it was revealed that BVD causes significant impairments in

spatial memory in rats, and that the effects of cannabinoid drugs in BVD animals are more

complex than expected. Since CB1 receptors are well known for their neuromodulatory effects

in the brain (see Section 1.2.2 Neuromodulatory Role of the Endocannabinoid System in

Chapter 1; also see Rodriguez de Fonseca et al., 2005; Pertwee, 2006; Breivogel and Sim-

Selley, 2009 for reviews), any changes in CB1 receptor density or affinity following BVD

may provide evidence for the involvement of the endocannabinoid system in brain

dysfunction observed following peripheral vestibular damage. As the hippocampus undergoes

significant synaptic changes following BVD, and high levels of CB1 receptors are found in

this area, the aim of the present study was to determine whether CB1 receptor density and

affinity in the hippocampus would change following BVD in rats. The results from the present

in vitro biochemical assays may provide some insight into the cellular and molecular

mechanisms that may underlie BVD-induced spatial memory deficits.

Although the involvement of the endocannabinoid system in various pathological

conditions has been well established, the primary focus of previous investigations has been on

changes in the number of CB1 receptors. Many believe that the effects of cannabinoids are

determined by the distribution of CB1 receptors in the brain (Herkenham et al., 1991, Jansen

et al., 1992; Sim et al., 1995), presumably assuming that these receptors have a uniform high

affinity, yet the binding affinity of CB1 receptors is not well characterised. Furthermore,

although numerous studies have been published investigating the efficacy of CB1 receptors

using a cannabinoid agonist-stimulated guanylyl 5‟-[-[35

S]thio]-triphosphate ([35

S]GTPS)

binding assay (Selley et al., 1996; Petitet et al., 1997; Breivogel et al., 1997; Vinod et al.,

2005; Castelli et al., 2007; see Childers, 2006 for review), there are very few studies

investigating the affinity of the CB1 receptor in the rat whole brain (Compton et al., 1993;

Hillard et al., 1995; Houtston and Howlett, 1993, 1998; Thomas et al., 1998) and even fewer

studies focusing specifically on the hippocampus (Romero et al., 1995; Gatley et al., 1997;

Castelli et al., 2007; Hill et al., 2010).

It is firmly established that the pharmacological actions of cannabinoids are mediated

through specific cannabinoid receptors (see Pertwee, 2006 for review). The CB1 receptors are

coupled to pertussis toxin-sensitive Gi/o proteins and their biochemical actions are effected by

the activation of these G-proteins (see Section 1.2.1 Cannabinoid Receptors and


Endocannabinoids in Chapter 1; Howlett et al., 1986; see Demuth and Molleman, 2006 for

review).

The first step in this signal transduction involves the binding of a ligand to a CB1

receptor. The degree to which this receptor-ligand binding occurs is measured as affinity,

expressed in molar units, normally in a nanomolar range (see Pertwee, 2005b for review). The

affinity of the cannabinoid receptor can be determined by displacement assays using tritiated

cannabinoids. The basic theory behind the radioligand binding assay is simple. A membrane

preparation containing the receptor of interest is incubated with a radioligand in conditions

favourable for the ligand to bind to the receptor. Then the radioactivity of the bound ligand is

detected. A saturation binding assay measures the specific binding at equilibrium at various

concentrations of radioligand to determine the receptor density (Bmax) and the affinity of the

receptor for the radioligand (Kd, the dissociation constant). If a single concentration of the

radioligand is used and the concentration of an unlabelled competing ligand is varied, then the

assay is defined as a competitive binding assay and the affinity of the competing ligand (Ki)

can be estimated accordingly. Furthermore, the concentration of the unlabelled ligand required

to block 50% of the specific binding of the radioligand (the IC50) can also be determined from

the competitive binding assay (see Bylund and Toews, 1993 for review).

Several different tritium-labelled ligands for cannabinoid receptors have been used in

previous studies. One of the most commonly used radioligands in these studies is [3H]-CP-

55,940, due to its high and approximately equal affinity for both the CB1 and CB2 receptors

(see Pertwee, 1999; Howlett et al., 2002 for reviews). Moreover, [3H]-CP-55,940 was the

radioligand used in the first cannabinoid receptor binding assay which characterised the CB1

receptors in the rat brain (Devane et al., 1988). Early studies revealed that [3H]-CP-55,940

binding is rapid, saturable, reversible, and has high affinity (Devane et al., 1988; Compton et

al., 1993; Houston and Howlett, 1993; Gatley et al., 1997; Kearn et al., 1999). Its use in

receptor binding assays in membrane preparations and tissue sections has facilitated the

characterisation and localisation of the cannabinoid receptors in the brain and in peripheral

tissues (Hekenham et al., 1991; Compton et al., 1993; Munro et al., 1993). Thus [3H]-CP-

55,940 was chosen for use in the present study.

To date, there has been no study investigating changes in CB1 receptor density, affinity,

or efficacy following BVD. Although a number of studies have investigated changes in the

level of the NMDA receptors (Li et al., 1997; King et al, 2002; Liu et al., 2003b), -amino-3-


hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (Li et al, 1996; King et al,

2002), glucocorticoid receptors (Lindsay et al., 2005; Zhang et al., 2005b), GABA receptors

(Horii et al., 2003; Zhang et al., 2005a), and CB1 receptors (Ashton et al., 2004b) following

vestibular lesions, it has all been following UVD. Given that the effects of UVD and BVD in

animals and in humans are distinctly different (Zheng et al., 2004, 2004, 2006, 2007, 2009b;

Brandt et al., 2005; Hüfner et al., 2007), the changes in receptor properties following BVD

may or may not be similar to what has been observed in animals following UVD.

Since the adverse effects of BVD on the hippocampus appear to be immediate and long-

lasting (results from Chapter 3; Stackman et al., 2002; Russell et al., 2003a, Zheng et al.,

2009b), it might be expected that the changes in CB1 receptor density and affinity would

occur shortly after BVD. The time following BVD at which changes are observed in CB1

receptor density and affinity in the hippocampus may give further indication of how

permanent the effects of BVD may be, and give further insight into interactions between the

vestibular and endocannabinoid systems. Therefore, the present study aimed to determine

hippocampal CB1 receptor density and affinity at 1, 3, and 7 days following BVD by using

western blotting and a [3H]CP-55,940 binding assay, respectively.




4.2.1.1 Animals

Thirty-nine naïve male Wistar rats were used, each weighing 250-300 g at the time of

surgery (see Chapter 3, section 3.2.2 for surgery details). There were 3 post-operative time

points: 1, 3, and 7 days. There were 6 sham and 7 BVD animals for each time point.

4.2.1.2 Tissue Collection

All animals were decapitated without anaesthesia at 1, 3, or 7 days after the surgery. The

whole brain was rapidly removed from the skull and immediately placed into ice-cold 0.9%

saline solution. The hippocampus was dissected into 3 sub-regions: CA1, CA3, and dentate

gyrus (DG) under a dissecting microscope. The dissection plate was placed on dry ice in order

to keep the tissue cold. Dissected tissue samples of individual structures were immediately

frozen on dry ice and stored separately at -80o C until use.

4.2.1.3 Tissue Homogenisation

Tissue buffer containing 50 mM Tris buffer (pH 7.6) and complete proteinase inhibitor

(1:25 dilution of stock; Roche) was added to the samples on ice, which were then

homogenised using ultrasonification (Sonic Vibra Cell, John Morris Scientific Ltd.).

Homogenates were centrifuged at 12,000 x g for 10 minutes at 4 o

C (Centrifuge 5810 R,

Eppendorf). Supernatant was collected and 5 L of each sample was taken for the Bradford

assay (see below) to determine the protein concentration.

4.2.1.4 Bradford Assay

Protein concentrations in the supernatant were measured using the Bradford method

(Bradford, 1976) using a Bio-Rad protein assay dye reagent concentrate. Serial dilution of the

bovine serum albumin (BSA) standard solution (1 mg/mL; Sigma) was carried out by

obtaining various concentrations of the standard solutions. These concentrations were: 500,

250, 125, 62.5, 31.25, 15.63, and 7.81 g/mL. Blank solution (0 g/mL) did not contain any

BSA solution. Supernatants of each sample were diluted by a factor of 60 using distilled water


(dH2O). Standard solution and samples were each loaded in triplicate into a 96-well plate. In

each well, 150 L of dH2O and 40 L of Bradford reagent (Bio-Rad) were added. The plate

was read using a microplate spectrophotometer (Benchmark Plus, Bio-Rad) set to a maximum

wavelength of 595 nm, and the standard curve was generated with the correlation coefficient

of > 0.98. The protein concentrations of the original samples were then calculated and the

sample concentrations were equalised for each hippocampal sub-region using the tissue buffer.

The equalised samples were mixed with the sample loading buffer (125 mM Tris-HCl, pH 6.8;

4% SDS, 20% glycerol, 10% 14.3 M mercaptoethanol, 2 mM EDTA, bromophenol blue

dissolved in dH2O) in a ratio of 1:1, then boiled for 5 minutes. The final concentration for

each sub-region was 3 mg/mL for CA1 and DG and 2 mg/mL for CA3. Samples were stored

at -20o C until use.

4.2.1.5 Western Blot

Gel Preparation

Both resolving and stacking gels were prepared at room temperature. A ten % resolving

gel (10% N,N‟-methylene-bis-acrylamide (Bio-Rad), 0.375 M Tris buffer (pH 8.8; Sigma),

0.1% sodium dodecylsulfate (SDS, Sigma), 5% glycerol (BDH), 1% N,N,N‟,N‟-

tetremethylethylenediamine (TEMED, Bio-Rad) and 0.036% ammonium persulfate (APS,

Bio-Rad)) was used in order to adequately separate the CB1 receptor protein (54 and 63 kDa).

TEMED and APS were added last into the gel mixture as they catalysed the polymerisation

and allowed the gel to set. Immediately after adding TEMED and APS, the gel mixture was

decanted in vertical mini-gel cassettes (Protean II, Bio-Rad). The cassettes consisted of two

glass plates separated by spacers (0.75 mm thick), which were held together by side clamps

and were sealed at the bottom. Methanol was used to flatten the surface of the resolving gel

during setting. Once the resolving gel was set, methanol was discarded and the top of the gel

was washed three times with dH2O before laying the stacking gel (5% N,N‟-methylene-bis-

acrylamide, 0.129 M Tris buffer (pH 6.8), 0.2% SDS, % TEMED, 0.11% APS). A plastic

comb was inserted into the stacking gel layer to create an individual well. The comb was

carefully removed after the stacking gel had completely set, and the cassettes were transferred

into the electrophoresis tank and filled with running buffer (0.3% Tris-Base, 1.44% glycine,

1% SDS w/v in dH2O).

Loading, Electrophoresis and Transfer

Dual colour protein marker (Bio-Rad) was loaded (5 L) in each gel as a molecular


weight standard and samples were loaded accordingly. The sample loading volume was

adjusted for the CB1 receptor antibody, as different antibodies show different levels of

sensitivity to the same samples. For each region of the hippocampus, 12 g of protein was

loaded in each well. In addition, a control tissue sample used as an internal standard was

loaded on each gel in order to control for the between gel variation.

Once all the samples were loaded, the electrophoresis tank was connected to a 400 W

power supply (PowerPack 3000; Bio-Rad) and the gel was run at 90 V until samples reached

the interface of the stacking and resolving gel, then at 180 V until samples reached the end of

the gel. Gels were removed from the glass plates, trimmed, and laid onto blot paper (7.5 x 10

cm; Bio-Rad). A polyvinylidene fluoride (PVDF; Bio-Rad) membrane, cut to the same size as

the gel, was pre-soaked in absolute methanol and then in transfer buffer at room temperature

(0.3% Tris-Base, 1.44% glycine, 20% absolute methanol) for 5 minutes each to equilibrate

before covering the gel. Each gel and PVDF membrane was sandwiched between blot papers

in a Trans-Blot Cassette (Bio-Rad) and inserted into a Trans-Blot Cell (Bio-Rad). Transfer

buffer was poured over the set up to completely cover the gel sandwich. Blotting took place

overnight at 10 V at room temperature.

Immunoblotting and Detection

Following completion of the transfer, the PVDF membrane was blocked for 6-7 hours at

4o C with blocking solution consisting of 0.1% BSA and 5% non-fat milk dissolved in TTBS

(10% 50 mM Tris buffer saline (TBS) pH 7.6, 0.1% Tween-20). The membrane was then

incubated with the CB1 receptor primary antibody (0.5% non-fat milk in TTBS; 1:1000

dilution, goat polyclonal antibody raised against a peptide mapping at the N-terminus of CB1

of human origin and recommended for detection of CB1 of mouse, rat, and human origin, sc-

10066, Santa Cruz Biotechnology, Inc) overnight at 4o

C. Following primary antibody

incubation, the membrane was washed four times with TTBS, for 5 minutes each time. The

membrane was then incubated with the horseradish peroxidase (HRP)-conjugated donkey

anti-goat secondary antibody (0.5% non-fat milk in TTBS; 1:5000 dilution, Santa Cruz

Biotechnology, Inc) for 4 hours at 4o C. After secondary antibody incubation, the membrane

was washed in TTBS four times, then twice in TBS for 5 minutes each time. The membrane

was lightly dried on tissue paper before immersing it in an enhanced chemiluminescence

(ECL) reagent (Amersham Biosciences) for 1 minute. ECL reagents allow the HRP-

conjugated secondary antibody to illuminate so that the membrane can be developed on a

photosensitive film. Following exposure to the ECL reagent, the membrane was placed in a


plastic envelope and taped inside a „Hypercassette‟ (Amersham). For immunodetection, all

procedures were carried out in a dark room. A sheet of photosensitive film (Amersham) was

placed on top of the luminescent membrane in the hypercassette. The exposure time of the

film to the membrane varied according to the strength of the signal, however, it was usually 1-

2 minutes. For developing, the film was first immersed in a developing solution (1:5 dilution;

Kodak) for 1 minute. The developing reaction was terminated by immersing the film in a stop

solution (1:25 dilution; Kodak) for 30 seconds. Photo-reactivity of the film was neutralised in

a fix solution (Kodak) for 4 minutes. The film was then rinsed under running tap water and

dried (Fc, JRC-33 air dryer). The molecular weight standards were marked on the film in

order to identify the target protein bands.

Stripping and Re-probing membranes

After developing, the PVDF membrane was submerged in a stripping buffer (20% SDS,

12.5% 0.5 M Tris HCl (pH 6.7), 0.8% -mercaptoethanol, 67.5% dH2O) for 30 minutes at 50o

C with constant agitation. This process was necessary for „stripping‟ off primary and

secondary antibody complexes from the membrane and to allow re-probing of the membrane

with different antibodies. Following stripping, the membrane was rinsed with TTBS four

times (5 minutes per rinse), and kept in TBS at 4o C until use. The membrane was stripped a

maximum of two times.

Blocking Peptide

The common problem with polyclonal antibodies is that non-specific binding of an

antibody (binding to proteins other than the antigen of interest) can sometimes occur.

Therefore, to determine which band was specific for the CB1 receptor protein, a blocking

peptide (sc-10066 P, Santa Cruz Biotechnology, Inc) was used at 1:1 ratio (primary antibody :

blocking peptide). The primary antibody was „neutralised‟ by the blocking peptide, that is, the

antibody was bound to the blocking peptide, and was therefore no longer available to bind to

the epitope present in the protein on the western blot. Thes neutralised antibody was then used

to simultaneously blot a membrane to the antibody alone, and the results were compared.

4.2.1.6 Data Acquisition

Protein Quantification from Western Blot

Developed film was analysed using a Calibrated Imaging Densitometer (Bio-Rad),

PowerPC Mac running OS 9.2 and Quantity One software. For each band, optical volume was


presented as a product of the frame area (mm2) and optical density (OD; average pixel

intensity per frame). Background volume was also taken for each lane to calculate specific

volume. The optical volume of CB1 receptor protein in each sample was normalised against

the optical volume of the internal standard for between gel comparison.

Normalisation to a „House Keeping‟ Protein

In order to control for any inconsistency in loading samples, „house-keeping‟ proteins

are used as internal controls to check whether the differences in the amount of protein of

interest could be due to an initial difference in the amount of sample loaded. The most

commonly used protein to serve this purpose is -actin, as actin is a cytoskeletal filament that

forms the internal scaffolding of a cell, and therefore its expression is ubiquitous in neuronal

and non-neuronal tissue (Lecuit and Lenne, 2007). Each membrane was therefore blotted for

actin (1:5000 dilution, Santa Cruz Biotechnology, Inc) to normalise the level of

immunolabelling of the CB1 receptor protein to actin, so that any potential variations in

protein loading could be eliminated.

4.2.1.7 Statistical Analysis

Statistical analyses were performed using SPSS 17. A series of two-way analyses of

variance (ANOVAs) followed by Bonferroni post hoc tests were performed in order to

determine whether BVD affected the levels of hippocampal CB1 receptor protein at different

post-operative time points. The data were tested for normality and homogeneity of variance

and were natural log (ln) transformed where necessary.


4.2.2.1 Animals

Forty-five naïve male Wistar rats were used, each weighing 250-300 g at the time of

surgery (see Chapter 3, Section 3.2.2 for surgery details). There were 3 post-operative time

points: 1, 3, and 7 days. For each time point, there were 7 sham and 8 BVD animals.

4.2.2.2 Cannabinoid Ligands

[3H] [1,2-(R)-5]-5-(1,1-dimethyleptyl)-2-[5-hydroxy-2-(3-hydroxy propyl)


cyclohexyl] phenol ([3H]-CP 55,940) (174.6 Ci/mmol) was purchased from Perkin Elmer Life

Sciences, Inc. WIN 55212-2 (WIN) was purchased from Tocris Bioscience (Bristol, UK). One

hundred mM of WIN (stock solution) was prepared in dimethyl sulfoxide (DMSO, Merck)

and stored at -20o C until use. When preparing the serial dilution solutions, loading buffer (50

mM Tris (pH 7.4), 1 mM EDTA, 3 mM MgCl2, 0.5% BSA) was added to make a final WIN

concentration of 1 mM (working solution).

4.2.2.3 Membrane Preparation

Animals were decapitated without anaesthesia. The whole brain was rapidly removed

from the skull and immediately placed into ice-cold 0.9% saline solution. The hippocampus

was dissected out from the forebrain and collected in 0.32 M of sucrose (Sigma) solution. The

hippocampus was then homogenised in 1.5 mL of 0.32 M sucrose solution using 10 gentle

strokes of a 2 mL hand-held glass teflon homogeniser (Kontes), which was kept cold in ice.

All of the membrane preparation procedures were carried out at 0 – 4° C. The homogenate

was centrifuged for 10 minutes at 1000 x g to remove unbroken cells and blood vessels. The

resulting supernatant was collected and centrifuged at 20,000 x g for 20 minutes, and the

pellet was then re-suspended in ice-cold dH2O and further centrifuged for 20 minutes at 8,000

x g. After centrifugation, a fluffy white-coloured band was obtained at the interface, and a

dark brown-coloured, mitochondria-rich pellet obtained at the bottom of the tube. Both the

supernatant fraction and buffy coat (the pale layer on top of the dark brown mitochondrial

pellet) were then collected and centrifuged for 10 minutes at 48,000 x g. The pellet was re-

suspended in Tris-HCl buffer (50 mM, pH 7.4; Sigma), and then centrifuged for 10 minutes at

48,000 x g. The pellet was immediately frozen on dry ice for 2 minutes in order to eliminate

endogenous ligand (i.e. endocannabinoids) within the membranes. The synaptosomal pellet

was resuspended in Tris-buffer (50 mM, pH 7.4) and centrifuged again at 48,000 x g for 10

minutes. These freezing, re-suspension, and centrifugation steps were repeated once. The

crude membrane receptor preparations were stored at -80o C until use.

The Bradford protein assay was performed in order to determine the protein

concentrations in each sample (see Section 5.2.4). The protein concentration in the samples

were then equalised to 1 mg/mL.


4.2.2.4 [3H]-CP 55,940 Binding Assay

The [3H]-CP 55,940 binding assay protocol used was modified from Sawant et al.

(2008). The concentration of [3H]-CP 55,940 was selected based on the Kd values of [

3H]-CP

55,940 from previous studies (Hillard et al., 1995; Castelli et al., 2007; Hill et al., 2008,

2010). In the present study, WIN 55212-2, which has been shown to be structurally different

to CP 55,940 (Georgieva et al., 2008), was selected to determine non-specific binding. Bylund

and Toews (1993) recommended that it is best to use a drug that is chemically distinct from

the radioligand, as it reduces the possibility of the unlabelled drug inhibiting „specific‟ but

non-receptor binding sites. Furthermore, the [3H]-CP 55,940 binding assay was carried out

using a whole hippocampus rather than separate sub-regions of the hippocampus as it would

not provide practical sample volume to run the assay.

One nM [3H]-CP 55,940 (126 Ci/mmol; Perkin Elmer Life Sciences, Inc) and 50 g of

membrane proteins were loaded in triplicate in a 96-well plate with increasing concentrations

of unlabelled competitive inhibitor, WIN. Dilutions of WIN were prepared to yield final

concentrations ranging from 100 fM to 10M. Non-specific binding was estimated in the

presence of 100 M WIN (Thomas et al., 1998). Samples were incubated for 70 minutes at

room temperature in a final volume of 200 μL. This period of incubation was sufficient to

ensure that equilibrium binding had been reached (Hillard et al., 1995).

A Unifilter GF/B glass fibre filter plate (Perkin Elmer Life Sciences, Inc., USA) was

pre-soaked in 40 μl of 0.1% polyethyleneimine (PEI; Sigma). The binding reaction was

terminated by rapid vacuum filtration onto the filter plate using a Unifilter Cell Harvestor

(Packard Instruments Inc.). The filter plate was washed 3 times with ice-cold 50 mM Tris

buffer, pH 7.4, containing 0.1% (w/v) BSA (washing buffer) to remove unbound [3H]-CP

55,940 and excess WIN. An important step was the rapid separation of bound radioligand

from free radioligand, which was achieved by reducing the buffer temperature in order to

slow the rate of dissociation. The filter plate was then dried at room temperature overnight.

On the following day, 40 μl of scintillation fluid (MicroScint 20, Perkin Elmer Life Sciences,

Inc., USA) was added to each well. The plate was then sealed and read by scintillation

spectroscopy (Packard TopCount, Packard Instruments Inc.). For each concentration, the non-

specific binding value was subtracted from total binding values to yield the specific binding.


4.2.2.5 Data Acquisition and Statistical Analysis

It has been shown that radioligand binding assays with [3H]-CP 55,940 have a Hill

coefficient of approximately 1 indicating a single binding site without any co-operativity

(Devane et al., 1988; Compton et al., 1993; Rodriguez de Fonseca et al., 1994; Kearn et al.,

1999; Vinod et al., 2005). Therefore, the data were fitted with a one site non-linear regression

to determine the IC50 value of each sample (Prism 5). Two-way ANOVAs followed by

Bonferroni post hoc comparisons were performed to determine whether there were

statistically significant differences between the IC50 values for the sham and BVD animals,

and between post-operative time points (SPSS 17). The data were tested for normality and

homogeneity of variance and were natural log (ln) transformed where necessary.


4.3 Results


4.3.1.1 Optimum Sample Amount

The CB1 receptor protein band intensity was proportional to the amount of protein

loaded into each well. Twelve g of protein was chosen as an optimum sample amount, as it

gave clear bands. The integrity of the band decreased as the amount of sample loaded

increased (Figure 4.1).

Figure 4.1

(a) The CB1 receptor band intensity increased as the amount of sample protein increased. This

change can be visualised in the western blots (b) for the CB1 receptor and its corresponding

actin.

CB1

Actin

6 12 18 24 30 36 42 48

Amount of protein (g)

a)

b)


4.3.1.2 CB1 Receptor Antibody Specificity

The specificity of the CB1 receptor antibody used in the present study (Santa Cruz

Biotechnology, Inc) was validated by using the blocking peptide. The CB1 receptor specific

bands (54 and 63 kDa) disappeared when blotted with a „neutralised‟ antibody (Figure 4.2).

Although the application of the blocking peptide led to the disappearance of some bands

specific to CB1 receptor, several other bands still remained on the blot after the treatment,

which raised a concern regarding the specificity of the CB1 receptor antibody. Based on these

results, non-specific CB1 receptor labelling would be expected using these antibodies in

immunochemistry (Appendices 1 and 2).

Figure 4.2

Western blot images showing the concerning lack of specificity of the CB1 receptor antibody

for CB1 receptors. Although the CB1 specific bands at 63 and 54 kDa (arrows) are absent in the

blot performed with the blocking peptide (BP) compared to the antibody alone, the other bands

remain.

4.3.1.3 Changes in CB1 Receptor Protein Density

The CB1 receptor protein level was significantly lower in the BVD animals compared to

sham animals, only in the CA3 area across the 3 time points (F(1,33) = 4.05, P = 0.027; Figure

4.3b), with no significant interaction between surgical group and time. However, post hoc

comparisons were significant only for BVD at 1 day and sham at 7 days (P = 0.02), and BVD

at 7 days and sham at 1 day (P = 0.0001) and 3 days (P = 0.006). The expression levels of the

CB1 receptor protein were significantly reduced in all regions of the hippocampus in a time-

dependent manner (CA1, F(2,33) = 104.25, P = 0.000; CA3, F(2,33) = 20.31, P = 0.000; DG,

F(2,33) = 324.27, P = 0.000; Figure 4.3a-c, respectively). Post hoc comparisons of the CA1 and

DG areas indicated significant differences between all time points, where 1 day post-operation

had the highest and 7 days post-operation had the lowest level of the CB1 receptor protein (P

= 0.000). In the CA3 region, the level of CB1 receptor protein was significantly lower in the 7

days post-operation group compared to the 1 and 3 day post-operation groups (P = 0.000 and

P = 0.001, respectively). However, there was no significant difference between 1 and 3 days

post-operation (P = 0.100). There were no significant interactions for any of the regions.

CB1 CB1 BP

75 kDa

50 kDa


Figure 4.3

The levels of CB1 receptor protein in the (a) CA1, (b) CA3, (c) DG of the hippocampus at 1, 3,

and 7 days following BVD compared to the sham controls. Data are represented as mean +

SEM. (d) A representative blot for the CB1 receptor protein (54 and 63 kDa, bottom and top

bands, respectively) and its corresponding actin in BVD (B) and sham (S) animals for CA1 at 7

days post-operation (n = 6 for sham and n = 7 for BVD animals).

CB1

Actin

`B S `B S B S `B S B S B S B

a) b)

c) d)



4.3.2.1 Optimisation of the Protein Amount and Radioligand Concentration

The specific binding of [3H]-CP 55,940 was higher in the wells containing 1 nM of

[3H]-CP 55,940 compared to 0.5 nM (Figure 4.4a). A 1 nM concentration was chosen because

the higher concentration of [3H]-CP 55,940 gave higher radioactivity, therefore any change in

the binding may be detected with greater sensitivity than with a lower concentration. Figure

4.4b shows that 100 L of WIN in non-specific binding wells was sufficient to inhibit [3H]-CP

55,940 binding. Although the total binding increased with the increase of the protein amount

in the well, the level of non-specific binding remained relatively unchanged. This was

demonstrated by a low R2 value for the non-linear regression analysis, in which the line of

best fit for the non-specific binding was almost horizontal (R2 = 0.872 for total binding and

0.160 for non-specific binding; Figure 4.4b).

Figure 4.4

(a) Saturation binding curve with increasing amount of hippocampal membrane preparation for

different concentrations of [3H]-CP 55,940. (b) Specific binding of [

3H]-CP 55,940 was calculated

from the difference between total and non-specific binding. Data are represented as mean

counts per minute (CPM) + SEM.

a)

b)


4.3.2.2 Changes in the CB1 Receptor Binding Affinity

There were no significant differences in the CB1 receptor binding affinities between the

sham and BVD animals at any post-operative time points, and there were no significant

interactions (Figure 4.5).

Figure 4.5

Displacement curves of the [3H]-CP 55,940 binding with WIN as a competitive inhibitor and the

IC50 values of WIN at (a, b) 1 day, (c, d) 3 days, (e, f) 7 days following sham or BVD surgery.

Data are represented as mean + SEM.

a) b)

c) d)

e) f)


4.4 Discussion

In the present study, changes in the density and affinity of CB1 receptors in the

hippocampus were investigated at specific time points following BVD. This was the first

study to show that BVD results in significant changes in the expression of the CB1 receptor in

a specific sub-region of the hippocampus. The density of CB1 receptor expression was

significantly lower in the BVD animals than in the sham animals in the CA3 region of the

hippocampus, but in no other hippocampal region investigated. Similar to the CB1 receptor

density decrease following BVD, a decrease in the level of CB1 receptors in the hippocampus

is generally observed following chronic administration of exogenous cannabinoids, as part of

the development of the animals‟ tolerance to the cannabinoid drugs (Romero et al., 1998;

Rubino et al., 2000a, b; Hill et al., 2004; Dalton et al., 2009) or in response to chronic stress

(Hill et al., 2005, 2008b, 2009; Reich et al., 2009; Suàrez et al., 2009). Furthermore, down-

regulation of CB1 receptor expression in other parts of the brain such as the basal ganglia and

the striatum can be observed in certain pathological conditions such as Huntington‟s disease

(Richfield and Herkenham, 1994; Glass et al., 1993, 2000; Allen et al., 2009; Dowie et al.,

2009; see Pazos et al., 2008 for review) and multiple sclerosis (Berrendero et al., 2001;

Cabranes et al., 2006; Cetonze et al., 2007; see Centonze et al., 2008 for review).

There are several possible explanations for the observed region-specific reduction of

CB1 receptor density in the BVD animals. First, it has recently been shown that the CA3

pyramidal neurons dynamically control their inhibitory inputs through the Group I

metabotropic glutamate receptor-mediated release of endocannabinoids (Inada et al., 2010).

Furthermore, Kim and Alger (2010) demonstrated the homeostatic mechanism of

endocannabinoid signaling in activity-deprived CA1 pyramidal neurons, where the afferent

excitation of the CA1 region was reduced by the surgical removal of the CA3 area. It has been

shown that neuronal networks are maintained by homeostatic compensation of synaptic

strength when long-lasting alterations in neuronal activity occur (see Turrigiano, 2008 for

review). Since studies have shown that the endocannabinoid system has a neuromodulatory

function in the hippocampus (see Section 1.2.2 of Chapter 1; Breivogel and Sim-Selley, 2009

for review) and that BVD results in severe hippocampal place cell dysfunction (Stackman et

al., 2002; Russell et al., 2003b, 2006), it can be hypothesised that decreased CB1 receptor

density in the CA3 region in BVD animals might be a part of a homeostatic mechanism for

changes neuronal activity in the hippocampus following BVD.


Second, previous studies have suggested that the CA3 region of the hippocampus is

important in memory formation (Hess et al., 1995; Gall et al., 1998; Zhao et al., 2000; He et

al., 2002). Since the endocannabinoid system is known to have an important role in learning

and memory (see Davies et al., 2002; Lichtman et al., 2002 for reviews) and many studies

have shown that BVD animals are cognitively impaired in a number of learning and memory

tasks (results from Chapter 3; Wallace et al., 2002c, Zheng et al., 2004, 2007, 2009b), this

decrease in CB1 receptor density in the CA3 region in BVD animals might be an initial

pathological indication of the functional deficits such as spatial memory impairment that are

associated with BVD. However, further studies will be required to determine the functional

significance of the changes in CB1 receptor expression in the CA3 region of the hippocampus

following BVD that were found in the present study.

Third, the decreased CB1 receptor density observed in the CA3 region in the current

study might be associated with degeneration in specific hippocampal structures, which might

include changes in synaptic proteins, dendrites, or even hippocampal cell death. Changes in

proteins that are related to synaptic transmission and neuronal plasticity in the hippocampus

have already been documented in BVD rats. These proteins are synaptophysin, synaptosomal-

associated protein 25 (SNAP-25), neurofilament light subunit (NF-L), and drebrin (Goddard

et al., 2008c). However, it has been observed that there is a significant increase in SNAP-25

only in the DG region of the hippocampus in BVD animals compared with sham animals,

with no further changes in other proteins (Goddard et al., 2008c). Although widespread

apoptosis has been observed in the hippocampus following BVD in rats (Smith et al., 2009),

this was a preliminary observation, and therefore more systematic quantification is required to

further clarify whether the decrease in CB1 receptor density found in the present investigation

is due to a decrease in cell number or instead due to a decrease in the number of CB1 receptors

expressed per cell.

In the present study, the density of CB1 receptor expression decreased in a time-

dependent manner in all sub-regions of the hippocampus following both the sham and BVD

surgeries. It was particularly interesting to note that even the sham surgery resulted in a

decrease in CB1 receptor density over time, to the same extent as for the BVD animals. An

age-related decrease in the density of hippocampal CB1 receptors has been reported in rats

(Berrendero et al., 1998; Marchalant et al., 2008; Canas et al., 2009) and in humans (Mato

and Pazos, 2004; Wong et al., 2010). However, in these studies, the effect of age on CB1

receptor density was investigated over a long period of time, which ranged from 2-24 months


for rats and 21-73 years for humans. The time-dependent decrease in CB1 receptor density

observed in the present study is unlikely to be due to aging because, not only were the animals

3 months old at the time of tissue collection, the particular time points used in the present

study (1, 3 and 7 days post-operation), are relatively short-term time points compared to those

of the above aging studies (Berrendero et al., 1998; Mato and Pazos, 2004; Marchalant et al.,

2008; Canas et al., 2009; Wong et al., 2010). For these reasons, future similar studies should

include a separate non-surgical control group for every time point to clarify whether the sham

surgery has any effect on the density of the CB1 receptors in the hippocampus.

Despite the number of studies showing a reduction in CB1 receptor density following

chronic stress or due to tolerance, studies investigating temporal changes in CB1 receptor

levels are scarce. A time-dependent decrease in CB1 receptor expression has been reported in

animal models of epilepsy (Falenski et al., 2009) and Parkinson‟s disease (Walsh et al., 2010),

with similar post-operative time points to the present study (4 and 7 days for Falenski et al.,

2009; 1, 3, 7, 14, 28 days for Walsh et al., 2010). This suggests the possibility that the

endocannabinoid system is highly sensitive to perturbations of any sort, and that even sham

surgery causes a transient up-regulation of CB1 receptor expression that gradually decreases

over the course of one week. Furthermore, it is possible that these temporal changes in CB1

receptor expression are not restricted to the hippocampus alone. Recently, it has been

hypothesised that effective performance in working memory tasks may involve prefrontal

cortical functionality in addition to the important role of the hippocampus. Furthermore, not

only are CB1 receptors expressed at a high level in the prefrontal cortex, it has been shown

that prefrontal cortex activity is altered by the administration of cannabinoids (see Egerton et

al., 2006 for review). For these reasons, the prefrontal cortex would be one of many areas to

target in future investigations into whether the expression of the CB1 receptor changes with

time following BVD.

The second step in determining whether BVD results in changes to CB1 receptors, was

to investigate possible changes in CB1 receptor binding affinity, using the [3H]-CP 55,940

binding assay. While there was no significant difference in CB1 receptor affinity between the

sham and BVD rats, it was interesting to find that the IC50 values obtained from the present

study varied between 16 – 43 nM. Astonishingly, no previous study has assessed the IC50 of

WIN in the hippocampal membrane preparation. Nonetheless, Houston and Howlett (1998)

reported an IC50 for WIN of 38 nM in a [3H]-CP-55,940 competitive binding assay using the

whole brain membrane preparation. Although Thomas et al. (1998) did not report an IC50 for


WIN, this value could be calculated from the data given, by using the Cheng-Prusoff equation

(Cheng and Prusoff, 1973), revealing an IC50 value of 27 nM, but again in the whole brain

membrane preparation. Using a single neuron or primary neuron culture, 47 and 14 nM were

reported as IC50 values for WIN, respectively (Pan et al., 1996; Shen and Thayer, 1998).

Taken together, although the IC50 values in the present study are within the range of results

from previous studies, a direct comparison cannot be made because the present study used a

hippocampal membrane preparation rather than a whole brain preparation. For this reason, a

non-surgical group should be included in future studies to determine whether the sham

surgery has any effect on CB1 receptor affinity.

Although there was no significant difference in the binding affinity of the hippocampal

CB1 receptors between the sham and BVD animals, the present study is the first study to

report the IC50 for WIN, and hence the binding affinity of the CB1 receptors for WIN, in the

rat hippocampus. In addition to a scarcity of literature on the IC50 for WIN in the brain, there

is also no study to date reporting the Ki for WIN in the hippocampus. However, there are two

studies that used the whole brain membrane preparation (Thomas et al., 1998; Gullapalli et al.,

2010). This was surprising as the endocannabinoid system and its involvement in many

pathological conditions has been studied thoroughly, yielding countless publications.

However, the majority of these studies have focused on the density or the efficacy of the

cannabinoid receptor, and only a very few studies have investigated the binding affinity.

In the present study, a competitive binding assay was performed, in which the

concentration of the [3H]-CP 55,940 remained constant (1 nM), while the concentration of the

unlabelled ligand (WIN) was varied. The binding affinity of a receptor can be expressed in

various ways, including measurements such as Kd, Ki, and IC50. The IC50 in the present study

was estimated instead of Ki, as the Prism 5 software required the use of limiting constraints

when performing the analysis to estimate Ki. These constraints were the concentration of the

radioligand used in the binding assay and more importantly, the Kd of the radioligand.

Without entering the values for these constraints, the programme would not allow non-linear

regression to fit a model to directly estimate Ki. Although it was simple to perform this

analysis, changing these constraints inevitably changed the regression, therefore, any estimate

from the line of best fit (that is, values for Ki) also changed. Therefore, it would have been

necessary to choose these constraints very cautiously and with considerable confidence in

their accuracy. The concentration of the [3H]-CP 55,940 used in the present study was 1 nM.

As a saturation binding assay was not performed in the current study, the Kd of the [3H]-CP


55,940 had to be obtained from the literature. However, the reported Kd of the [3H]-CP 55,940

in the hippocampus varied between 0.5 – 1.7 nM (Romero et al., 1995; Gatley et al., 1997;

Castelli et al., 2007; Hill et al., 2005, 2008a, 2010), which consequently resulted in a large

variation in Ki estimation. For this reason, it was necessary to consider approaches other than

Ki to estimate the affinity of the CB1 receptor.

Fortunately, the non-linear regression analysis to estimate IC50 did not require the use of

any limiting constraints. Since Ki is proportional to IC50 (Motulsky, 1995), the IC50 for WIN

could also be used to quantify the binding affinity of the CB1 receptors in the hippocampus.

However, a disadvantage of estimating the IC50 is that by changing the experimental

conditions, such as changing the radioligand used or changing its concentration, the IC50

would also change (Motulsky, 1995). In other words, increasing the radioligand concentration

used or using a radioligand with a lower Kd (that is, a ligand with higher affinity) would

increase the IC50, as a larger concentration of unlabelled ligand would be required to compete

for the binding site when the radioligand concentration is high, or more unlabelled ligand

would be required to compete for a high affinity radioligand (low Kd) than for a low affinity

radioligand (high Kd).

Ideally, a saturation binding assay with the [3H]-CP 55,940 should have been performed

in order to obtain the CB1 receptor affinity more directly. However, due to a greater amount of

radioligand required to perform the saturation binding assay compared to the competitive

binding assay, it would have been prohibitively expensive to run 45 separate saturation

binding assays for the present study. Nevertheless, another option might have been to perform

the saturation binding assays for a small number of the samples to obtain the Kd of the [3H]-

CP-55,940, and then to carry out the competitive binding assays to obtain the Ki. Such

possibilities should be considered in future studies of a similar nature.

Numerous studies have shown the distribution and localisation of the CB1 receptors in

the brain (Pettit et al., 1998; Tsou et al., 1998; Ong and Mackie, 1999; Moldrich and Wenger,

2000; Egertová and Elphick, 2000; Suárez et al., 2008, 2009). In order to provide a complete

picture of effects of BVD on CB1 hippocampal receptors, the expression of hippocampal CB1

receptors following BVD was also examined in the present study by means of

immunohistochemistry (Appendix 2), using the same CB1 receptor antibody that was used for

western blotting in this Chapter. However, since multiple protein bands were observed with

this CB1 receptor „specific‟ antibody in western blotting, it is possible that these other bands


that are recognised by the CB1 antibody may similarly produce non-specific CB1 receptor

labelling in immunohistochemistry. The lack of the specificity of the CB1 receptor antibody

has also been observed with other commercial CB1 receptor antibodies (Sigma, Affinity

BioReagents, Cayman, and BoiSource International; Grimsey et al., 2008; Jelsing et al.,

2008). Although the use of tissues from the CB1 receptor knockout mice would have been the

easiest way to clarify the specificity of the CB1 receptor antibody, such tissues were not

available for the present study. Taken together, although the specificity of the CB1 receptor

antibody was tested by varying antibody concentrations and incubation durations (Appendix

1), due to considerable doubt regarding the binding specificity of the CB1 receptor antibody,

the labelling obtained in immunohistochemical study is considered questionable (Appendix 2).

The particular time points that were chosen for the present study (1, 3 and 7 days post-

operation) were relatively short post-operation time points compared to a 14 months post-

operation time point in the foraging task (Chapter 3), and therefore it is possible that

behavioural results from Chapter 3 are not directly comparable with the results presented in

the present Chapter. Furthermore, because these are acute time points, the changes observed

in CB1 receptor density and affinity in the present study might be related to the immediate

effects of the loss of vestibular function rather than to its long-term consequences. However, it

has been shown that hippocampal place cell function is disrupted immediately following BVD

(1 hr post-BVD induction, Stackman et al., 2002), and there is no evidence of recovery from

BVD-induced spatial memory deficits (see Chapter 3; Zheng et al., 2009b). Thus, it was

logical to analyse CB1 receptor density and affinity at acute time points following BVD,

rather than at long-term time points. Nevertheless, since CB1 receptor density and affinity did

not change substantially at 1, 3 and 7 days post-operation, longer post-operative time intervals

should be analysed in future studies to determine whether there are long-term changes in the

endocannabinoid system following BVD.

116

Chapter 5

Discussion and Conclusions

Chapter 5: Discussion and Conclusions 117

There has been an accumulation of evidence suggesting that damage to the peripheral

vestibular system results in the disruption of hippocampal synaptic transmission and cognitive

function. Over the past several years, it has been firmly established that the endocannabinoid

system is of fundamental importance in hippocampal synaptic plasticity and cognitive

function. The key aim of this thesis was to determine the involvement of the endocannabinoid

system in the cognitive deficits caused by bilateral loss of vestibular function, in order to

elucidate the potential mechanism(s) that may underlie this impairment. The results from the

present study suggest that cognitive deficits following BVD may be permanent and provide

some preliminary evidence that the role of the hippocampal endocannabinoid system in the

BVD-induced cognitive deficits may be limited.

Previous studies involving rats with BVD have demonstrated spatial memory deficits

that appear to be long-lasting, suggesting long-term adverse effects on the hippocampus

(Russell et al., 2003b; Zheng et al., 2009b). However, the longest post-operative time interval

that had been studied before the present investigation was approximately 5-7 months post-

surgery (Zheng et al., 2009b). The results from the present study suggest that rats with BVD

probably do not recover from spatial memory deficits even at very long time intervals

following the surgery, such as 14 months (Chapter 3), suggesting that deficits enduring at this

long time point may well be permanent. These apparently permanent learning and memory

impairments following BVD are of significance as approximately 80% of patients with

bilateral vestibulopathy do not recover significantly (Zingler et al., 2008) and ongoing

memory problems may contribute to this phenomenon. Although the effects of cannabinoid

drugs on BVD animals‟ spatial memory were more complicated than expected, some

speculation could be made based on the observed changes in CB1 receptor density and affinity

following BVD (Chapter 4).

Although a two-way ANOVA showed that BVD significantly decreased CB1 receptor

density in the CA3 region of the hippocampus across 3 post-operation time points – 1, 3 and 7

days, post hoc tests revealed that none of the comparisons for the same individual post-

operation time points were significant (Chapter 4). Therefore, although there was evidence for

a decrease in CB1 receptor density in BVD animals, it was not as convincing as it might have

been. The observed down-regulation of CB1 receptor density following BVD may reflect

either a decrease in synthesis rate or an increase in degradation rate of receptors. One possible

explanation for the down-regulation of CB1 receptors in CA3 is that this reduction is due to a

homeostatic response to increased levels of endocannabinoids in the hippocampus. Not only


has the enhancement of endocannabinoid levels been observed following various pathogenic

insults in the brain (Panikashvili et al., 2001; Marsicano et al., 2003; Wallace et al., 2003; de

Lago et al., 2006; van der Stelt et al., 2006), endocannabinoids have been shown to provide

protection against these neuronal insults (see Fowler et al., 2010 for review).

There is increasing evidence that the integrity of the hippocampus relies on input from

the vestibular system. The impaired performance of BVD animals in the foraging task

(Chapter 3), together with results from previous studies (Stackman and Herbert, 2002; Russell

et al., 2003a; Zheng et al., 2006, 2007, 2009b), have shown that BVD results in impairment in

various spatial memory tasks that are known to be sensitive to disruption of hippocampal

functions in animals. In addition, neurophysiological studies have shown that BVD causes

abnormalities in the function of place cells and in theta rhythm in the hippocampus (Stackman

et al., 2002; Russell et al., 2003b, 2006). In humans, bilateral hippocampal atrophy has been

observed following BVD (Brandt et al., 2005), while in animals, BVD appears to induce

apoptosis in the hippocampus (Smith et al., 2009). Taken together, it is evident that BVD

adversely affects the hippocampus, and therefore, it is conceivable that endocannabinoid

levels in the hippocampus might be elevated following BVD as observed in other

neuropathological conditions. For this reason, it will be interesting in future studies to

determine whether the level of endocannabinoids in the hippocampus changes following BVD.

The consequence(s) of decreased CB1 receptor density in the CA3 region of the

hippocampus in BVD animals (Chapter 4) and whether this decrease might be relevant to

BVD-induced cognitive deficits, remains to be elucidated. Why the CB1 receptor should be

affected only in the CA3 region of the hippocampus is also unclear at present. It is possible

that the reduction in CB1 receptor expression in CA3 following BVD might be related either

to the adverse effects of vestibular loss on the hippocampus, or to compensatory processes

initiated in response to the injury. However, despite the significant difference from the sham

controls, the possibility that any sensory loss might have caused similar changes, must be

considered.

Numerous pharmacological and brain lesion studies have shown that the CA3 region of

the hippocampus is important for spatial information processing (Routtenberg, 1984; Stubley-

Weatherly et al., 1996; Roozendaal et al., 2001; Steffenach et al., 2002; Stupien et al., 2003;

Kesner, 2007; Holahan and Routtenberg, 2010). Similar to the heading angle measurements in

the foraging task of the present study (Chapter 3), Holahan and Routtenberg (2010) also


measured rats‟ heading angles towards a hidden platform from the starting point in the water

maze task, where the ideal heading was a straight line from the launch point to the platform.

In their study, lidocaine was injected directly into the CA3 region of the hippocampus, which

locally inactivated the neural activity in the granule cell-mossy fibre CA3 circuit. Interestingly,

like BVD animals in the present study, animals treated with lidocaine showed a significant

deviation from the ideal heading angle in the water maze task (Holahan and Routtenberg,

2010). These results suggest that the CA3 region of the hippocampus may be important for

processing vestibular information, which in turn could contribute to the cognitive impairments

induced by BVD. This hypothesis leads to a potential future investigation involving

intrahippocampal administration of cannabinoids to BVD animals, which would provide

direct evidence of whether the cannabinoid receptors in the hippocampus are involved in

BVD-induced cognitive deficits.

It is currently unknown which specific regions of the hippocampus receive vestibular

information first. However, it may be reasonably supposed that a decrease in CB1 receptors in

the CA3 region is likely to interfere with CA3 output to CA1 via the Schaffer collateral

pathway. It has been shown that the hippocampal input pathway from the DG to the CA1

region is unidirectional. Although the granule cells in the DG project to the pyramidal cells in

the CA3 region via mossy fibre projections, pyramidal cells of CA3 do not project back to the

granule cells. Likewise, the pyramidal cells of the CA3 region project to CA1 via Schaffer

collaterals, but CA1 does not project back to CA3 (Amaral and Lavenex, 2007). Since the

CB1 receptors are known for modulating synaptic transmission by regulating the release of

major neurotransmitters such as glutamate and GABA (see Turu and Hunyady, 2010 for

review), it is possible that a decrease of the CB1 receptors in the CA3 region, as seen in the

BVD rats, results in a decrease in the release of glutamate and GABA, which in turn reduces

synaptic transmission to CA1. Thus, a reduction in CB1 receptor density in the CA3 region of

the hippocampus in BVD animals might be a part of the initial stages of the pathogenesis

which contributes to the emergence of cognitive deficits following BVD. Given that the

responsiveness of place cells in the CA1 region is severely disrupted following BVD

(Stackman et al., 2002; Russell et al., 2003b), it is conceivable that the down-regulation of

CB1 receptors in the CA3 region may contribute to the impairment of place cell function and

spatial memory following vestibular lesions.

Ashton et al. (2004b) showed that there were no significant changes in CB1 receptor

density in the hippocampus at 10 hours and 2 weeks following UVD. However, given that


UVD causes an imbalance in activity between the two brainstem vestibular nuclei (Smith and

Curthoys, 1989), the effects of UVD on the endocannabinoid system in the hippocampus are

likely to be different compared to BVD. Therefore, further studies will be required to

determine the functional significance of the changes in CB1 receptor expression in the CA3

region of the hippocampus following BVD that were found in the present study (Chapter 4).

As the method of western blotting was used to determine the density of CB1 receptors in

the hippocampus, it is not possible to know whether this reduction occurred in neurons or

glial cells, or both. In the present study, immunohistochemistry was carried out to further

examine the change in CB1 receptor expression following BVD (Appendix 2). However, one

of the major shortcomings of the present study was the lack of specificity of the CB1 and CB2

receptor antibodies (Chapter 4; Appendix 3). Although the optimum protocols for labelling

CB1 (Appendix 1) and CB2 (Appendix 3) receptors were determined, due to the lack of

specificity of the commercially available CB1 and CB2 receptor antibodies (Chapter 4;

Appendix 3; Grimsey et al., 2008; Jelsing et al., 2008), concerns were raised regarding the

reliability of the immunohistochemical data obtained in the present study to determine

changes in CB1 receptor density (Appendix 2).

To completely characterise changes in CB1 receptors following BVD and their

involvement in BVD-related cognitive deficits, further studies will be required to determine

the functional significance of these changes (Chapter 4). It is possible that they are related to

the impairment of place cell function and theta rhythm following BVD. It is also conceivable

that changes take place in the G-protein coupling of the CB1 receptor. Possible experiments to

investigate these possibilities include using a [35

S]GTPS binding assay to measure the level

of G-protein activation upon CB1 receptor stimulation, and some biochemical assays which

measure the level of cAMP and protein kinase A activity. The latter assays are logical as the

cAMP cascade is one of the major intracellular signaling pathways implicated in the

biological actions of cannabinoids. As well as investigating the CB1 receptor and its related

protein expression, CB1 receptor mRNA expression should also be studied using in situ

hybridization to determine whether the BVD changes the genetic expression of CB1 receptors

in the hippocampus. Furthermore, other components that comprise the endocannabinoid

system, that is, the endocannabinoids and the enzymes that break down endocannabinoids,

could also be assessed. Knowledge of the changes in the level of endocannabinoids and the

activity of FAAH and MAGL (enzymes responsible for the breakdown of endocannabinoids)

in the brain following BVD, would further indicate whether the endocannabinoid system is


involved in the functional changes in the hippocampus that occur following BVD.

Many believe that the effects of cannabinoids are influenced by the expression of CB1

receptors in the brain (Herkenham et al., 1991, Jansen et al., 1992; Sim et al., 1995), but the

possibility of changes in the affinity of these receptors are often neglected. It was interesting

to observe that while there was a change in receptor expression following BVD, there was no

change in the affinity of the CB1 receptors for WIN, as IC50 values for WIN were not different

between the sham and BVD animals (Chapter 4). Similar results were found by Hill et al.

(2005, 2008a), in which both chronic stress and repeated administration of corticosterone in

rats significantly decreased hippocampal CB1 receptor density without affecting affinity for

the CB1 receptor agonist, [3H]-CP 55,940. The results from the present study, together with

the Hill et al. studies, suggest that one cannot assume that the changes in receptor expression

would result in changes in affinity.

Much of our understanding of vestibular-hippocampal connections comes from clinical

and experimental observations in humans and animals with unilateral and bilateral vestibular

lesions. However, there have been some studies suggesting that the stimulation of the

vestibular system either by cold water (i.e. caloric stimulation; Bachtold et al., 2001) or by

low intensity electrical stimulation (i.e. galvanic stimulation; Wilkinson et al., 2008, 2010)

may improve some aspects of memory in humans, although this has not been investigated for

spatial memory as yet (see Smith et al., 2010 in press for review). Similarly, McGauran et al.

(2005) showed that vestibular stimulation by rotation, enhanced rats‟ performance in the water

maze task. Since the present study investigated the effects of vestibular lesions on the

endocannabinoid system in the brain, it would be interesting to also investigate whether or not

the endocannabinoid system is affected by vestibular stimulation.

Clearly, the mechanisms contributing to the effects of BVD on cognitive functioning

require further investigation. Nevertheless, the results from the foraging task have confirmed

that inputs from the vestibular system are crucial for spatial navigation and memory in

animals. The BVD animals were unable to use not only self-movement cues, but also

allothetic cues in navigation (Chapter 3). This result is consistent with the results of Brandt et

al. (2005) in humans, and suggests that spatial memory deficits in patients with vestibular

dysfunction may become worse over time.

In the present study, although it was expected that a cannabinoid receptor agonist would


impair spatial memory (Suenaga and Ichitani, 2008; Robbe and Buzsaki, 2009; Wise et al.,

2009), WIN did not seem to impair spatial memory even in the sham animals, as would be

expected (Chapter 3). In fact, WIN at 2 mg/kg, significantly improved BVD animals‟

performance in the dark, but not in sham animals, suggesting that BVD might have resulted in

changes in the brain‟s endocannabinoid system. Furthermore, the pre-treatment with the

cannabinoid receptor inverse agonist, AM251, demonstrated that the involvement of the

endocannabinoid system in the BVD-induced cognitive deficits is complicated.

In conclusion, evidence presented in this thesis indicates that bilateral vestibular loss

may cause permanent spatial memory deficits in animals, and lays a foundation for further

investigation of the involvement of the endocannabinoid system in the debilitating effects of

vestibular damage.

123

Appendices

Appendix 1: Specificity of CB1 Receptor Labelling in the Hippocampus 124

Appendix 1

The Specificity of Cannabinoid CB1 Receptor Labelling in

the Rat Hippocampus

1.1 Introduction

In order to reliably compare the effects of surgery or drug treatment on the expression of

CB1 receptors in the brain, it is essential to have sufficient controls to convince a sceptical

observer that the staining patterns obtained using antibodies in the present study, really do

represent the CB1 receptor. Confirming the specificity of CB1 receptor antibodies was

therefore the first step necessary before investigating the effects of vestibular damage and

cannabinoid treatment in the rat.

A considerable amount of work has been done to clarify the molecular mechanisms

responsible for the effects of cannabinoids on cell function and behaviour in animals. Since

the discovery of the CB1 (Devane et al., 1988) and CB2 receptors (Munro et al., 1993), the

focus of cannabinoid research has been on the molecular events that alter the behaviour of

cannabinoid receptors. An important issue that needs to be dealt with before studying CB1

receptor function is to confirm where and how these receptors are expressed and distributed.

Many rat studies have been published on the distribution and localisation of the CB1 and CB2


receptors in the brain and in the immune system, respectively. Numerous conventional

methods such as receptor autoradiography, in situ hybridisation, and immunohistochemistry

have been utilised to clarify the distribution and the localisation of these two receptors in the

rat brain. The common principle behind these methods is the formation of the receptor-ligand

complex. Here, the ligand can be a radiolabelled agonist or antagonist, a complementary DNA

(deoxyribonucleic acid) or RNA (ribonucleic acid) probe, or a specific antibody that

recognises a particular protein, the CB1 receptor in this case. Antibodies are large protein

molecules that are able to identify an epitope found on a protein. The epitope may be in a

certain molecular configuration in the native protein, or it can be exposed in the fixed

molecule that is not present in the native configuration (see Saper and Sawchenko, 2003 for

review). When the antibody recognises an epitope, it binds to that specific epitope and the

bound antibody can be visualised in various ways by using an enzyme-conjugated secondary

antibody that catalyses a colour-producing reaction or using a secondary antibody that has

been labelled with a fluorophore.

A crucial issue in studying endogenous cannabinoid receptor proteins in the brain and in

other areas, is the availability and specificity of the antibodies used. For the past decade, CB1

receptor antibodies, targeting different regions of the receptor, have become indispensable

research tools and have been manufactured by numerous research groups around the world. A

diverse range of CB1 antibodies can also be purchased through a variety of commercial

suppliers. However, concerns have been raised regarding the specificity of the antibodies in

labelling CB1 receptors, since it is rare that an antibody is exclusively specific to the desired

target protein. Due to this unreliable antibody specificity, immunohistochemistry has

sometimes yielded variable and even flawed results with various antibody preparations. It is

important to realise that the antibodies used in immunohistochemistry are biological agents,

not ordinary chemical reagents. Therefore, it is highly likely that antibodies may recognise a

broad range of antigens, rather than only the antigen they were raised to recognise, and thus

one should always consider that specific antibody labelling is limited and that even the most

specific antibody available may label not only the actual antigen, but also other “antigen-like”

molecules.

In order to minimise misrepresentation of antigens, many investigators have chosen to

produce their own antibodies rather than to buy them from commercial companies. However,

unsatisfactory immunohistochemical results do not solely depend on whether the antibody

was made privately or commercially bought. There are many other possible factors that may


affect the end results. These include preparation of the tissue, tissue thickness, the host of the

antibody, antibody incubation time, the antibody‟s binding site, whether the antibody binds to

the N-terminal or C-terminal domains of the receptor, and the antibody concentration (Lorincz

and Nusser, 2008; see Goldstein et al., 2007 for review). Considering each of the above

factors, it is clear that a series of steps must be taken to control for these variables. Previous

immunohistochemical studies have shown surprising discrepancies in the specificity of CB1

receptor antibodies, in which the results obtained were contradictory to one another.

Furthermore, a standard method for carrying out immunohistochemistry to label CB1

receptors in the brain was absent in these studies. In order to control for this inconsistency, at

least to some extent, Grimsey and others have recently tested the ability of a series of CB1

antibodies to detect endogenous CB1 receptors in cultured cells and in brain slices (Grimsey et

al., 2008). In this study, four commercially available (Sigma, Affinity BioReagents, Cayman,

and BoiSource International) and two privately made CB1 antibodies were examined.

Theoretically, all CB1 receptor antibodies should recognise the same CB1 receptor antigen,

and hence give a similar labelling pattern. However, the CB1 receptor antibodies from

different providers showed notably different CB1 receptor labelling patterns (Grimsey et al.,

2008). This study raised even more doubts about the specificity of the CB1 receptor antibodies

and whether the staining patterns observed in any study truly represent the CB1 receptor.

Thus it is necessary to validate CB1 receptor antibody specificity in any study where

immunohistochemistry for CB1 receptors is employed. For this reason, it was crucial to

optimise the protocol for immunohistochemical labelling of the CB1 receptors before

investigating the effects of BVD on the expression of the CB1 receptors.



1.2.1 Removal of Brain

Three male Wistar rats, each weighing 250-300 g, were decapitated without anaesthesia.

The whole brain was rapidly removed from the skull and immediately placed into ice-cold

0.9 % saline solution. The brain was dissected on a cold plate into two parts, i.e. the forebrain

and the hindbrain. The forebrain was further divided in half in the sagittal plane. The brains

were completely covered with O.C.T. compound (BDH Laboratory Supplies), and were then

immersed in n-hexane solution (Biolab) that had been placed in liquid nitrogen. After the

O.C.T. compound had turned to white, the tissue was wrapped in aluminium foil and stored at

-20o

C until sectioning. CB1 and CB2 receptor labelling was also trialed using

paraformaldehyde-fixed tissue. However, the CB2 receptor labeling quality was very poor

when fixed tissue was used, which prohibited using the fixed tissue. In order to equalise the

tissue conditions for both CB1 and CB2 receptor labeling, fresh tissue was used for CB1

receptor labeling as well. Hydrogen peroxide was used to quench any endogenous peroxidase.

1.2.2 Tissue Sectioning

Prior to tissue sectioning, the glass slides were coated with a Poly-L-Lysine solution

(Sigma) in order to improve adherence of the frozen sections. Briefly, slides were immersed

in Poly-L-Lysine solution that had been diluted with distilled water (1:10) for 5 minutes. The

slides were then drained and dried in a drying oven at 60o C for 1 hr. Once the slides were

completely dried, they were cooled and kept at room temperature until use.

All tissue sectioning was performed at -18oC with a 16 m section thickness using a

Cryostat (Leica CM 1850). Each brain was cut in the sagittal plane using a stereological

method. Briefly, after a random start using a random number generated by a calculator, a

systematic subset of these sections separated by a fixed distance (d), was collected. Twelve

levels of the hippocampus were taken per animal where each section in each level was

separated by 464 m. Systematic random sampling was used to achieve a lower coefficient of

error compared with random sampling. The sections were thaw-mounted on Poly-L-Lysine

coated slides and stored at -20o C until staining.


1.2.3 Immunohistochemistry

Sections were air-dried briefly at room temperature to eliminate moisture that appeared

immediately after the sections were removed from -20o

C. Hydrophobic boundaries were

drawn around the sections using a DAKO pen (DAKO), and the sections were dried for a

further 5 minutes. Each step was carried out at room temperature unless otherwise stated.

When the hydrophobic ring was completely dried, sections were washed with 0.01 M

phosphate-buffered saline (PBS, Sigma), then fixed in 4% paraformaldehyde (PFA, Sigma)

for 10 minutes. To terminate PFA fixation, 0.1 M glycine (Sigma) was applied to sections for

15 minutes. Sections were treated with 0.3% H2O2 (Merck) in methanol (BDH) for 20

minutes to block endogenous peroxidase activity, and then washed with 0.01 M PBS for 5

minutes twice. In order to block non-specific binding, sections were incubated with 1.5%

heat-inactivated donkey serum (Sigma) for 1 hr. This blocking solution was prepared in 0.01

M PBS containing 0.2% bovine serum albumin (BSA, Gibco). The concentration of the

blocking solution used in the present study was utilised from the previous studies (Ashton et

al., 2004a, b; Fusco et al., 2004). After blocking was completed, various concentrations of

polyclonal CB1 receptor goat primary antibody (Santa Cruz Biotechnology, Inc.) were applied

and incubated for either 18 hrs (overnight) or 41 hrs (2 nights) at 4o C (Table 1.1). Both

primary and secondary antibody solutions were diluted in antibody-diluting buffer containing

1% BSA and 0.5% Triton X-100 (BDH) in 0.01 M PBS.

Following the primary antibody incubation, the sections were washed four times for 15

minutes each wash, with the antibody washing buffer (1% BSA and 0.5% Triton X-100 in

0.01 M PBS) to reduce non-specific binding prior to the secondary antibody application.

The sections were then incubated with an HRP-conjugated polyclonal donkey anti-goat

antibody (Santa Cruz Biotechnology, Inc.), diluted at various concentrations (Table 1.1). The

secondary antibody was either incubated for 1 or 2 hrs at room temperature. The

immunohistochemical complex was visualised by exposure to diaminobenzidine (DAB,

Sigma) for 20 minutes. Positive immunoreactivity to the CB1 receptor was expressed as

brown staining in the cytoplasm, cell body, axons, dendrites, and fibres. Negative controls

consisted of sections treated as detailed above without the primary antibodies. The stained

sections were rinsed with distilled water (dH2O) twice and then twice with tap water, for 5

minutes each time. After rinsing, the sections were dehydrated in ascending concentrations of

alcohol; 5 minutes in each of 70% and 95% ethanol, 10 minutes in 100% ethanol and 5

minutes in xylene, and cover-slipped with DPX mounting medium (a mixture of xylene and

dibutyl phthalate, BDH).


Table 1.1

Various combinations of CB1 receptor primary (1o) and subsequent secondary (2

o) antibody

titres trialled and their incubation durations.

1o antibody

concentration

2o antibody

concentration

1o / 2

o antibody incubation

time (hrs)

1:50 1:500

18 / 1

1:100 1:200

1:100 1:500

1:200 1:500

1:200 1:1000

1:50 1:500

41 / 1

1:50 1:1000

1:50 1:1500

1:100 1:200

1:100 1:500

1:100 1:1000

1:100 1:1500

1:200 1:500

1:400 1:500

1:50 1:200 41 / 2

1:50 1:500

1.2.4 Double Label Immunofluorescence

Double immunofluorescent labelling of the CB1 receptor with an antibody for NeuN, a

neuronal marker (monoclonal raised in mouse; Chemicon), was carried out for two reasons.

Firstly, the labelling pattern observed with immunohistochemical DAB staining needed to be

confirmed. Secondly, it was necessary to confirm that the CB1 receptor is expressed by

neurons. This protocol was the same as for the DAB immunohistochemistry, except for the

use of a fluorescent secondary antibody. The secondary antibodies utilised here were an Alexa

Fluor 488-conjugated anti-mouse IgG raised in goat (Invitrogen) for NeuN labelling and

Alexa Fluor 594-conjugated anti-goat IgG raised in donkey (Invitrogen) for the CB1 receptor.

The dilution of the CB1 receptor primary antibody was 1:100 and 1:1000 for the secondary

antibody. For NeuN, the primary antibody concentration was 1:200 and the secondary

antibody concentration was 1:500. Immunofluorescence reinforces the results obtained from

DAB immunohistochemistry as it completely eliminates the possibility of endogenous


peroxidase labelling within the tissue. Therefore, it reduces the possibility of non-specific

labelling to some extent, which in turn gives better end results. The CB1 receptor antibody

was incubated for two nights (41 hrs) whereas the NeuN antibody was incubated overnight

(both at 4o C). Because NeuN was a monoclonal antibody, blocking with serum was not

required. For both the CB1 receptor and NeuN primary antibodies, the secondary antibodies

were incubated for 1 hr at room temperature. As the secondary antibodies are light sensitive,

this procedure was carried out in the dark.


Unless stated otherwise, a Zeiss Axioplan MC80 BX microscope, Zeiss AxioCam HRc

digital camera and Zeiss Axiovision 3.1 software (Carl Zeiss Vision GmBH, Germany) were

employed for all morphological analyses and photography of immunohistochemical data.

Identical fields on each section stained with different antibody preparations were evaluated. In

this study, the dentate gyrus (DG) of the hippocampus was assessed. For visualising

fluorescent labelling, a Zeiss 510 LSM confocal microscope (Carl Zeiss GmbH, Germany)

and LSM 510 control software (version 3.2) were used.


1.3 Results

1.3.1 Optimisation of the CB1 Receptor Primary Antibody

In the first part of this study, CB1 receptor labelling with an antibody purchased from

Santa Cruz Biotechnology, Inc. was examined. Figure 1.1 shows the staining pattern obtained

with two different CB1 receptor primary antibody incubation times, to highlight the

differences in labelling observed. The incubation time window was selected on the basis of

previous immunohistochemical studies. From the literature, the standard CB1 receptor

primary antibody incubation time seems to be either overnight or 48 hrs (Tsou et al., 1998;

Ong and Mackie, 1999; Ashton et al., 2004; Fusco et al., 2004; Cristino et al., 2006; Grimsey

et al., 2008; Jelsing et al., 2008; Malone et al., 2008; Suàrez et al., 2008). In the present study,

18 hr (overnight) incubation appeared to be not sufficient to give CB1 receptor labelling,

therefore 41 hr incubation was chosen as an optimal primary antibody incubation duration

(Figure 1.1).

Figure 1.1

CB1 receptor labelling with different primary antibody incubation durations. The antibody

concentrations for both conditions were the same, i.e. 1:200 dilution for the primary antibody

and 1:500 dilution for the secondary antibody. Primary incubation durations were: (a) 18 and (b)

41 hrs. The secondary antibody was incubated for 1 hr for both primary incubation times. Scale

bars represent 100 m.

Figure 1.2 reveals how different antibody titres affect the CB1 receptor labelling. Similar

to antibody incubation time, there is a greater possibility of non-specific binding with higher

concentrations of the antibody. The staining with the 1:50 titre showed what may have been

some positively labelled cells for the CB1 receptor (Figure 1.2a). However, with a high

background this labelling cannot be guaranteed to be truly positive. On the other hand, the

1:400 titre showed too little CB1 receptor labelling (Figure 1.2d). Between 1:100 (Figure 1.2b)

a) 18 hrs b) 41 hrs


and 1:200 (Figure 1.2c) dilutions, the quality of the apparent CB1 receptor specific labelling

was equal, while the 1:100 titre had less background labelling. Therefore, a 1:100 dilution of

the CB1 antibody was selected as an optimal primary antibody concentration.

Figure 1.2

CB1 receptor labelling with different primary antibody concentrations. The secondary antibody

concentration (1:500 dilution) for all conditions remained constant. The dilutions for the

primary antibody were: (a) 1:50, (b) 1:100, (c) 1:200, (d) 1:400. The primary antibody was

incubated for 41 hrs and the secondary antibody for 1 hr. Scale bars represent 100 m.

a) 1:50 b) 1:100

c) 1:200 d) 1:400


1.3.2 Optimisation of the Secondary Antibody

The incubation time for the secondary antibody was much shorter than for the primary

antibody. In most studies, the secondary antibody is incubated for either 1 or 2 hrs at room

temperature (Tsou et al., 1998; Ong and Mackie, 1999; Fusco et al., 2004; Cristino et al.,

2006; De March et al., 2008; Grimsey et al., 2008; Jelsing et al., 2008; Suàrez et al., 2008).

Unlike the primary antibody, no particular change in labelling intensity or pattern was

observed when the two durations were tested (Figure 1.3). However, Figure 1.4 highlights the

fact that when compared to alteration of the primary antibody dilution, greater changes in the

intensity and the extent of labelling were observed as the secondary antibody dilution was

altered in various ways. Figure 1.4 shows that diluting the secondary antibody reduces the

level of background staining. When the 1:200 and 1:500 titres are compared to the 1:1000 and

1:1500 titres, it is apparent that there is less fine thread-like labelling in the background in the

1:1000 and 1:1500 titre group (Figure 1.4). However, when the antibody was diluted to

1:1500, it gave a negligible signal (Figure 1.4d). Therefore, a 1:1000 dilution was chosen as

an optimum secondary antibody concentration.

Figure 1.3

CB1 receptor labelling with different secondary antibody incubation durations. The primary and

the secondary antibody concentrations for both conditions were the same (1:50 and 1:500

dilution, respectively). Secondary antibody incubation durations were: (a) 1 and (b) 2 hrs. The

primary antibody was incubated for 41 hrs. Scale bars represent 100 m.

a) 1 hr b) 2 hrs


Figure 1.4

CB1 receptor labelling with different secondary antibody concentrations. The primary antibody

concentration remained constant for all conditions (1:100 dilution). The dilutions for the

secondary antibody were: (a) 1:200, (b) 1:500, (c) 1:1000, (d) 1:1500. The primary antibody was

incubated for 41 hrs and the secondary antibody for 1 hr. Scale bars represent 100 m.

a) 1:200 b) 1:500

c) 1:1000 d) 1:1500


1.3.3 Double Labelling Immunofluorescence

Double labelling of the CB1 receptor with a NeuN antibody suggested that the CB1

receptor is expressed by some of neurons in various regions of the hippocampus. The green

fluorescence represents neurons labelled with the NeuN antibody and red fluorescence

corresponds to cells apparently expressing the CB1 receptor (Figure 1.5). Apparent labelling

of the CB1 receptor was also observed in axons and dendrites (white arrows in Figure 1.5),

which was consistent in the DAB staining (Figure 1.6).

Figure 1.5

Double labelling immunofluorescence of the CB1 receptor antibody with an antibody for NeuN,

a neuronal marker, in the (a) CA1, (b) CA3, (c) hilus, (d) DG regions of the hippocampus. Green

fluorescence represents neurons and red fluorescence corresponds to cells expressing the

CB1 receptor. The white arrows indicate the axons and dendrites expressing the CB1 receptor.

The yellow arrows indicate neurons expressing the CB1 receptor. All scale bars represent 50

m.

a) CA1 b) CA3

c) Hilus d) DG


Figure 1.6

Immunohistochemical labelling of the CB1 receptor in axons and dendrites in the (a) stratum

oriens and (b) DG regions of the hippocampus. The primary antibody concentration was 1:100

and secondary antibody, 1:1000.

a) Stratum b) DG


1.4 Discussion

From the present study, an optimum protocol was obtained for using the Santa Cruz CB1

receptor antibody in the rat hippocampus. The optimum concentrations were 1:100 for the

CB1 receptor primary antibody and 1:1000 for the secondary antibody. Moreover, the

optimum incubation times for the primary and secondary antibodies were 41 hrs and 1 hr,

respectively. This study also proved that the antibody concentration and incubation time are

critical for the immunohistochemical experiments reported in this thesis (Appendix 2).

However, the western blot results (Chapter 4) suggested that the CB1 receptor antibody used

in the present study is not very specific. Therefore, although the present study showed

credible labelling of the CB1 receptors in the rat hippocampus, one cannot be confident that

this labelling is CB1 receptor-specific.

It was startling to see the difference in the apparent labelling of the CB1 receptors using

various conditions with the same batch of antibody from one commercial company. Should

the labelling be specific, the intensity of the staining must vary across the section for two

reasons: (1) the CB1 receptor antigen is not distributed evenly across the brain; and (2) the

brain tissue is sectioned at various levels of the cell. Under high magnification, the intensity

of false positive cells did not change throughout different regions of the brain tissue (data not

shown). With low CB1 primary antibody concentrations, the CB1 receptor antigen was not

recognised by the antibody, mainly because there were fewer available antibodies that could

bind to the antigen. The results from the present study suggest that while there would be

sufficient antibodies to recognise and bind to the antigen at the high concentration, it also

increases the level of the non-specific binding. Conversely, using a low concentration of the

primary antibody might decrease the non-specific binding, but the probability of obtaining

specific binding also decreases. Taken together, these results outline the importance of

determining an optimum primary antibody concentration for immunohistochemistry prior to

investigating changes in the CB1 receptor expression.

Optimising incubation duration is as important as finding the optimum antibody

concentration. Goldstein et al. (2007) demonstrated an altered immunohistochemical staining

percentage and intensity of estrogen receptor antibody labelling in invasive breast carcinomas,

by varying the antibody incubation time and the type of chromogen detection system.

Adequate time must be given for antibodies to bind to their specific antigen, yet if incubated

for too long, the chances of non-specific binding increase, which in turn will contribute to the


elevation of staining intensity and the occurrence of false positive labelling. Typically, the

CB1 receptor antibody is incubated overnight or for 48 hrs. However, there is little published

in the literature regarding standardisation of the incubation time for the CB1 receptor antibody.

For this reason, the incubation time varies greatly between different immunohistochemical

studies. For example, one study stated that the CB1 receptor antibody was incubated for only

one hour (Pettit et al., 1998) and other studies did not even mention the antibody incubation

time (Egertova and Elphick, 2000; Moldrech and Wenger, 2000).

The present study clearly demonstrated that in immunohistochemistry, it is not only the

primary antibody which can cause non-specific binding, but also the secondary antibody to

some extent. Variations in the secondary antibody concentration resulted in variation in

immunohistochemical labelling. One possible explanation as to why the secondary antibody

contributes to greater non-specific binding than the primary antibody when the concentrations

of each were manipulated, is that when immunohistochemical staining is visualised, it is the

secondary antibody that converts or reacts with the chromogen to produce a visible coloured

product. Therefore, even if there is non-specific binding of the primary antibody, as long as it

is not bound by a secondary antibody, it cannot be observed. On the other hand, if the

secondary antibody is non-specifically bound to a protein it will give rise to coloured product

in the visualisation process. The current literature does not take into consideration how the

secondary antibody concentration can affect the immunohistochemical results. Because few

people are aware of how the secondary antibody concentration can affect the end result, many

of the immunohistochemical studies only control for the primary antibody. From the results of

this study, it is clear that the secondary antibody concentration must be taken into account as

an additional control when designing controls for antibody staining.

Although double labelling with a neuronal marker suggested that some of hippocampal

neurons express CB1 receptors, given the lack of specificity of the CB1 receptor primary

antibody, further studies are required to validate this result. Overall, the results from the

present study, together with the Grimsey et al (2008) study, cast considerable doubt upon the

binding specificity of the CB1 antibodies and indicate the need for standardising the

immunohistochemical procedure.

Appendix 2: Changes in CB1 Receptor Expression Following BVD 139

Appendix 2

Changes in CB1 Receptor Expression Following Bilateral

Vestibular Deafferentation in Rats

2.1 Introduction

A significant role of the endocannabinoid system in modulating hippocampal

neurotransmission was first suggested by the abundance of CB1 receptors observed in this

brain region using radioligand binding assays and autoradiography (Devane et al., 1988;

Herkenham et al., 1990, 1991) and in situ hybridisation of receptor mRNA (Mailleux and

Vanderhaeghen, 1992; Matsuda et al., 1993). To date, there have been several

immunohistochemical studies evaluating the distribution and localisation of the CB1 receptors

in the brain (Pettit et al., 1998; Tsou et al., 1998; Ong and Mackie, 1999; Moldrich and

Wenger, 2000; Egertová and Elphick, 2000; Suárez et al., 2008). Although current findings

demonstrate both the presence and the level of CB1 receptor expression in the hippocampus,

there is a lack of information regarding the spatial distribution of the CB1 receptors across the

hippocampus.

The most accurate method for measuring the spatial distribution of biological objects

within an anatomical structure is unbiased stereology (see Glaser and Glaser, 2000;


Mamdarim de Lacerda et al., 2010 for review). This method is currently the most advanced

and the most modern for quantifying immunohistochemical labeling, as it generates accurate

cell numbers within anatomically defined structures (West and Gundersen, 1990; West et al.,

1991; Abusaad et al., 1999; Keuker et al., 2001; Hosseini-Sharifabad and Nyengaard, 2007).

However, this approach involves the counting of immuno-positive cells from sampled

sections, which is both labour-intensive and time-consuming. For this reason, the number of

studies using stereological approaches to determine the spatial distribution of a particular

receptor type (Miettinen et al., 2002; Zhang et al., 2005a, b; Price et al., 2009), is relatively

small compared with studies reporting the presence of that receptor by using other

biochemical assays such as western blotting.

There are several methods other than stereology by which immunohistochemical

staining may be meaningfully analysed and quantified. A scoring system has been used by

some researchers (Detre et al., 1995; Koethe et al., 2007; Suàrez et al., 2008; Sutherland et al.,

2009), while others simply report the extent and intensity of the immunohistochemical

labeling by visually identifying qualitative changes in receptor expression (Mitrirattanakul et

al., 2007; Ludanyi et al., 2008; Falenski et al., 2007, 2009; Araujo et al., 2010). However,

these arbitrary interpretations of immunohistochemical staining by classifying labelling as

„weak, moderate or strong‟ are observer-based, and therefore, subjective. No matter how

clearly the criteria are set forth, the application of such criteria inevitably results in variation

in outcomes between observers. In order to overcome this variability and to ensure more

objective analysis, computer-assisted image analyses have been utilised (Kim et al., 1997;

Simantov et al., 1999; Williams et al., 2005; Malone et al., 2008; Dowie et al., 2009; Suàrez

et al., 2009; Walsh et al., 2010).

To date, there has been no study investigating changes in CB1 receptor expression in the

hippocampus or in other brain regions following BVD. While Ashton and others have

examined CB1 receptor expression in the hippocampus, this investigation was following UVD

and there were no statistically significant differences in UVD animals compared to the control

groups (Ashton et al., 2004b). Furthermore, the majority of the studies which have

investigated the effects of peripheral vestibular damage on the expression of various receptor

types in the brain have used western blotting to quantify the level of expression (Li et al.,

1997; King et al, 2002; Horii et al., 2003; Lindsay et al., 2005). Although western blotting

analysis provides some measure of receptor density, the spatial distribution of the expression

cannot be obtained. Therefore, the aim of the present study was to utilise


immunohistochemistry and computer-assisted image analysis to determine whether BVD

surgery alters the spatial distribution of the CB1 receptors in different sub-regions of the rat

hippocampus. However, due to concerns about the specificity of the CB1 receptor antibody

(Grimsey et al., 2008; also see Chapter 4), the results obtained from the present study are

uncertain.



2.2.1 Animals

Thirty-six naïve male Wistar rats were used, each weighing 250-300 g at the time of the

surgery. There were 3 post-operative time points; 1, 3, and 7 days. For each time point, 6

sham and 6 BVD animals were used. All animals were decapitated without anaesthesia. The

brains were rapidly removed, fixed and sectioned as described in Sections 1.2.1 and 1.2.2 in

Appendix 1, respectively.


The sagittal hippocampal sections were labelled for the CB1 receptors as described in

Section 1.2.3 of Appendix 1. Briefly, the concentrations of the CB1 receptor primary antibody

and donkey anti-goat secondary antibody used were 1:100 and 1:1000, respectively.


A Zeiss Axioplan MC80 BX microscope, Zeiss AxioCam HRc digital camera and Zeiss

Axiovision 3.1 software (Carl Zeiss Vision GmBH, Germany) were employed to obtain

immunohistochemical images. The CB1 receptor labelling quantification was carried out by

measuring the grey level density from digital images using the ImageJ 1.38x analysis

programme (Wayne Rasband, National Institutes of Health, USA, http://rsb.info.nih.gov/ij/).


Statistical analyses were performed in SPSS 17. Two-way ANOVAs with repeated

measures were carried out to determine changes in the spatial distribution of the CB1

receptors across the hippocampus in a sagittal direction. The area under the curve (AUC) was

calculated from the spatial distribution graphs (Prism 5) to determine the effect of post-

operative time (1, 3, or 7 days) on CB1 receptor expression. In order to determine whether

CB1 receptor expression was different between the dorsal and ventral hippocampus, the AUC

was calculated in a dorsal-ventral direction. Then two-way ANOVAs were performed with

surgery and anatomical position (that is, dorsal and ventral) as factors.


2.3 Results

2.3.1 Spatial Distribution of the CB1 Receptors in the Hippocampus

Representative immunohistochemical sections from the sham and BVD animals in

Figure 2.1 exemplify changes observed in CB1 receptor expression in the hippocampus. For

the analysis in which sagittal sections were compared in a medio-lateral direction, there was a

significant surgery effect at 1 day post-operation in the CA1 and CA3 areas of the

hippocampus, where BVD animals‟ CB1 receptor expression was higher in the CA1 area and

lower in the CA3 area compared to the sham animals (F(1,123) = 7.54; P = 0.007 for CA1;

F(1,116) = 7.18; P = 0.008 for CA3; Figure 2.2a, b). Furthermore, the CB1 receptor expression

decreased across the DG region of the hippocampus at 7 days post-operation in the medio-

lateral plane (F(8,90) = 2.86; P = 0.007; Figure 2.2i). There were no significant interactions

between the surgery and anatomical positions in any regions or at any time points (Figure 2.2).

Figure 2.1

Representative immunohistochemical sections of the CB1 receptor labelling in the

hippocampus of (a) a sham and (b) a BVD animal. The scale bars represent 100 m.

a) SHAM b) BVD


Figure 2.2

The spatial distribution of the CB1 receptors across the CA1, CA3, and DG of the hippocampus

at (a – c) 1, (d – f) 3, and (g – i) 7 days following BVD compared to the sham controls. Sections

were analysed in a medio-lateral direction. Data are represented as mean + SEM. P values are

shown where there was a significant effect.

1 day post-operation 3 days post-operation 7 days post-operation

CA

1

CA

3

DG

a) b) c)

d) e) f)

g) h) i)


The ventral hippocampus of both the sham and BVD animals showed significantly

lower CB1 receptor expression compared to the dorsal region at 1 and 7 days post-operative

time points (1 day, F(1,68) = 11.57, P = 0.001, Figure 2.3a; 7 days, F(1,68) = 4.26, P = 0.043,

Figure 2.3c), but not at 3 days (F(1,68) = 0.78, P = 0.381, Figure 2.3b). There was no significant

surgery effect or interaction between surgery and anatomical position at any post-operative

time points (Figure 2.3).

Figure 2.3

The AUC of the spatial distribution of CB1 receptors in the dorsal and ventral hippocampus at

(a) 1, (b) 3, and (c) 7 days following BVD compared to the sham controls. Data are represented

as mean + SEM.

a) b)

c)


2.4 Discussion

The results from the present study suggest apparent changes in the expression of the

CB1 receptors following BVD in specific sub-regions of the hippocampus. These changes

include a significant increase in CB1 receptor expression in the CA1 region in BVD rats

compared to sham controls at 1 day post-operation, concurrent with a significant reduction in

the CA3 region of BVD compared to sham animals, with no change in the DG. Although a

reduction in the expression of the CB1 receptors in the CA3 region observed in the present

study is superficially consistent with the western blot data (Chapter 4), due to the lack of CB1

receptor antibody specificity shown by the western blotting (Chapter 4; Grimsey et al., 2008),

no definite conclusion cannot be drawn other than that the CB1 receptor antibody is in doubt.


Appendix 3

The Specificity of Cannabinoid CB2 Receptor Labelling in

the Rat Hippocampus

3.1 Introduction

In 1993, Munro et al. discovered a second cannabinoid receptor subtype, the so-called

CB2 receptor, that does not mediate the psychoactive effects of cannabinoids. The initial

studies that explored the distribution of this „peripheral cannabinoid receptor‟ indicated that

CB2 receptors are present exclusively in the periphery, particularly in tissues and cells of the

immune system, such as spleen macrophages, tonsils, B cells and natural killer cells,

monocytes, neutrophils and T cells (see Fernández-Ruiz et al., 2007 for review). Being absent

from the CNS (Lynn and Herkenham, 1994), the distribution of this second cannabinoid

receptor was in contrast with the well-known distribution of the CB1 receptor. However,

further studies investigating the expression of CB2 receptors have raised significant

controversy regarding their presence in the mammalian brain. While CB2 receptors have not

been found in the intact CNS by some researchers (Derocq et al., 1995; Galiègue et al., 1995;

Schatz et al., 1997; Griffin et al., 1999; Sugiura et al., 2000), others have shown that CB2

receptor expression might be induced in glial cells, in particular reactive microglia, in

response to diseases, such as Alzheimer‟s disease (Benito et al., 2003; Ramirez et al., 2005),


brain tumours (Ellert-Miklaszewska et al., 2007), multiple sclerosis (Benito et al., 2007), and

Parkinson‟s disease (Price et al., 2009).

Despite the fact that numerous studies have failed to detect CB2 receptors in healthy

brains, recent studies have reported the expression of CB2 receptors in both cultured neural

cells and in the nervous system of several mammals under normal conditions. For example,

CB2 receptors have been reported to be expressed in glial cells, including microglia and

astrocytes (Núñez et al., 2004; Arévalo-Martín et al., 2007), endothelial cells (Ashton et al.,

2006), neural progenitors (Palazuelos et al., 2006), neural stem/precursor cells (Molina-

Holgado et al., 2007), and most recently in the sub-regions of the hippocampus (Suàrez et al.,

2009). Only a few years ago, the answer to the question of whether CB2 receptors are

expressed in neurons was clearly „no‟. However, recent evidence has opened this field to

further discussion.

CB2 receptor mRNA expression has been reported in cerebellar neurons (Skaper et al.,

1996) and more recently, Van Sickle et al. (2005) reported CB2 receptor mRNA and protein in

the cerebellum, cortex and brainstem neurons of the rat, the mouse, and the ferret. In addition,

Gong and others have demonstrated abundant expression of CB2 receptor mRNA and protein

in numerous regions of the intact brain (Gong et al., 2006), and Suàrez et al. (2008) identified

CB2 receptor expression in neurons of the cerebellum and the brainstem vestibular nucleus,

which was replicated by Baek et al. (2008). Furthermore, evidence for a functional role of

CB2 receptors was reported by Jhaveri et al. (2008) in thalamic neurons and by Morgan et al.

(2009) in hippocampal neurons.

Nevertheless, the expression of CB2 receptors in the brain remains controversial due to

uncertainty about the experimental approaches used, and the specificity of some

methodological tools such as CB2 receptor antibodies. At present, no previous study has

compared the specificity of CB2 receptor antibodies from different commercial companies. As

has been shown by Grimsey et al. (2008) and Jelsing et al. (2008), the specificity of

antibodies can vary immensely, and these authors emphasise the importance of thorough

validation of the specificity of antibodies for each particular application before use. There are

many possible factors which could affect the labelling pattern of the same target antigen by

different „specific‟ antibodies. Some of these include the method of tissue fixation, tissue

thickness, antigen retrieval, antibody concentration, incubation duration and temperature.


Although Gong et al. (2006) compared different CB2 receptor primary antibodies from

three commercial companies, Cayman, Sigma, and Santa Cruz Biotechnology (Santa Cruz),

they did not attempt to alter antibody dilution or the incubation time for any of the primary or

secondary antibodies. Even though they used controls such as a blocking peptide, a brain

section from CB2 receptor knockout mice, and a control section incubated without the primary

antibody, the use of antibodies from different companies was not consistent throughout the

experimental design. For example, only the antibody from Santa Cruz, but from no other

manufacturer, was used to analyse the specificity of the antibody in knockout mice brain

tissue sections. Also, a blocking peptide was used only with the antibody from Cayman.

Further controversy regarding CB2 receptor labelling was revealed when Suàrez et al. (2008)

reported that cerebellar Purkinje cell bodies were not CB2 receptor immunopositive, when the

same type of cells had been shown to be intensely stained for CB2 receptors in the Gong et al.

(2006) study. The astounding differences in the labelling pattern of CB2 receptors observed in

the above studies raise serious concerns about the specificity of the CB2 receptor antibodies,

not only for the above studies, but for any study which has used CB2 receptor antibodies to

localise CB2 receptor expression in the brain using immunohistochemistry.

Numerous studies using immunohistochemistry may have reached invalid conclusions

due to misinterpretation of non-specific staining patterns. Moreover, the limitations of using

antibodies to perform immunohistochemistry, especially on brain tissue sections, are said to

be greater than for other biochemical methods (Rhodes and Trimmer, 2006). The main reason

for this is that the brain has a relatively complex and heterogeneous cellular and molecular

composition compared to other tissues. Because immunoreactivity in immunohistochemistry

reveals all sites to which the antibodies bind, it leaves considerable room for misinterpretation

and confusion as to which labelling is specific and which is non-specific. One approach to

obtain concordant data, suggested by Rhodes and Trimmer (2006), is to compare the labelling

patterns revealed by two different antibody preparations raised against distinct, non-

overlapping antigenic sequences on the same target protein. This is important because

different antibodies binding to different epitopes of the target protein may result in different

labelling patterns. At present, many commercial companies are manufacturing CB2 receptor

primary antibodies. All of the CB2 receptor primary antibodies that are currently available

bind to either the N- or C-terminus of the CB2 receptor protein (Figure 3.1). Furthermore,

since there are no monoclonal CB2 receptor antibodies that are commercially available at

present, one should be cautious obtaining data using currently available polyclonal CB2

receptor antibodies, because the specificity of such polyclonal antibodies may vary over time


depending on the breed and on the individual animals being immunised during antibody

production (Leenaars & Hendriksen, 2005).

Figure 3.1

A CB2 receptor diagram showing the locations of different commercial and privately made CB2

receptor primary antibodies targeting different epitopes of the CB2 receptor protein. All except

the Ken Mackie antibody are manufactured by commercial companies.

Overall, it has been increasingly recognised by many researchers how difficult it is to

obtain a true representation of an antigen of interest using a specific antibody in

immunohistochemistry. Furthermore, the precise conditions for the immunohistochemical

assay can make an enormous difference to the results (Goldstein et al., 2007; Lorincz and

Nusser, 2008). Therefore, it is very important to have sufficient controls in order to convince a

sceptical observer. The present study aimed to investigate the specificity of two commercially

available polyclonal CB2 receptor antibodies by varying the concentrations and durations of

exposure used for the primary and secondary antibodies. Although it was initially intended to

analyse the expression of CB2 receptors in the hippocampus following BVD, concerns about

antibody specificity resulted in these experiments being discontinued.

Abcam (aa 1-32)

Sigma (aa 1-33)

Affinity BioReagents (aa 1-33) Cayman

(aa 20-33)

Alpha Diagnostics

& Santa Cruz

(C-terminus)

Ken Mackie

(aa 326-342)

Santa Cruz

(N-terminus)



3.2.1 Animals

Three male Wistar rats were used for immunohistochemistry and 2 for the western

blotting. Each animal weighed 250-300 g. The procedure of harvesting and sectioning the

brain tissue was the same as described in Sections 1.2.1 and 1.2.2 of Appendix 1. The tissue

dissection and homogenisation procedures for western blotting are described in Sections

4.2.1.2 and 4.2.1.3 of Chapter 4.

3.2.2 Western blotting

Western blotting procedures for the CB2 receptor were the same as described in Section

4.1.2.3 of Chapter 4. The CB2 receptor primary antibody concentration for both the Santa

Cruz and the Abcam antibodies was 1:1000 and their corresponding secondary antibody

concentration was 1:5000. The HRP-conjugated donkey anti-goat secondary antibody was

used for the Santa Cruz CB2 receptor primary antibody and the HRP-conjugated goat anti-

rabbit secondary antibody was used for the Abcam CB2 receptor primary antibody.


Immunohistochemical procedures for the labelling of the CB2 receptor were the same as

for labelling the CB1 receptors, as described in Section 1.2.3 of Appendix 1. Both the primary

(goat polyclonal CB2 receptor) and the secondary (HRP-conjugated donkey anti-goat)

antibodies used in this experiment were from Santa Cruz, Inc.

In order to obtain the best possible immunohistochemical labelling of the CB2 receptors,

various combinations of both primary and secondary antibody concentrations and incubation

durations were tested (Table 2.1). Briefly, the primary antibody dilutions ranged from 1:50 to

1:400 and the incubation was either overnight (18 hrs) or for 2 nights (41 hrs). The secondary

antibody concentrations varied from 1:500 to 1:1000 dilutions, and the incubation durations

were either 1 or 2 hrs.


Table 3.1

Various combinations of CB2 receptor primary (1o) and subsequent secondary (2

o) antibody

titres trialled and their incubation durations.

1o antibody

concentration

2o antibody

concentration

1o / 2

o antibody incubation

time (hrs)

1:200 1:500 41 / 1

1:400

1:50

1:1000 41 / 1 1:100

1:200

1:200 1:500

41 / 1 1:1000

1:200 1:500

18 / 1

41 / 1

41 / 2

3.2.4 Double Label Immunofluorescence

Double immunofluorescent labelling of the CB2 receptor (Santa Cruz) with a neuronal

marker, NeuN (mouse monoclonal, Chemicon) was carried out in order to further characterise

the types of cell(s) in which CB2 receptors are expressed in the brain. The primary antibody

concentration for both the CB2 receptor and NeuN was 1:200, and for subsequent secondary

antibodies, 1:500. The Alexa Fluor 594-conjugated donkey anti-goat IgG (Invitrogen) resulted

in red fluorescence for CB2 receptor positive cells and the Alexa Fluor 488-conjugated goat

anti-mouse IgG (Invitrogen) resulted in green fluorescence for NeuN labelling. The CB2

receptor antibody was incubated for 41 hrs and NeuN was incubated overnight, both at 4o C.

Because NeuN is a monoclonal antibody, blocking with serum was not required. For both the

CB2 receptor and NeuN antibodies, the secondary antibodies were incubated for 1 hr at room

temperature. As the secondary antibodies are light sensitive, this procedure was carried out in

the dark.

Due to the use of a different CB2 receptor primary antibody in double labelling (Abcam)

compared with the single labelling (Santa Cruz), double labelling using both of these CB2

receptor primary antibodies was also performed. An Alexa Fluor 488-conjugated donkey anti-

goat IgG antibody (Invitrogen) was used as a secondary antibody for the CB2 goat polyclonal

antibody from Santa Cruz. Both primary and secondary antibody concentrations and


incubation durations were kept the same as for the single labelling protocol. The purpose of

this experiment was to determine whether there were any differences in the labelling pattern

with antibodies from two different commercial companies, which nominally target the same

antigen (both N-terminus).


Unless stated otherwise, a Zeiss Axioplan MC80 BX microscope, Zeiss AxioCam HRc

digital camera and Zeiss Axiovision 3.1 software (Carl Zeiss Vision GmBH, Germany) were

employed for all morphological analyses and photography of immunohistochemical data.

Identical fields on each section stained with different antibody preparations were evaluated. In

this study, various regions of the hippocampus were assessed. For visualising fluorescent

labelling, a Zeiss 510 LSM confocal microscope (Carl Zeiss GmbH, Germany) and LSM 510

control software (version 3.2) running on a Windows PC was used.


3.3 Results


In spleen tissue, the CB2 antibody from Santa Cruz produced two very weak bands

around 30 kDa and the CB2 antibody from Abcam produced multiple bands within the 30 – 45

kDa range (Figure 3.2, rectangle box). In the hippocampal tissues, both the Santa Cruz and

Abcam CB2 receptor antibodies labelled multiple protein bands in the molecular weight range

of 32 – 45 kDa (Figure 3.2, rectangle box). However, the molecular weight of the bands in the

hippocampal tissue did not match that in the spleen tissue. Furthermore, labelling of many

other protein bands could be seen with both antibodies in both the spleen and the hippoampal

tissues (Figure 3.2).

Figure 3.2

Western blots of hippocampal tissue (Hp1 and Hp2) and spleen (sp, as a positive control),

using the Santa Cruz and Abcam CB2 receptor antibodies. The rectangle indicates the

molecular weight range for the CB2 receptor protein.

3.3.2 Immunohistochemical Analysis of the CB2-Specific Antibody

Figure 3.3 shows the staining pattern obtained with two different CB2 receptor primary

antibody concentrations to highlight the differences in the specificity. In order to examine the

effect of altering the primary antibody concentrations, the secondary antibody concentration

was maintained at the dilution of 1:500 for both sections. It was clear that compared to a

1:200 concentration (Figure 3.3a), a 1:400 dilution of the CB2 receptor primary antibody was


too weak to obtain any specific labelling in the hippocampus (Figure 3.3b). Although when

compared to a 1:200 concentration, staining with a 1:400 concentration could be mistaken for

a negative control, when compared to a true negative control section (Figure 3.3c), there was

clearly less staining in the negative control than with a 1:400 antibody concentration.

Figure 3.3

Immunohistochemical comparison of CB2 receptor labelling with different primary antibody

concentrations in the dentate gyrus (DG) of the rat hippocampus. The secondary antibody

concentration for all conditions was the same, i.e. 1:500. The dilutions for the primary antibody

were: (a) 1:200, (b) 1:400, (c) negative control. The primary antibody was incubated for 41 hrs,

the secondary antibody for 1 hr. Scale bars indicate 100 m.

A lower secondary antibody concentration (1:1000) was tested with various

concentrations of the primary antibody (1:50, 1:100, or 1:200; Figure 3.4). Similar results to

those shown in Figure 3.3 were obtained: as the concentration of the primary antibody

decreased, the antibody specificity decreased also. Although 1:50 primary and 1:1000

secondary antibody dilution combinations gave similar results to a 1:200 primary and 1:500

secondary combination, overall, less antibody was required for the latter combination. These

results indicate that it is very important to determine the optimum primary antibody

concentration.

a) 1:200

b) 1:400 c) Negative control


Figure 3.4

Immunohistochemical comparison of CB2 receptor labelling with different primary antibody

concentrations in the DG of the rat hippocampus. The secondary antibody concentration was a

1:1000 dilution. The dilutions for the primary antibody were: (a) 1:50, (b) 1:100, (c) 1:200. The

primary antibody was incubated for 41 hrs, the secondary antibody for 1 hr. Scale bars indicate

100 m.

Having investigated the optimal primary antibody concentration, the secondary antibody

concentrations were then investigated. Figure 3.5 shows that varying the concentration of the

secondary antibody alters the final outcome. The primary antibody concentration was kept

constant at a 1:200 titre, whereas two different concentrations of the secondary antibody were

tested: 1:500 and 1:1000 dilutions. A 1:500 dilution gave apparent specific labelling of the

CB2 receptor (Figure 3.5a), whereas a 1:1000 dilution was inadequate (Figure 3.5b).

Figure 3.5

Immunohistochemical comparison of CB2 receptor labelling with different secondary antibody

concentrations in the DG of the rat hippocampus. The primary antibody concentration for all

conditions was constant at a 1:200 dilution. The dilutions for the secondary antibody were: (a)

1:500, (b) 1:1000. The primary antibody was incubated for 41 hrs and the secondary antibody

for 1 hr. Scale bars indicate 100 m.

a) 1:50

b) 1:100 c) 1:200

a) 1:500 b) 1:1000


Not only the antibody concentration, but also the incubation duration, is important for

specific labelling in immunohistochemistry (Goldstein et al., 2007). Figure 3.6a and b show

the effect of different primary antibody incubation durations, while the primary and secondary

antibody concentrations were kept the same. Surprisingly, an overnight (18 hr) incubation

duration for the CB2 receptor primary antibody (Figure 3.6a) was not sufficient to allow what

appears to be specific binding to occur. However, when incubated for 2 nights (41 hrs),

apparent specific labelling was achieved (Figure 3.6b). Unlike primary antibodies, alteration

of the secondary antibody incubation duration did not alter the apparent specific binding

(Figure 3.6b, c). Figure 3.7 shows apparent specific labelling of the CB2 receptors in different

areas of the hippocampus. CB2 receptor-positive labelling is clearly visible in the cytoplasm,

nucleus, nuclear membrane, and cell membrane. Dendrites were also labelled for CB2

receptors, particularly in the CA3 area of the hippocampus (Figure 3.7c).

Figure 3.6

Immunohistochemical comparison of CB2 receptor labelling with different antibody incubation

durations in the DG of the rat hippocampus. The primary and secondary antibody

concentrations in all conditions were the same at 1:200 for the primary antibody and 1:500 for

the secondary antibody. Antibody incubation durations were (primary/secondary): (a) 18/1 hrs,

(b) 41/1 hrs, (c) 41/2 hrs. Scale bars indicate 100 m.

a) 18/1 hrs

c) 41/2 hrs

b) 41/1 hrs


Figure 3.7

Immunohistochemical labelling of CB2 receptors in the hippocampus. Positive

immunoreactivity to the CB2 receptor was expressed as brown staining in the cytoplasm

(purple arrow), nucleus (blue arrow), nuclear membrane (red arrow), and cell membrane (green

arrow). Low magnification image of the entire hippocampus is shown in (a). Higher

magnification images of various regions of the hippocampus: (b) CA1, (c) CA3, (d) DG. The

dilution for the CB2 receptor primary antibody was 1:200 and for the secondary antibody 1:500.

The primary antibody was incubated for 41 hrs at 4 o

C and the secondary for 1 hr at room

temperature. Scale bars for (a) and (b-d) indicate 200 and 20 m, respectively.

3.3.3 Double Labelling Immunofluorescence

Double immunofluorescence labelling showed the apparent co-localisation of the CB2

receptor and the NeuN antibodies in the CA3 region of the hippocampus (Figure 3.8a, b).

However, there was no apparent co-localisation of the CB2 receptor and neurons in the dentate

gyrus (DG) area of the hippocampus (Figure 3.8c).

Double labelling of two CB2 receptor antibodies from different commercial companies

(Abcam and Santa Cruz) showed surprising results. The overall labelling pattern of these two

antibodies appeared to be surprisingly similar with abundant immunofluorescence observed in

a) Hippocampus b) CA1

c) CA3 d) DG


CA3 (Figure 3.9a, b). In the same section, the Santa Cruz antibody labelled the cytoplasm,

nucleus, nuclear membrane, and cell membrane (Figure 3.9) which matched the result

obtained from DAB single labelling of the CB2 receptors (Figure 3.7), while the Abcam

antibody labelled only the cytoplasm of the same cell (Figure 3.9c).

Figure 3.8

Double labelling immunofluorescence of the CB2 receptor antibody with a NeuN antibody, a

neuronal marker in various regions of the hippocampus: (a) CA3, (b) CA3 at higher

magnification, (c) DG. Green fluorescence represents neurons and red fluorescence

corresponds to cells expressing the CB2 receptor. The white arrows indicate NeuN-positive

neurons, yellow arrows indicate neurons expressing the CB2 receptor, and blue arrows indicate

cells expressing only the CB2 receptor. Scale bars for (a, c) and (b) indicate 50 and 20 m,

respectively.

a) CA3 b) CA3

c) DG


Figure 3.9

Immunofluorescent labelling showing the distribution of the CB2 receptors in the CA3 region of

the hippocampus using antibodies from the (a) Abcam (rabbit polyclonal) and (b) Santa Cruz

(goat polyclonal). (c) Double labelling of CB2 receptors using antibodies from the Santa Cruz

(Green) and Abcam (Red). Scale bars for (a, b) and (c) indicate 50 and 20 m, respectively.

a) Abcam b) Santa Cruz

c) Abcam/Santa Cruz


3.4 Discussion

From the present study, the optimum procedures for the use of the Santa Cruz CB2

receptor antibody in immunohistochemistry were determined, which were a 1:200 dilution for

the CB2 receptor primary antibody, and a 1:500 dilution for the secondary antibody

concentrations, with 41 hrs and 1 hr as the optimum incubation times for the primary and

secondary antibodies, respectively. However, consistent with the CB1 receptor antibodies

(Appendix 1), variation in the CB2 receptor primary antibody and secondary antibody

concentrations and incubation durations did affect the CB2 receptor antibody labelling in the

brain. Fortunately, the same Santa Cruz antibody used in this study was previously used by

Ashton and others (Ashton et al., 2006, 2007). In Ashton et al. (2006), the primary antibody

concentration was 1:50 and the secondary antibody concentration was 1:1000. This

combination was tested in the current study and it was found to give equivalent labelling to

that reported by Ashton et al. (2006).

Unlike the CB1 receptor (Grimsey et al., 2008; Jelsing et al., 2008), the specificity of

the CB2 receptor antibodies used in immunohistochemical studies has not been investigated

thoroughly. In addition, because most of the antibodies are capable of producing non-specific

binding, there is still controversy surrounding the specificity of the CB2 receptor antibodies.

Therefore, it is crucial to systematically evaluate the specificity of an antibody in order to

obtain reliable data. The western blotting showed that both the Santa Cruz and Abcam CB2

receptor antibodies labelled multiple protein bands in addition to those in the molecular

weight range for the CB2 receptor protein. This was similar to the results obtained for

commercial CB1 receptor antibodies by Grimsey et al. (2008). Most importantly, the spleen

tissue which is known to have high levels of CB2 receptors failed to show consistent band(s)

when probed with these two antibodies. Based on these results, non-specific labelling using

immunochemistry with these antibodies would be expected.

In order to address this problem, the current study used not only two different CB2

receptor antibodies, but also carried out double labelling of the CB2 receptor antibody with a

neuronal marker, NeuN. It was shown that the CB2 receptor antibody from Abcam appears to

label neurons in the hippocampus. The specificity was further investigated by the double

labelling of two CB2 receptor antibodies which were from Abcam and Santa Cruz. It was

interesting to see the difference in the labelling pattern. The Abcam antibody labelled the

cytoplasm and cell membrane, whereas the Santa Cruz antibody stained the entire cell. The


Santa Cruz antibody labelling was consistent with Ashton et al. (2006, 2007). On the other

hand, the cytoplasmic labelling pattern of the Abcam antibody was observed in the Van Sickle

et al. (2005) study, in which the CB2 receptor antibody used was from Alpha Diagnostics.

Taken together, the results from the present study suggest that CB2 receptor “specific”

antibodies are likely to produce non-specific labelling in immunohistochemistry. This

undermines the idea of CB2 receptor expression in the hippocampus. Therefore, great caution

needs to be taken when interpreting the results of brain immunohistochemistry using some

commercial CB2 receptor antibodies.

163

References

References 164

Abel, E.L. (1971) Retrieval of information after use of marihuana. Nature, 231, 58.

Abusaad, I., MacKay, D., Zhao, J., Stanford, P., Collier, D.A. & Everall, I.P. (1999) Stereological estimation of

the total number of neurons in the murine hippocampus using the optical disector. J Comp Neurol, 408,

560-566.

Abush, H. & Akirav, I. (2009) Cannabinoids modulate hippocampal memory and plasticity. Hippocampus, 20,

1126-1138.

Adams, I.B. & Martin, B.R. (1996) Cannabis: pharmacology and toxicology in animals and humans. Addiction,

91, 1585-1614.

Adermark, L. & Lovinger, D.M. (2007) Combined activation of L-type Ca2+ channels and synaptic transmission

is sufficient to induce striatal long-term depression. J Neurosci, 27, 6781-6787.

Adermark, L., Talani, G. & Lovinger, D.M. (2009) Endocannabinoid-dependent plasticity at GABAergic and

glutamatergic synapses in the striatum is regulated by synaptic activity. Eur J Neurosci, 29, 32-41.

Aggleton, J.P. (1985) One-trial object recognition by rats. Q J Exp Psychol B, 37, 279-294.

Agu, R.U., Valiveti, S., Paudel, K.S., Klausner, M., Hayden, P.J. & Stinchcomb, A.L. (2006) Permeation of WIN

55,212-2, a potent cannabinoid receptor agonist, across human tracheo-bronchial tissue in vitro and rat

nasal epithelium in vivo. J Pharm Pharmacol, 58, 1459-1465.

Aigner, T.G. (1988) Delta-9-tetrahydrocannabinol impairs visual recognition memory but not discrimination

learning in rhesus monkeys. Psychopharmacology (Berl), 95, 507-511.

Allen, K., Potvin, O., Thibaudeau, G., Dore, F.Y. & Goulet, S. (2007) Processing idiothetic cues to remember

visited locations: hippocampal and vestibular contributions to radial-arm maze performance. Hippocampus,

17, 642-653.

Allen, K.L., Waldvogel, H.J., Glass, M. & Faull, R.L. (2009) Cannabinoid (CB(1)), GABA(A) and GABA(B)

receptor subunit changes in the globus pallidus in Huntington's disease. J Chem Neuroanat, 37, 266-281.

Allum, J.H. & Pfaltz, C.R. (1985) Visual and vestibular contributions to pitch sway stabilization in the ankle

muscles of normals and patients with bilateral peripheral vestibular deficits. Exp Brain Res, 58, 82-94.

Altemus, K.L. & Almli, C.R. (1997) Neonatal hippocampal damage in rats: long-term spatial memory deficits

and associations with magnitude of hippocampal damage. Hippocampus, 7, 403-415.

Amaral, D. & Lavenex, P. (2007) Hippocampal neuroanatomy. In Anderson, P., Morris, R., Amaral, D., Bliss, T.

& O'Keefe, J. (ed) The Hippocampus Book. Oxford University Press, New York, pp. 37-114.

Ameri, A. (1999) The effects of cannabinoids on the brain. Prog Neurobiol, 58, 315-348.

Ames, F. (1958) A clinical and metabolic study of acute intoxication with Cannabis sativa and its role in the

model psychoses. J Ment Sci, 104, 972-999.

Angelaki, D.E. & Cullen, K.E. (2008) Vestibular system: the many facets of a multimodal sense. Annu Rev

Neurosci, 31, 125-150.

Angelaki, D.E., Klier, E.M. & Snyder, L.H. (2009) A vestibular sensation: probabilistic approaches to spatial

perception. Neuron, 64, 448-461.

Araujo, B.H., Torres, L.B., Cossa, A.C., Naffah-Mazzacoratti Mda, G. & Cavalheiro, E.A. (2010) Hippocampal

expression and distribution of CB1 receptors in the Amazonian rodent Proechimys: an animal model of

resistance to epilepsy. Brain Res, 1335, 35-40.

Arenos, J.D., Musty, R.E. & Bucci, D.J. (2006) Blockade of cannabinoid CB1 receptors alters contextual

References 165

learning and memory. Eur J Pharmacol, 539, 177-183.

Arévalo-Martín, A., Garcia-Ovejero, D., Rubio-Araiz, A., Gomez, O., Molina-Holgado, F. & Molina-Holgado, E.

(2007) Cannabinoids modulate Olig2 and polysialylated neural cell adhesion molecule expression in the

subventricular zone of post-natal rats through cannabinoid receptor 1 and cannabinoid receptor 2. Eur J

Neurosci, 26, 1548-1559.

Ashton, J.C. & Glass, M. (2007) The cannabinoid CB2 receptor as a target for inflammation-dependent

neurodegeneration. Curr Neuropharmacol, 5, 73-80.

Ashton, J.C., Appleton, I., Darlington, C.L. & Smith, P.F. (2004a) Immunohistochemical localization of

cannabinoid CB1 receptor in inhibitory interneurons in the cerebellum. Cerebellum, 3, 222-226.

Ashton, J.C., Friberg, D., Darlington, C.L. & Smith, P.F. (2006) Expression of the cannabinoid CB2 receptor in

the rat cerebellum: an immunohistochemical study. Neurosci Lett, 396, 113-116.

Ashton, J.C., Rahman, R.M., Nair, S.M., Sutherland, B.A., Glass, M. & Appleton, I. (2007) Cerebral hypoxia-

ischemia and middle cerebral artery occlusion induce expression of the cannabinoid CB2 receptor in the

brain. Neurosci Lett, 412, 114-117.

Ashton, J.C., Zheng, Y., Liu, P., Darlington, C.L. & Smith, P.F. (2004b) Immunohistochemical characterisation

and localisation of cannabinoid CB1 receptor protein in the rat vestibular nucleus complex and the effects

of unilateral vestibular deafferentation. Brain Res, 1021, 264-271.

Astur, R.S., Taylor, L.B., Mamelak, A.N., Philpott, L. & Sutherland, R.J. (2002) Humans with hippocampus

damage display severe spatial memory impairments in a virtual Morris water task. Behav Brain Res, 132,

77-84.

Bachtold, D., Baumann, T., Sandor, P.S., Kritos, M., Regard, M. & Brugger, P. (2001) Spatial- and verbal-

memory improvement by cold-water caloric stimulation in healthy subjects. Exp Brain Res, 136, 128-132.

Baek, J.H., Zheng, Y., Darlington, C.L. & Smith, P.F. (2008) Cannabinoid CB2 receptor expression in the rat

brainstem cochlear and vestibular nuclei. Acta Otolaryngol, 128, 961-967.

Baek, J.H., Zheng, Y., Darlington, C.L. & Smith, P.F. (2009) The CB1 receptor agonist, WIN 55,212-2, dose-

dependently disrupts object recognition memory in adult rats. Neurosci Lett, 464, 71-73.

Bailey, C.H. & Kandel, E.R. (1993) Structural changes accompanying memory storage. Annu Rev Physiol, 55,

397-426.

Bajo, M., Roberto, M. & Schweitzer, P. (2009) Differential alteration of hippocampal excitatory synaptic

transmission by cannabinoid ligands. J Neurosci Res, 87, 766-775.

Baker, H.J., Lindsey, J. R. & Weisbroth, S. H. (ed) (1979) The Laboratory Rat. Academic Press, New York.

Baloh, R.W. (1979) Clinical neurophysiology of the vestibular system. F. A. Davis Co., Philadelphia.

Barmack, N.H. (2003) Central vestibular system: vestibular nuclei and posterior cerebellum. Brain Res Bull, 60,

511-541.

Barna, I., Soproni, K., Arszovszki, A., Csabai, K. & Haller, J. (2007) WIN-55,212-2 chronically implanted into

the CA3 region of the dorsal hippocampus impairs learning: a novel method for studying chronic, brain-

area-specific effects of cannabinoids. Behav Pharmacol, 18, 515-520.

Barnes, C.A. (1979) Memory deficits associated with senescence: a neurophysiological and behavioral study in

the rat. J Comp Physiol Psychol, 93, 74-104.

Barnes, C.A. (1979) Memory deficits associated with senescence: a neurophysiological and behavioral study in

References 166

the rat. J Comp Physiol Psychol, 93, 74-104.

Barnes, C.A. (2004) Spatial memory loss of normal aging: Animal models and neural mechanisms. In Smelser,

N.J., Baltes, P. B. (ed) International Encyclopedia of the Social and Behavioural Sciences. Elsevier, pp.

14804-14807.

Bassett, J.P., Tullman, M.L. & Taube, J.S. (2007) Lesions of the tegmentomammillary circuit in the head

direction system disrupt the head direction signal in the anterior thalamus. J Neurosci, 27, 7564-7577.

Bastschelet, E. (1981) Circular statistics in biology. Academic, London.

Baude, A., Nusser, Z., Roberts, J.D., Mulvihill, E., McIlhinney, R.A. & Somogyi, P. (1993) The metabotropic

glutamate receptor (mGluR1 alpha) is concentrated at perisynaptic membrane of neuronal subpopulations

as detected by immunogold reaction. Neuron, 11, 771-787.

Beltramo, M. & Piomelli, D. (2000) Carrier-mediated transport and enzymatic hydrolysis of the endogenous

cannabinoid 2-arachidonylglycerol. Neuroreport, 11, 1231-1235.

Benito, C., Núñez, E., Tolon, R.M., Carrier, E.J., Rabano, A., Hillard, C.J. & Romero, J. (2003) Cannabinoid

CB2 receptors and fatty acid amide hydrolase are selectively overexpressed in neuritic plaque-associated

glia in Alzheimer's disease brains. J Neurosci, 23, 11136-11141.

Benito, C., Romero, J.P., Tolon, R.M., Clemente, D., Docagne, F., Hillard, C.J., Guaza, C. & Romero, J. (2007)

Cannabinoid CB1 and CB2 receptors and fatty acid amide hydrolase are specific markers of plaque cell

subtypes in human multiple sclerosis. J Neurosci, 27, 2396-2402.

Bense, S., Deutschlander, A., Stephan, T., Bartenstein, P., Schwaiger, M., Brandt, T. & Dieterich, M. (2004)

Preserved visual-vestibular interaction in patients with bilateral vestibular failure. Neurology, 63, 122-128.

Beraneck, M., McKee, J.L., Aleisa, M. & Cullen, K.E. (2008) Asymmetric recovery in cerebellar-deficient mice

following unilateral labyrinthectomy. J Neurophysiol, 100, 945-958.

Bergman, J., Delatte, M.S., Paronis, C.A., Vemuri, K., Thakur, G.A. & Makriyannis, A. (2008) Some effects of

CB1 antagonists with inverse agonist and neutral biochemical properties. Physiol Behav, 93, 666-670.

Bergquist, F., Ludwig, M. & Dutia, M.B. (2008) Role of the commissural inhibitory system in vestibular

compensation in the rat. J Physiol, 586, 4441-4452.

Berrendero, F., Romero, J., Garcia-Gil, L., Suarez, I., De la Cruz, P., Ramos, J.A. & Fernandez-Ruiz, J.J. (1998)

Changes in cannabinoid receptor binding and mRNA levels in several brain regions of aged rats. Biochim

Biophys Acta, 1407, 205-214.

Berrendero, F., Sanchez, A., Cabranes, A., Puerta, C., Ramos, J.A., Garcia-Merino, A. & Fernandez-Ruiz, J.

(2001) Changes in cannabinoid CB(1) receptors in striatal and cortical regions of rats with experimental

allergic encephalomyelitis, an animal model of multiple sclerosis. Synapse, 41, 195-202.

Bhatti, A.S., Aydin, C., Oztan, O., Ma, Z., Hall, P., Tao, R. & Isgor, C. (2009) Effects of a cannabinoid receptor

(CB) 1 antagonist AM251 on behavioral sensitization to nicotine in a rat model of novelty-seeking

behavior: correlation with hippocampal 5HT. Psychopharmacology (Berl), 203, 23-32.

Bisogno, T., MacCarrone, M., De Petrocellis, L., Jarrahian, A., Finazzi-Agro, A., Hillard, C. & Di Marzo, V.

(2001) The uptake by cells of 2-arachidonoylglycerol, an endogenous agonist of cannabinoid receptors.

Eur J Biochem, 268, 1982-1989.

Bisogno, T., Melck, D., Bobrov, M., Gretskaya, N.M., Bezuglov, V.V., De Petrocellis, L. & Di Marzo, V. (2000)

N-acyl-dopamines: novel synthetic CB(1) cannabinoid-receptor ligands and inhibitors of anandamide

References 167

inactivation with cannabimimetic activity in vitro and in vivo. Biochem J, 351 Pt 3, 817-824.

Bisogno, T., Sepe, N., Melck, D., Maurelli, S., De Petrocellis, L. & Di Marzo, V. (1997) Biosynthesis, release

and degradation of the novel endogenous cannabimimetic metabolite 2-arachidonoylglycerol in mouse

neuroblastoma cells. Biochem J, 322 ( Pt 2), 671-677.

Blanks, R.H., Curthoys, I.S. & Markham, C.H. (1972) Planar relationships of semicircular canals in the cat. Am J

Physiol, 223, 55-62.

Blanks, R.H., Curthoys, I.S. & Markham, C.H. (1975) Planar relationships of the semicircular canals in man.

Acta Otolaryngol, 80, 185-196.

Bockisch, C.J., Straumann, D., Hess, K. & Haslwanter, T. (2004) Enhanced smooth pursuit eye movements in

patients with bilateral vestibular deficits. Neuroreport, 15, 2617-2620.

Bohme, G.A., Laville, M., Ledent, C., Parmentier, M. & Imperato, A. (2000) Enhanced long-term potentiation in

mice lacking cannabinoid CB1 receptors. Neuroscience, 95, 5-7.

Bosier, B., Muccioli, G.G., Hermans, E. & Lambert, D.M. (2010) Functionally selective cannabinoid receptor

signalling: therapeutic implications and opportunities. Biochem Pharmacol, 80, 1-12.

Bouaboula, M., Poinot-Chazel, C., Bourrie, B., Canat, X., Calandra, B., Rinaldi-Carmona, M., Le Fur, G. &

Casellas, P. (1995) Activation of mitogen-activated protein kinases by stimulation of the central

cannabinoid receptor CB1. Biochem J, 312 ( Pt 2), 637-641.

Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein

utilizing the principle of protein-dye binding. Anal Biochem, 72, 248-254.

Bradshaw, A.P., Curthoys, I.S., Todd, M.J., Magnussen, J.S., Taubman, D.S., Aw, S.T. & Halmagyi, G.M. A

mathematical model of human semicircular canal geometry: a new basis for interpreting vestibular

physiology. J Assoc Res Otolaryngol, 11, 145-159.

Brady, A.M., Saul, R.D. & Wiest, M.K. Selective deficits in spatial working memory in the neonatal ventral

hippocampal lesion rat model of schizophrenia. Neuropharmacology.

Brandt, T. (1996) Bilateral vestibulopathy revisited. Eur J Med Res, 1, 361-368.

Brandt, T., Schautzer, F., Hamilton, D.A., Bruning, R., Markowitsch, H.J., Kalla, R., Darlington, C., Smith, P. &

Strupp, M. (2005) Vestibular loss causes hippocampal atrophy and impaired spatial memory in humans.

Brain, 128, 2732-2741.

Breivogel, C.S. & Childers, S.R. (1998) The functional neuroanatomy of brain cannabinoid receptors. Neurobiol

Dis, 5, 417-431.

Breivogel, C.S. & Sim-Selley, L.J. (2009) Basic neuroanatomy and neuropharmacology of cannabinoids. Int Rev

Psychiatry, 21, 113-121.

Breivogel, C.S., Sim, L.J. & Childers, S.R. (1997) Regional differences in cannabinoid receptor/G-protein

coupling in rat brain. J Pharmacol Exp Ther, 282, 1632-1642.

Brenowitz, S.D. & Regehr, W.G. (2003) Calcium dependence of retrograde inhibition by endocannabinoids at

synapses onto Purkinje cells. J Neurosci, 23, 6373-6384.

Broadbent, N.J., Gaskin, S., Squire, L.R. & Clark, R.E. (2010) Object recognition memory and the rodent

hippocampus. Learn Mem, 17, 5-11.

Broadbent, N.J., Squire, L.R. & Clark, R.E. (2004) Spatial memory, recognition memory, and the hippocampus.

Proc Natl Acad Sci U S A, 101, 14515-14520.

References 168

Brodal, A. & Pompeiano, O. (1957) The vestibular nuclei in cat. J Anat, 91, 438-454.

Brodal, A. (1974) Handbook of sensory physiology, vestibular system, morphology. Springer, Berlin, Heidelberg,

New York.

Brodal, A. (1981) Neurological anatomy. Oxford University Press, New York.

Brodkin, J. & Moerschbaecher, J.M. (1997) SR141716A antagonizes the disruptive effects of cannabinoid

ligands on learning in rats. J Pharmacol Exp Ther, 282, 1526-1532.

Bromberg, W. (1934) Marijuana intoxication. Am J Psychiatry, 91, 303-330.

Brookes, G.B., Gresty, M.A., Nakamura, T. & Metcalfe, T. (1993) Sensing and controlling rotational orientation

in normal subjects and patients with loss of labyrinthine function. Am J Otol, 14, 349-351.

Brusco, A., Tagliaferro, P., Saez, T. & Onaivi, E.S. (2008a) Postsynaptic localization of CB2 cannabinoid

receptors in the rat hippocampus. Synapse, 62, 944-949.

Brusco, A., Tagliaferro, P.A., Saez, T. & Onaivi, E.S. (2008b) Ultrastructural localization of neuronal brain CB2

cannabinoid receptors. Ann N Y Acad Sci, 1139, 450-457.

Buckley, M.J., Booth, M.C., Rolls, E.T. & Gaffan, D. (2001) Selective perceptual impairments after perirhinal

cortex ablation. J Neurosci, 21, 9824-9836.

Buffalo, E.A., Ramus, S.J., Clark, R.E., Teng, E., Squire, L.R. & Zola, S.M. (1999) Dissociation between the

effects of damage to perirhinal cortex and area TE. Learn Mem, 6, 572-599.

Burgess, N., Maguire, E.A. & O'Keefe, J. (2002) The human hippocampus and spatial and episodic memory.

Neuron, 35, 625-641.

Bussey, T.J., Saksida, L.M. & Murray, E.A. (2003) Impairments in visual discrimination after perirhinal cortex

lesions: testing 'declarative' vs. 'perceptual-mnemonic' views of perirhinal cortex function. Eur J Neurosci,

17, 649-660.

Buttner-Ennever, J.A. (1999) A review of otolith pathways to brainstem and cerebellum. Ann N Y Acad Sci, 871,

51-64.

Bylund, D.B. & Toews, M.L. (1993) Radioligand binding methods: practical guide and tips. Am J Physiol, 265,

L421-429.

Cabranes, A., Pryce, G., Baker, D. & Fernandez-Ruiz, J. (2006) Changes in CB1 receptors in motor-related brain

structures of chronic relapsing experimental allergic encephalomyelitis mice. Brain Res, 1107, 199-205.

Campolongo, P., Roozendaal, B., Trezza, V., Hauer, D., Schelling, G., McGaugh, J.L. & Cuomo, V. (2009)

Endocannabinoids in the rat basolateral amygdala enhance memory consolidation and enable

glucocorticoid modulation of memory. Proc Natl Acad Sci U S A, 106, 4888-4893.

Canas, P.M., Duarte, J.M., Rodrigues, R.J., Kofalvi, A. & Cunha, R.A. (2009) Modification upon aging of the

density of presynaptic modulation systems in the hippocampus. Neurobiol Aging, 30, 1877-1884.

Caprioli, A., Ghirardi, O., Giuliani, A., Ramacci, M.T. & Angelucci, L. (1991) Spatial learning and memory in

the radial maze: a longitudinal study in rats from 4 to 25 months of age. Neurobiol Aging, 12, 605-607.

Caprioli, A., Ghirardi, O., Ramacci, M.T. & Angelucci, L. (1990) Age-dependent deficits in radial maze

performance in the rat: effect of chronic treatment with acetyl-L-carnitine. Prog Neuropsychopharmacol

Biol Psychiatry, 14, 359-369.

Carleton, S.C. & Carpenter, M.B. (1984) Distribution of primary vestibular fibers in the brainstem and

cerebellum of the monkey. Brain Res, 294, 281-298.

References 169

Carlini, E.A., Hamaoui, A., Bieniek, D. & Korte, F. (1970) Effects of (--) delta-9-trans-tetrahydrocannabinol and

a synthetic derivative on maze performance of rats. Pharmacology, 4, 359-368.

Carlisle, S.J., Marciano-Cabral, F., Staab, A., Ludwick, C. & Cabral, G.A. (2002) Differential expression of the

CB2 cannabinoid receptor by rodent macrophages and macrophage-like cells in relation to cell activation.

Int Immunopharmacol, 2, 69-82.

Cartwright, B.A. & Collett, T. S. (1982) How honey-bees use landmarks to guide their return to a food source.

Nature, 295, 560-564.

Cartwright, B.A. & Collett, T. S. (1983) Landmark learning in bees: experiments and models. J Comp Physiol A,

151, 521-543.

Castelli, M.P., Paola Piras, A., D'Agostino, A., Pibiri, F., Perra, S., Gessa, G.L., Maccarrone, M. & Pistis, M.

(2007) Dysregulation of the endogenous cannabinoid system in adult rats prenatally treated with the

cannabinoid agonist WIN 55,212-2. Eur J Pharmacol, 573, 11-19.

Caulfield, M.P. & Brown, D.A. (1992) Cannabinoid receptor agonists inhibit Ca current in NG108-15

neuroblastoma cells via a pertussis toxin-sensitive mechanism. Br J Pharmacol, 106, 231-232.

Centonze, D., Bari, M., Rossi, S., Prosperetti, C., Furlan, R., Fezza, F., De Chiara, V., Battistini, L., Bernardi, G.,

Bernardini, S., Martino, G. & Maccarrone, M. (2007) The endocannabinoid system is dysregulated in

multiple sclerosis and in experimental autoimmune encephalomyelitis. Brain, 130, 2543-2553.

Centonze, D., Battistini, L. & Maccarrone, M. (2008) The endocannabinoid system in peripheral lymphocytes as

a mirror of neuroinflammatory diseases. Curr Pharm Des, 14, 2370-2342.

Cha, Y.M., White, A.M., Kuhn, C.M., Wilson, W.A. & Swartzwelder, H.S. (2006) Differential effects of delta9-

THC on learning in adolescent and adult rats. Pharmacol Biochem Behav, 83, 448-455.

Chait, L.D. & Perry, J.L. (1992) Factors influencing self-administration of, and subjective response to, placebo

marijuana. Behav Pharmacol, 3, 545-552.

Chambers, A.P., Koopmans, H.S., Pittman, Q.J. & Sharkey, K.A. (2006) AM 251 produces sustained reductions

in food intake and body weight that are resistant to tolerance and conditioned taste aversion. Br J

Pharmacol, 147, 109-116.

Chambers, A.P., Sharkey, K.A. & Koopmans, H.S. (2004) Cannabinoid (CB)1 receptor antagonist, AM 251,

causes a sustained reduction of daily food intake in the rat. Physiol Behav, 82, 863-869.

Chan, P.K., Chan, S.C. & Yung, W.H. (1998) Presynaptic inhibition of GABAergic inputs to rat substantia nigra

pars reticulata neurones by a cannabinoid agonist. Neuroreport, 9, 671-675.

Chaperon, F. & Thiébot, M.H. (1999) Behavioral effects of cannabinoid agents in animals. Crit Rev Neurobiol,

13, 243-281.

Chaperon, F., Soubrie, P., Puech, A. J. & Thiebot, M. (1998) Involvement of central cannabinoids (CB1)

receptors in the establishment of place conditioning in rats. Psychopharmacology (Berl), 135, 324-332.

Cheng, K. (1988) Some psychophysics of the pigeon's use of landmarks. J Comp Physiol A, 162, 815-826.

Cheng, K. (1989) The vector sum model of pigeon landmark use. J Exp Psychol Anim Behav Process, 15, 366-

375.

Cheng, Y. & Prusoff, W.H. (1973) Relationship between the inhibition constant (K1) and the concentration of

inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol, 22,

3099-3108.

References 170

Chevaleyre, V. & Castillo, P.E. (2003) Heterosynaptic LTD of hippocampal GABAergic synapses: a novel role of

endocannabinoids in regulating excitability. Neuron, 38, 461-472.

Chevaleyre, V., Heifets, B.D., Kaeser, P.S., Sudhof, T.C. & Castillo, P.E. (2007) Endocannabinoid-mediated

long-term plasticity requires cAMP/PKA signaling and RIM1alpha. Neuron, 54, 801-812.

Chevaleyre, V., Takahashi, K.A. & Castillo, P.E. (2006) Endocannabinoid-mediated synaptic plasticity in the

CNS. Annu Rev Neurosci, 29, 37-76.

Childers, S.R. & Breivogel, C.S. (1998) Cannabis and endogenous cannabinoid systems. Drug Alcohol Depend,

51, 173-187.

Chiu, C.Q. & Castillo, P.E. (2008) Input-specific plasticity at excitatory synapses mediated by endocannabinoids

in the dentate gyrus. Neuropharmacology, 54, 68-78.

Choi, K.D., Oh, S.Y., Kim, H.J., Koo, J.W., Cho, B.M. & Kim, J.S. (2007) Recovery of vestibular imbalances

after vestibular neuritis. Laryngoscope, 117, 1307-1312.

Ciccocioppo, R., Antonelli, L., Biondini, M., Perfumi, M., Pompei, P. & Massi, M. (2002) Memory impairment

following combined exposure to delta(9)-tetrahydrocannabinol and ethanol in rats. Eur J Pharmacol, 449,

245-252.

Clark, R.E., Zola, S.M. & Squire, L.R. (2000) Impaired recognition memory in rats after damage to the

hippocampus. J Neurosci, 20, 8853-8860.

Clarke, J.R., Rossato, J.I., Monteiro, S., Bevilaqua, L.R., Izquierdo, I. & Cammarota, M. (2008) Posttraining

activation of CB1 cannabinoid receptors in the CA1 region of the dorsal hippocampus impairs object

recognition long-term memory. Neurobiol Learn Mem, 90, 374-381.

Cohen, H. & Keshner, E.A. (1989) Current concepts of the vestibular system reviewed: 2. Visual/vestibular

interaction and spatial orientation. Am J Occup Ther, 43, 331-338.

Collett, T.S. (1975) Visual spatial memory in a hover-fly. J Comp Physiol A, 100, 59-84.

Collett, T.S. (1987) The use of visual landmarks by gerbils: reaching a goal when landmarks are displaced. J

Comp Physiol A, 160, 109-113.

Collins, D.R., Pertwee, R.G. & Davies, S.N. (1994) The action of synthetic cannabinoids on the induction of

long-term potentiation in the rat hippocampal slice. Eur J Pharmacol, 259, R7-8.

Collins, D.R., Pertwee, R.G. & Davies, S.N. (1995) Prevention by the cannabinoid antagonist, SR141716A, of

cannabinoid-mediated blockade of long-term potentiation in the rat hippocampal slice. Br J Pharmacol,

115, 869-870.

Compton, D.R., Rice, K.C., De Costa, B.R., Razdan, R.K., Melvin, L.S., Johnson, M.R. & Martin, B.R. (1993)

Cannabinoid structure-activity relationships: correlation of receptor binding and in vivo activities. J

Pharmacol Exp Ther, 265, 218-226.

Cristino, L., de Petrocellis, L., Pryce, G., Baker, D., Guglielmotti, V. & Di Marzo, V. (2006)

Immunohistochemical localization of cannabinoid type 1 and vanilloid transient receptor potential

vanilloid type 1 receptors in the mouse brain. Neuroscience, 139, 1405-1415.

Cullen, K.E., Minor, L.B., Beraneck, M. & Sadeghi, S.G. (2009) Neural substrates underlying vestibular

compensation: contribution of peripheral versus central processing. J Vestib Res, 19, 171-182.

Curthoys, I.S., Blanks, R.H. & Markham, C.H. (1977) Semicircular canal functional anatomy in cat, guinea pig

and man. Acta Otolaryngol, 83, 258-265.

References 171

Curthoys, I.S., Curthoys, E.J., Blanks, R.H. & Markham, C.H. (1975) The orientation of the semicircular canals

in the guinea pig. Acta Otolaryngol, 80, 197-205.

Curthoys, I.S., Uzun-Coruhlu, H., Wong, C.C., Jones, A.S. & Bradshaw, A.P. (2009) The configuration and

attachment of the utricular and saccular maculae to the temporal bone. New evidence from

microtomography-CT studies of the membranous labyrinth. Ann N Y Acad Sci, 1164, 13-18.

Cuthbert, P.C., Gilchrist, D.P., Hicks, S.L., MacDougall, H.G. & Curthoys, I.S. (2000) Electrophysiological

evidence for vestibular activation of the guinea pig hippocampus. Neuroreport, 11, 1443-1447.

D'Ambra, T.E., Estep, K.G., Bell, M.R., Eissenstat, M.A., Josef, K.A., Ward, S.J., Haycock, D.A., Baizman, E.R.,

Casiano, F.M., Beglin, N.C. & et al. (1992) Conformationally restrained analogues of pravadoline:

nanomolar potent, enantioselective, (aminoalkyl)indole agonists of the cannabinoid receptor. J Med Chem,

35, 124-135.

Da Silva, G.E. & Takahashi, R.N. (2002) SR 141716A prevents delta 9-tetrahydrocannabinol-induced spatial

learning deficit in a Morris-type water maze in mice. Prog Neuropsychopharmacol Biol Psychiatry, 26,

321-325.

Dalton, V.S., Wang, H. & Zavitsanou, K. (2009) HU210-induced downregulation in cannabinoid CB1 receptor

binding strongly correlates with body weight loss in the adult rat. Neurochem Res, 34, 1343-1353.

Daniel, H., Rancillac, A. & Crepel, F. (2004) Mechanisms underlying cannabinoid inhibition of presynaptic

Ca2+ influx at parallel fibre synapses of the rat cerebellum. J Physiol, 557, 159-174.

Darlington, C.L. & Smith, P.F. (2000) Molecular mechanisms of recovery from vestibular damage in mammals:

recent advances. Prog Neurobiol, 62, 313-325.

Darlington, C.L., Dutia, M.B. & Smith, P.F. (2002) The contribution of the intrinsic excitability of vestibular

nucleus neurons to recovery from vestibular damage. Eur J Neurosci, 15, 1719-1727.

Darmani, N.A. (2001) The cannabinoid CB1 receptor antagonist SR 141716A reverses the antiemetic and motor

depressant actions of WIN 55, 212-2. Eur J Pharmacol, 430, 49-58.

Davies, S.N., Pertwee, R.G. & Riedel, G. (2002) Functions of cannabinoid receptors in the hippocampus.

Neuropharmacology, 42, 993-1007.

Day, T.A., Rakhshan, F., Deutsch, D.G. & Barker, E.L. (2001) Role of fatty acid amide hydrolase in the transport

of the endogenous cannabinoid anandamide. Mol Pharmacol, 59, 1369-1375.

de Lago, E., Fernandez-Ruiz, J., Ortega-Gutierrez, S., Cabranes, A., Pryce, G., Baker, D., Lopez-Rodriguez, M.

& Ramos, J.A. (2006) UCM707, an inhibitor of the anandamide uptake, behaves as a symptom control

agent in models of Huntington's disease and multiple sclerosis, but fails to delay/arrest the progression of

different motor-related disorders. Eur Neuropsychopharmacol, 16, 7-18.

de March, Z., Zuccato, C., Giampa, C., Patassini, S., Bari, M., Gasperi, V., De Ceballos, M.L., Bernardi, G.,

Maccarrone, M., Cattaneo, E. & Fusco, F.R. (2008) Cortical expression of brain derived neurotrophic

factor and type-1 cannabinoid receptor after striatal excitotoxic lesions. Neuroscience, 152, 734-740.

de Oliveira Alvares, L., de Oliveira, L.F., Camboim, C., Diehl, F., Genro, B.P., Lanziotti, V.B. & Quillfeldt, J.A.

(2005) Amnestic effect of intrahippocampal AM251, a CB1-selective blocker, in the inhibitory avoidance,

but not in the open field habituation task, in rats. Neurobiol Learn Mem, 83, 119-124.

de Oliveira Alvares, L., Genro, B.P., Diehl, F. & Quillfeldt, J.A. (2008) Differential role of the hippocampal

endocannabinoid system in the memory consolidation and retrieval mechanisms. Neurobiol Learn Mem, 90,

References 172

1-9.

de Oliveira Alvares, L., Genro, B.P., Vaz Breda, R., Pedroso, M.F., Da Costa, J.C. & Quillfeldt, J.A. (2006)

AM251, a selective antagonist of the CB1 receptor, inhibits the induction of long-term potentiation and

induces retrograde amnesia in rats. Brain Res, 1075, 60-67.

Deadwyler, S.A. & Hampson, R.E. (1997) The significance of neural ensemble codes during behavior and

cognition. Annu Rev Neurosci, 20, 217-244.

Deadwyler, S.A., Goonawardena, A.V. & Hampson, R.E. (2007) Short-term memory is modulated by the

spontaneous release of endocannabinoids: evidence from hippocampal population codes. Behav Pharmacol,

18, 571-580.

Deadwyler, S.A., Hampson, R.E., Mu, J., Whyte, A. & Childers, S. (1995) Cannabinoids modulate voltage

sensitive potassium A-current in hippocampal neurons via a cAMP-dependent process. J Pharmacol Exp

Ther, 273, 734-743.

Della Santina, C.C., Potyagaylo, V., Migliaccio, A.A., Minor, L.B. & Carey, J.P. (2005) Orientation of human

semicircular canals measured by three-dimensional multiplanar CT reconstruction. J Assoc Res

Otolaryngol, 6, 191-206.

Demuth, D. G. & Molleman, A. (2006) Cannabinoid signaling. Life Sci, 78, 549-563.

Dere, E., Huston, J.P. & De Souza Silva, M.A. (2007) The pharmacology, neuroanatomy and neurogenetics of

one-trial object recognition in rodents. Neurosci Biobehav Rev, 31, 673-704.

Derocq, J.M., Segui, M., Marchand, J., Le Fur, G. & Casellas, P. (1995) Cannabinoids enhance human B-cell

growth at low nanomolar concentrations. FEBS Lett, 369, 177-182.

Detre, S., Saclani Jotti, G. & Dowsett, M. (1995) A "quickscore" method for immunohistochemical

semiquantitation: validation for oestrogen receptor in breast carcinomas. J Clin Pathol, 48, 876-878.

Deutsch, D.G., Glaser, S.T., Howell, J.M., Kunz, J.S., Puffenbarger, R.A., Hillard, C.J. & Abumrad, N. (2001)

The cellular uptake of anandamide is coupled to its breakdown by fatty-acid amide hydrolase. J Biol Chem,

276, 6967-6973.

Devane, W.A., Dysarz, F.A., 3rd, Johnson, M.R., Melvin, L.S. & Howlett, A.C. (1988) Determination and

characterization of a cannabinoid receptor in rat brain. Mol Pharmacol, 34, 605-613.

Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Stevenson, L.A., Griffin, G., Gibson, D., Mandelbaum, A.,

Etinger, A. & Mechoulam, R. (1992) Isolation and structure of a brain constituent that binds to the

cannabinoid receptor. Science, 258, 1946-1949.

Di Marzo, V., Fontana, A., Cadas, H., Schinelli, S., Cimino, G., Schwartz, J.C. & Piomelli, D. (1994) Formation

and inactivation of endogenous cannabinoid anandamide in central neurons. Nature, 372, 686-691.

Di Marzo, V., Hill, M.P., Bisogno, T., Crossman, A.R. & Brotchie, J.M. (2000) Enhanced levels of endogenous

cannabinoids in the globus pallidus are associated with a reduction in movement in an animal model of

Parkinson's disease. FASEB J, 14, 1432-1438.

Diana, M.A. & Marty, A. (2004) Endocannabinoid-mediated short-term synaptic plasticity: depolarization-

induced suppression of inhibition (DSI) and depolarization-induced suppression of excitation (DSE). Br

J Pharmacol, 142, 9-19.

Diana, M.A., Levenes, C., Mackie, K. & Marty, A. (2002) Short-term retrograde inhibition of GABAergic

synaptic currents in rat Purkinje cells is mediated by endogenous cannabinoids. J Neurosci, 22, 200-208.

References 173

Dieringer, N. (1995) 'Vestibular compensation': neural plasticity and its relations to functional recovery after

labyrinthine lesions in frogs and other vertebrates. Prog Neurobiol, 46, 97-129.

Dieterich, M., Bauermann, T., Best, C., Stoeter, P. & Schlindwein, P. (2007) Evidence for cortical visual

substitution of chronic bilateral vestibular failure (an fMRI study). Brain, 130, 2108-2116.

Dieterich, M., Bense, S., Stephan, T., Yousry, T.A. & Brandt, T. (2003) fMRI signal increases and decreases in

cortical areas during small-field optokinetic stimulation and central fixation. Exp Brain Res, 148, 117-127.

Dowie, M.J., Bradshaw, H.B., Howard, M.L., Nicholson, L.F., Faull, R.L., Hannan, A.J. & Glass, M. (2009)

Altered CB1 receptor and endocannabinoid levels precede motor symptom onset in a transgenic mouse

model of Huntington's disease. Neuroscience, 163, 456-465.

Drai, D., Benjamini, Y. & Golani, I. (2000) Statistical discrimination of natural modes of motion in rat

exploratory behavior. J Neurosci Methods, 96, 119-131.

Drews, E., Schneider, M. & Koch, M. (2005) Effects of the cannabinoid receptor agonist WIN 55,212-2 on

operant behavior and locomotor activity in rats. Pharmacol Biochem Behav, 80, 145-150.

Dutia, M.B. (2010) Mechanisms of vestibular compensation: recent advances. Curr Opin Otolaryngol Head

Neck Surg, 18, 420-424.

Egashira, N., Mishima, K., Iwasaki, K. & Fujiwara, M. (2002) Intracerebral microinjections of delta 9-

tetrahydrocannabinol: search for the impairment of spatial memory in the eight-arm radial maze in rats.

Brain Res, 952, 239-245.

Egerton, A., Allison, C., Brett, R.R. & Pratt, J.A. (2006) Cannabinoids and prefrontal cortical function: insights

from preclinical studies. Neurosci Biobehav Rev, 30, 680-695.

Egertova, M. & Elphick, M.R. (2000) Localisation of cannabinoid receptors in the rat brain using antibodies to

the intracellular C-terminal tail of CB. J Comp Neurol, 422, 159-171.

Egertova, M., Giang, D.K., Cravatt, B.F. & Elphick, M.R. (1998) A new perspective on cannabinoid signalling:

complementary localization of fatty acid amide hydrolase and the CB1 receptor in rat brain. Proc Biol

Sci, 265, 2081-2085.

Eilam, D. & Golani, I. (1989) Home base behavior of rats (Rattus norvegicus) exploring a novel environment.

Behav Brain Res, 34, 199-211.

Ellert-Miklaszewska, A., Grajkowska, W., Gabrusiewicz, K., Kaminska, B. & Konarska, L. (2007) Distinctive

pattern of cannabinoid receptor type II (CB2) expression in adult and pediatric brain tumors. Brain Res,

1137, 161-169.

Elphick, M.R. & Egertova, M. (2001) The neurobiology and evolution of cannabinoid signalling. Philos Trans R

Soc Lond B Biol Sci, 356, 381-408.

Ennaceur, A. & Delacour, J. (1988) A new one-trial test for neurobiological studies of memory in rats. 1:

Behavioral data. Behav Brain Res, 31, 47-59.

Etienne, A.S. (1980) The orientation of the golden hamster to its nest-site after the elimination of various sensory

cues. Experientia, 36, 1048-1050.

Etienne, A.S., Maurer, R., Saucy, F. & Teroni, E. (1986) Short-distance homing in the golden hamster after a

passive outward journey. Anim Behav, 34, 696-715.

Falenski, K.W., Blair, R.E., Sim-Selley, L.J., Martin, B.R. & DeLorenzo, R.J. (2007) Status epilepticus causes a

long-lasting redistribution of hippocampal cannabinoid type 1 receptor expression and function in the rat

References 174

pilocarpine model of acquired epilepsy. Neuroscience, 146, 1232-1244.

Falenski, K.W., Carter, D.S., Harrison, A.J., Martin, B.R., Blair, R.E. & DeLorenzo, R.J. (2009) Temporal

characterization of changes in hippocampal cannabinoid CB(1) receptor expression following pilocarpine-

induced status epilepticus. Brain Res, 1262, 64-72.

Fan, N., Yang, H., Zhang, J. & Chen, C. (2010) Reduced expression of glutamate receptors and phosphorylation

of CREB are responsible for in vivo Delta9-THC exposure-impaired hippocampal synaptic plasticity. J

Neurochem, 112, 691-702.

Faraji, J., Lehmann, H., Metz, G.A. & Sutherland, R.J. (2008) Rats with hippocampal lesion show impaired

learning and memory in the ziggurat task: a new task to evaluate spatial behavior. Behav Brain Res, 189,

17-31.

Fedrowitz, M., Lindemann, S., Loscher, W. & Gernert, M. (2003) Altered spontaneous discharge rate and pattern

of basal ganglia output neurons in the circling (ci2) rat mutant. Neuroscience, 118, 867-878.

Fegley, D., Kathuria, S., Mercier, R., Li, C., Goutopoulos, A., Makriyannis, A. & Piomelli, D. (2004)

Anandamide transport is independent of fatty-acid amide hydrolase activity and is blocked by the

hydrolysis-resistant inhibitor AM1172. Proc Natl Acad Sci U S A, 101, 8756-8761.

Ferbinteanu, J. & McDonald, R.J. (2001) Dorsal/ventral hippocampus, fornix, and conditioned place preference.

Hippocampus, 11, 187-200.

Ferbinteanu, J., Ray, C. & McDonald, R.J. (2003) Both dorsal and ventral hippocampus contribute to spatial

learning in Long-Evans rats. Neurosci Lett, 345, 131-135.

Fernandez-Ruiz, J. (2009) The endocannabinoid system as a target for the treatment of motor dysfunction. Br J

Pharmacol, 156, 1029-1040.

Fernández-Ruiz, J., Romero, J., Velasco, G., Tolon, R.M., Ramos, J.A. & Guzman, M. (2007) Cannabinoid CB2

receptor: a new target for controlling neural cell survival? Trends Pharmacol Sci, 28, 39-45.

Fernandez, M.M., E. R. (1962) Inertial guidance engineering. Prentice Hall, Englewood Cliffs, New Jersey.

Ferrari, F., Ottani, A., Vivoli, R. & Giuliani, D. (1999) Learning impairment produced in rats by the cannabinoid

agonist HU 210 in a water-maze task. Pharmacol Biochem Behav, 64, 555-561.

Fetter, M., Diener, H.-C. & Dichgans, J. (1991) Recovery of postural control after an acute unilateral vestibular

lesion in humans. J Vestib Res, 1, 373-383.

Fetter, M., Heimberger, J., Black, R., Hermann, W., Sievering, F. & Dichgans, J. (1996) Otolith-semicircular

canal interaction during postrotatory nystagmus in humans. Exp Brain Res, 108, 463-472.

Foldy, C., Neu, A., Jones, M.V. & Soltesz, I. (2006) Presynaptic, activity-dependent modulation of cannabinoid

type 1 receptor-mediated inhibition of GABA release. J Neurosci, 26, 1465-1469.

Forwood, S.E., Winters, B.D. & Bussey, T.J. (2005) Hippocampal lesions that abolish spatial maze performance

spare object recognition memory at delays of up to 48 hours. Hippocampus, 15, 347-355.

Fowler, C.J. & Jacobsson, S.O. (2002) Cellular transport of anandamide, 2-arachidonoylglycerol and

palmitoylethanolamide--targets for drug development? Prostaglandins Leukot Essent Fatty Acids, 66, 193-

200.

Fowler, C.J., Rojo, M.L. & Rodriguez-Gaztelumendi, A. (2010) Modulation of the endocannabinoid system:

neuroprotection or neurotoxicity? Exp Neurol, 224, 37-47.

Fox, K.M., Sterling, R.C. & Van Bockstaele, E.J. (2009) Cannabinoids and novelty investigation: influence of

References 175

age and duration of exposure. Behav Brain Res, 196, 248-253.

Freund, T.F., Katona, I. & Piomelli, D. (2003) Role of endogenous cannabinoids in synaptic signaling. Physiol

Rev, 83, 1017-1066.

Frick, K.M., Baxter, M.G., Markowska, A.L., Olton, D.S. & Price, D.L. (1995) Age-related spatial reference and

working memory deficits assessed in the water maze. Neurobiol Aging, 16, 149-160.

Frisk, V. & Milner, B. (1990) The role of the left hippocampal region in the acquisition and retention of story

content. Neuropsychologia, 28, 349-359.

Fukudome, Y., Ohno-Shosaku, T., Matsui, M., Omori, Y., Fukaya, M., Tsubokawa, H., Taketo, M.M., Watanabe,

M., Manabe, T. & Kano, M. (2004) Two distinct classes of muscarinic action on hippocampal inhibitory

synapses: M2-mediated direct suppression and M1/M3-mediated indirect suppression through

endocannabinoid signalling. Eur J Neurosci, 19, 2682-2692.

Furman, J.M. & Jacob, R.G. (2001) A clinical taxonomy of dizziness and anxiety in the otoneurological setting. J

Anxiety Disord, 15, 9-26.

Fusco, F.R., Martorana, A., Giampa, C., De March, Z., Farini, D., D'Angelo, V., Sancesario, G. & Bernardi, G.

(2004) Immunolocalization of CB1 receptor in rat striatal neurons: a confocal microscopy study. Synapse,

53, 159-167.

Galiègue, S., Mary, S., Marchand, J., Dussossoy, D., Carriere, D., Carayon, P., Bouaboula, M., Shire, D., Le Fur,

G. & Casellas, P. (1995) Expression of central and peripheral cannabinoid receptors in human immune

tissues and leukocyte subpopulations. Eur J Biochem, 232, 54-61.

Gall, C.M., Hess, U.S. & Lynch, G. (1998) Mapping brain networks engaged by, and changed by, learning.

Neurobiol Learn Mem, 70, 14-36.

Galve-Roperh, I., Aguado, T., Palazuelos, J. & Guzman, M. (2007) The endocannabinoid system and

neurogenesis in health and disease. Neuroscientist, 13, 109-114.

Gaoni, Y. & Mechoulam, R. (1964) Isolation, structure and partial synthesis of an active constituent of hashish. J

Am Chem Soc, 86, 1646-1647.

Gatley, S.J., Lan, R., Volkow, N.D., Pappas, N., King, P., Wong, C.T., Gifford, A.N., Pyatt, B., Dewey, S.L. &

Makriyannis, A. (1998) Imaging the brain marijuana receptor: development of a radioligand that binds to

cannabinoid CB1 receptors in vivo. J Neurochem, 70, 417-423.

Gavrilov, V.V., Wiener, S.I. & Berthoz, A. (1995) Enhanced hippocampal theta EEG during whole body rotations

in awake restrained rats. Neurosci Lett, 197, 239-241.

Gavrilov, V.V., Wiener, S.I. & Berthoz, A. (1996) Whole-body rotations enhance hippocampal theta rhythmic

slow activity in awake rats passively transported on a mobile robot. Ann N Y Acad Sci, 781, 385-398.

Georgieva, T., Devanathan, S., Stropova, D., Park, C.K., Salamon, Z., Tollin, G., Hruby, V.J., Roeske, W.R.,

Yamamura, H.I. & Varga, E. (2008) Unique agonist-bound cannabinoid CB1 receptor conformations

indicate agonist specificity in signaling. Eur J Pharmacol, 581, 19-29.

Gerdeman, G.L. & Lovinger, D.M. (2003) Emerging roles for endocannabinoids in long-term synaptic plasticity.

Br J Pharmacol, 140, 781-789.

Gerdeman, G.L., Ronesi, J. & Lovinger, D.M. (2002) Postsynaptic endocannabinoid release is critical to long-

term depression in the striatum. Nat Neurosci, 5, 446-451.

Gessa, G.L., Mascia, M.S., Casu, M.A. & Carta, G. (1997) Inhibition of hippocampal acetylcholine release by

References 176

cannabinoids: reversal by SR 141716A. Eur J Pharmacol, 327, R1-2.

Gifford, A.N. & Ashby, C.R., Jr. (1996) Electrically evoked acetylcholine release from hippocampal slices is

inhibited by the cannabinoid receptor agonist, WIN 55212-2, and is potentiated by the cannabinoid

antagonist, SR 141716A. J Pharmacol Exp Ther, 277, 1431-1436.

Gifford, A.N., Bruneus, M., Gatley, S.J. & Volkow, N.D. (2000) Cannabinoid receptor-mediated inhibition of

acetylcholine release from hippocampal and cortical synaptosomes. Br J Pharmacol, 131, 645-650.

Gilbert, P.E. & Kesner, R.P. (2002) Role of the rodent hippocampus in paired-associate learning involving

associations between a stimulus and a spatial location. Behav Neurosci, 116, 63-71.

Gilchrist, D.P., Curthoys, I.S., Cartwright, A.D., Burgess, A.M., Topple, A.N. & Halmagyi, M. (1998) High

acceleration impulsive rotations reveal severe long-term deficits of the horizontal vestibulo-ocular reflex in

the guinea pig. Exp Brain Res, 123, 242-254.

Gill, E.W., Paton, W.D. & Pertwee, R.G. (1970) Preliminary experiments on the chemistry and pharmacology of

cannabis. Nature, 228, 134-136.

Glaser, J.R. & Glaser, E.M. (2000) Stereology, morphometry, and mapping: the whole is greater than the sum of

its parts. J Chem Neuroanat, 20, 115-126.

Glaser, S.T., Abumrad, N.A., Fatade, F., Kaczocha, M., Studholme, K.M. & Deutsch, D.G. (2003) Evidence

against the presence of an anandamide transporter. Proc Natl Acad Sci U S A, 100, 4269-4274.

Glass, M. & Felder, C.C. (1997) Concurrent stimulation of cannabinoid CB1 and dopamine D2 receptors

augments cAMP accumulation in striatal neurons: evidence for a Gs linkage to the CB1 receptor. J

Neurosci, 17, 5327-5333.

Glass, M., Dragunow, M. & Faull, R.L. (1997) Cannabinoid receptors in the human brain: a detailed anatomical

and quantitative autoradiographic study in the fetal, neonatal and adult human brain. Neuroscience, 77,

299-318.

Glass, M., Dragunow, M. & Faull, R.L. (2000) The pattern of neurodegeneration in Huntington's disease: a

comparative study of cannabinoid, dopamine, adenosine and GABA(A) receptor alterations in the human

basal ganglia in Huntington's disease. Neuroscience, 97, 505-519.

Glass, M., Faull, R.L. & Dragunow, M. (1993) Loss of cannabinoid receptors in the substantia nigra in

Huntington's disease. Neuroscience, 56, 523-527.

Glover, J.C. (2009) Vestibular System. In Squire, L.R. (ed) Encyclopedia of Neuroscience. Academic Press, pp.

127-132.

Goble, T.J, Møller, A.R. & Thompson, L.T. (2009) Acute high-intensity sound exposure alters responses of place

cells in hippocampus. Hear Res, 253, 52-59.

Goddard, M., Zheng, Y., Darlington, C. L., Smith, P. F. (2008a) Locomotor and exploratory behaviour in the rat

following bilateral vestibular deafferentation. Behavioural Neuroscience, 122, 448-459.

Goddard, M., Zheng, Y., Darlington, C.L. & Smith, P.F. (2008b) Monoamine transporter and enzyme expression

in the medial temporal lobe and frontal cortex following chronic bilateral vestibular loss. Neurosci Lett,

437, 107-110.

Goddard, M., Zheng, Y., Darlington, C.L. & Smith, P.F. (2008c) Synaptic protein expression in the medial

temporal lobe and frontal cortex following chronic bilateral vestibular loss. Hippocampus, 18, 440-444.

Golani, I., Benjamini, Y. & Eilam, D. (1993) Stopping behavior: constraints on exploration in rats (Rattus

References 177

norvegicus). Behav Brain Res, 53, 21-33.

Goldberg, M.E. & Hudspeth, A. J. (2000) The vestibular system. In Kandel, E.R., Schwartz, J. H. & Jessell, T. M.

(ed) Principles of Neural Science. McGraw-Hill, pp. 801-805.

Goldstein, N.S., Hunter, S., Forbes, S., Odish, E. & Tehrani, M. (2007) Estrogen receptor antibody incubation

time and extent of immunoreactivity in invasive carcinoma of the breast: the importance of optimizing

antibody avidity. Appl Immunohistochem Mol Morphol, 15, 203-207.

Gong, J.P., Onaivi, E.S., Ishiguro, H., Liu, Q.R., Tagliaferro, P.A., Brusco, A. & Uhl, G.R. (2006) Cannabinoid

CB2 receptors: immunohistochemical localization in rat brain. Brain Res, 1071, 10-23.

Gonzalez, S., Cebeira, M. & Femandez-Ruiz, J. (2005) Cannabinoid tolerance and dependence: A review of

studies in laboratory animals. Pharmacol Biochem Behav, 81, 300-318.

Goodrich-Hunsaker, N.J., Livingstone, S.A., Skelton, R.W. & Hopkins, R.O. (2010) Spatial deficits in a virtual

water maze in amnesic participants with hippocampal damage. Hippocampus, 20, 481-491.

Goonawardena, A.V., Riedel, G. & Hampson, R.E. (2010) Cannabinoids alter spontaneous firing, bursting, and

cell synchrony of hippocampal principal cells. Hippocampus.

Goparaju, S.K., Ueda, N., Yamaguchi, H. & Yamamoto, S. (1998) Anandamide amidohydrolase reacting with 2-

arachidonoylglycerol, another cannabinoid receptor ligand. FEBS Lett, 422, 69-73.

Gould, J.L. (1987) Landmark learning by honey-bees. J Comp Physiol A, 35, 26-34.

Griffin, G., Wray, E.J., Tao, Q., McAllister, S.D., Rorrer, W.K., Aung, M.M., Martin, B.R. & Abood, M.E. (1999)

Evaluation of the cannabinoid CB2 receptor-selective antagonist, SR144528: further evidence for

cannabinoid CB2 receptor absence in the rat central nervous system. Eur J Pharmacol, 377, 117-125.

Grimm, R.J., Hemenway, W.G., Lebray, P.R. & Black, F.O. (1989) The perilymph fistula syndrome defined in

mild head trauma. Acta Otolaryngol Suppl, 464, 1-40.

Grimsey, N.L., Goodfellow, C.E., Scotter, E.L., Dowie, M.J., Glass, M. & Graham, E.S. (2008) Specific

detection of CB1 receptors; cannabinoid CB1 receptor antibodies are not all created equal! J Neurosci

Methods, 171, 78-86.

Grossman, G.E., Leigh, R.J., Abel, L.A., Lanska, D.J. & Thurston, S.E. (1988) Frequency and velocity of

rotational head perturbations during locomotion. Exp Brain Res, 70, 470-476.

Grossman, G.E., Leigh, R.J., Bruce, E.N., Huebner, W.P. & Lanska, D.J. (1989) Performance of the human

vestibuloocular reflex during locomotion. J Neurophysiol, 62, 264-272.

Grunfeld, Y. & Edery, H. (1969) Psychopharmacological activity of the active constituents of hashish and some

related cannabinoids. Psychopharmacologia, 14, 200-210.

Gullapalli, S., Amrutkar, D., Gupta, S., Kandadi, M.R., Kumar, H., Gandhi, M., Karande, V. & Narayanan, S.

(2010) Characterization of active and inactive states of CB1 receptor and the differential binding state

modulation by cannabinoid agonists, antagonists and inverse agonists. Neuropharmacology, 58, 1215-1219.

Hain, T.C. & Helminski, J. O. (2007) Vestibular rehabilitation. F. A. Davis Company, Philadelphia.

Haj-Dahmane, S. & Shen, R.Y. (2010) Regulation of plasticity of glutamate synapses by endocannabinoids and

the cyclic-AMP/protein kinase A pathway in midbrain dopamine neurons. J Physiol, 588, 2589-2604.

Hájos, N., Katona, I., Naiem, S.S., MacKie, K., Ledent, C., Mody, I. & Freund, T.F. (2000) Cannabinoids inhibit

hippocampal GABAergic transmission and network oscillations. Eur J Neurosci, 12, 3239-3249.

Hajos, N., Ledent, C. & Freund, T.F. (2001) Novel cannabinoid-sensitive receptor mediates inhibition of

References 178

glutamatergic synaptic transmission in the hippocampus. Neuroscience, 106, 1-4.

Hall, W. & Degenhardt, L. (2009) Adverse health effects of non-medical cannabis use. Lancet, 374, 1383-1391.

Hamann, K.F., Reber, A., Hess, B.J. & Dieringer, N. (1998) Long-term deficits in otolith, canal and optokinetic

ocular reflexes of pigmented rats after unilateral vestibular nerve section. Exp Brain Res, 118, 331-340.

Hampson, R.E. & Deadwyler, S.A. (2000) Cannabinoids reveal the necessity of hippocampal neural encoding for

short-term memory in rats. J Neurosci, 20, 8932-8942.

Hanes, D.A. & McCollum, G. (2006) Cognitive-vestibular interactions: a review of patient difficulties and

possible mechanisms. J Vestib Res, 16, 75-91.

Hanus, L., Abu-Lafi, S., Fride, E., Breuer, A., Vogel, Z., Shalev, D.E., Kustanovich, I. & Mechoulam, R. (2001)

2-arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB1 receptor. Proc Natl Acad Sci

U S A, 98, 3662-3665.

Harker, K.T. & Whishaw, I.Q. (2004) Impaired place navigation in place and matching-to-place swimming pool

tasks follows both retrosplenial cortex lesions and cingulum bundle lesions in rats. Hippocampus, 14, 224-

231.

He, J., Yamada, K. & Nabeshima, T. (2002) A role of Fos expression in the CA3 region of the hippocampus in

spatial memory formation in rats. Neuropsychopharmacology, 26, 259-268.

Heifets, B.D. & Castillo, P.E. (2009) Endocannabinoid signaling and long-term synaptic plasticity. Annu Rev

Physiol, 71, 283-306.

Heimbrand, S., Muller, M., Schweigart, G. & Mergner, T. (1991) Perception of horizontal head and trunk

rotation in space in patients with loss of vestibular functions. J Vestib Res, 1, 291-298.

Heishman, S.J., Arasteh, K. & Stitzer, M.L. (1997) Comparative effects of alcohol and marijuana on mood,

memory, and performance. Pharmacol Biochem Behav, 58, 93-101.

Helmchen, C., Klinkenstein, J., Machner, B., Rambold, H., Mohr, C. & Sander, T. (2009) Structural changes in

the human brain following vestibular neuritis indicate central vestibular compensation. Ann N Y Acad Sci,

1164, 104-115.

Herdman, S.J., Hall, C.D., Schubert, M.C., Das, V.E. & Tusa, R.J. (2007) Recovery of dynamic visual acuity in

bilateral vestibular hypofunction. Arch Otolaryngol Head Neck Surg, 133, 383-389.

Herkenham, M. (1995) Cannabinoid receptors. Academic Press, London.

Herkenham, M., Lynn, A.B., Johnson, M.R., Melvin, L.S., de Costa, B.R. & Rice, K.C. (1991) Characterization

and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. J

Neurosci, 11, 563-583.

Herkenham, M., Lynn, A.B., Little, M.D., Johnson, M.R., Melvin, L.S., de Costa, B.R. & Rice, K.C. (1990)

Cannabinoid receptor localization in brain. Proc Natl Acad Sci U S A, 87, 1932-1936.

Heskin-Sweezie, R., Titley, H.K., Baizer, J.S. & Broussard, D.M. (2010) Type B GABA receptors contribute to

the restoration of balance during vestibular compensation in mice. Neuroscience, 169, 302-314.

Hess, U.S., Lynch, G. & Gall, C.M. (1995) Changes in c-fos mRNA expression in rat brain during odor

discrimination learning: differential involvement of hippocampal subfields CA1 and CA3. J Neurosci, 15,

4786-4795.

Heyser, C.J., Hampson, R.E. & Deadwyler, S.A. (1993) Effects of delta-9-tetrahydrocannabinol on delayed

match to sample performance in rats: alterations in short-term memory associated with changes in task

References 179

specific firing of hippocampal cells. J Pharmacol Exp Ther, 264, 294-307.

Hicks, S. L., Gilchrist, D. P. D., Curthbert, P. & Curthoys, I. S. (2004) Hippocampal field responses to direct

electrical stimulation of the vestibular system in awake or anesthetised guinea pigs. Proc Aust Neurosci Soc,

15, 87.

Higgs, S., Barber, D.J., Cooper, A.J. & Terry, P. (2005) Differential effects of two cannabinoid receptor agonists

on progressive ratio responding for food and free-feeding in rats. Behav Pharmacol, 16, 389-393.

Highstein, S.M. & Holstein, G.R. (2006) The anatomy of the vestibular nuclei. Prog Brain Res, 151, 157-203.

Hill, A.J. & Best, P.J. (1981) Effects of deafness and blindness on the spatial correlates of hippocampal unit

activity in the rat. Exp Neurol, 74, 204-217.

Hill, M.N., Carrier, E.J., Ho, W.S., Shi, L., Patel, S., Gorzalka, B.B. & Hillard, C.J. (2008a) Prolonged

glucocorticoid treatment decreases cannabinoid CB1 receptor density in the hippocampus. Hippocampus,

18, 221-226.

Hill, M.N., Carrier, E.J., McLaughlin, R.J., Morrish, A.C., Meier, S.E., Hillard, C.J. & Gorzalka, B.B. (2008b)

Regional alterations in the endocannabinoid system in an animal model of depression: effects of concurrent

antidepressant treatment. J Neurochem, 106, 2322-2336.

Hill, M.N., Froc, D.J., Fox, C.J., Gorzalka, B.B. & Christie, B.R. (2004) Prolonged cannabinoid treatment results

in spatial working memory deficits and impaired long-term potentiation in the CA1 region of the

hippocampus in vivo. Eur J Neurosci, 20, 859-863.

Hill, M.N., Hunter, R.G. & McEwen, B.S. (2009) Chronic stress differentially regulates cannabinoid CB1

receptor binding in distinct hippocampal subfields. Eur J Pharmacol, 614, 66-69.

Hill, M.N., Patel, S., Carrier, E.J., Rademacher, D.J., Ormerod, B.K., Hillard, C.J. & Gorzalka, B.B. (2005)

Downregulation of endocannabinoid signaling in the hippocampus following chronic unpredictable stress.

Neuropsychopharmacology, 30, 508-515.

Hill, M.N., Titterness, A.K., Morrish, A.C., Carrier, E.J., Lee, T.T., Gil-Mohapel, J., Gorzalka, B.B., Hillard, C.J.

& Christie, B.R. (2010) Endogenous cannabinoid signaling is required for voluntary exercise-induced

enhancement of progenitor cell proliferation in the hippocampus. Hippocampus, 20, 513-523.

Hillard, C.J. & Jarrahian, A. (2000) The movement of N-arachidonoylethanolamine (anandamide) across cellular

membranes. Chem Phys Lipids, 108, 123-134.

Hillard, C.J., Edgemond, W.S. & Campbell, W.B. (1995) Characterization of ligand binding to the cannabinoid

receptor of rat brain membranes using a novel method: application to anandamide. J Neurochem, 64, 677-

683.

Hillard, C.J., Manna, S., Greenberg, M.J., DiCamelli, R., Ross, R.A., Stevenson, L.A., Murphy, V., Pertwee, R.G.

& Campbell, W.B. (1999) Synthesis and characterization of potent and selective agonists of the neuronal

cannabinoid receptor (CB1). J Pharmacol Exp Ther, 289, 1427-1433.

Him, A. & Dutia, M.B. (2001) Intrinsic excitability changes in vestibular nucleus neurons after unilateral

deafferentation. Brain Res, 908, 58-66.

Hines, D.J. & Whishaw, I.Q. (2005) Home bases formed to visual cues but not to self-movement (dead

reckoning) cues in exploring hippocampectomized rats. Eur J Neurosci, 22, 2363-2375.

Hodos, W. (1961) Progressive ratio as a measure of reward strength. Science, 134, 943-944.

Hoffman, A.F. & Lupica, C.R. (2000) Mechanisms of cannabinoid inhibition of GABA(A) synaptic transmission

References 180

in the hippocampus. J Neurosci, 20, 2470-2479.

Hoffman, A.F., Oz, M., Yang, R., Lichtman, A.H. & Lupica, C.R. (2007) Opposing actions of chronic Delta9-

tetrahydrocannabinol and cannabinoid antagonists on hippocampal long-term potentiation. Learn Mem, 14,

63-74.

Holahan, M.R. & Routtenberg, A. (2010) Lidocaine injections targeting CA3 hippocampus impair long-term

spatial memory and prevent learning-induced mossy fiber remodeling. Hippocampus.

Hollister, L.E. (1974) Structure-activity relationships in man of cannabis constituents, and homologs and

metabolites of delta9-tetrahydrocannabinol. Pharmacology, 11, 3-11.

Honrubia, V., Jenkins, H.A., Baloh, R.W., Yee, R.D. & Lau, C.G. (1984) Vestibulo-ocular reflexes in peripheral

labyrinthine lesions: I. Unilateral dysfunction. Am J Otolaryngol, 5, 15-26.

Honrubia, V., Marco, J., Andrews, J., Minser, K., Yee, R.D. & Baloh, R.W. (1985) Vestibulo-ocular reflexes in

peripheral labyrinthine lesions: III. Bilateral dysfunction. Am J Otolaryngol, 6, 342-352.

Horak, F.B. (2009) Postural compensation for vestibular loss. Ann N Y Acad Sci, 1164, 76-81.

Horak, F.B., Nashner, L.M. & Diener, H.C. (1990) Postural strategies associated with somatosensory and

vestibular loss. Exp Brain Res, 82, 167-177.

Horii, A., Kitahara, T., Smith, P.F., Darlington, C.L., Masumura, C. & Kubo, T. (2003) Effects of unilateral

labyrinthectomy on GAD, GAT1 and GABA receptor gene expression in the rat vestibular nucleus.

Neuroreport, 14, 2359-2363.

Horii, A., Russell, N.A., Smith, P.F., Darlington, C.L. & Bilkey, D.K. (2004) Vestibular influences on CA1

neurons in the rat hippocampus: an electrophysiological study in vivo. Exp Brain Res, 155, 245-250.

Horii, A., Takeda, N., Mochizuki, T., Okakura-Mochizuki, K., Yamamoto, Y. & Yamatodani, A. (1994) Effects of

vestibular stimulation on acetylcholine release from rat hippocampus: an in vivo microdialysis study. J

Neurophysiol, 72, 605-611.

Horii, A., Takeda, N., Mochizuki, T., Okakura-Mochizuki, K., Yamamoto, Y., Yamatodani, A. & Kubo, T. (1995)

Vestibular modulation of the septo-hippocampal cholinergic system of rats. Acta Otolaryngol Suppl, 520 Pt

2, 395-398.

Horii, A., Takeda, N., Yamatodani, A. & Kubo, A.T. (1996) Vestibular influences on the histaminergic and

cholinergic systems in the rat brain. Ann N Y Acad Sci, 781, 633-634.

Hosseini-Sharifabad, M. & Nyengaard, J.R. (2007) Design-based estimation of neuronal number and individual

neuronal volume in the rat hippocampus. J Neurosci Methods, 162, 206-214.

Houston, D.B. & Howlett, A.C. (1993) Solubilization of the cannabinoid receptor from rat brain and its

functional interaction with guanine nucleotide-binding proteins. Mol Pharmacol, 43, 17-22.

Houston, D.B. & Howlett, A.C. (1998) Differential receptor-G-protein coupling evoked by dissimilar

cannabinoid receptor agonists. Cell Signal, 10, 667-674.

Howlett, A.C. & Fleming, R.M. (1984) Cannabinoid inhibition of adenylate cyclase. Pharmacology of the

response in neuroblastoma cell membranes. Mol Pharmacol, 26, 532-538.

Howlett, A.C. (1984) Inhibition of neuroblastoma adenylate cyclase by cannabinoid and nantradol compounds.

Life Sci, 35, 1803-1810.

Howlett, A.C. (1985) Cannabinoid inhibition of adenylate cyclase. Biochemistry of the response in

neuroblastoma cell membranes. Mol Pharmacol, 27, 429-436.

References 181

Howlett, A.C. (2002) The cannabinoid receptors. Prostaglandins Other Lipid Mediat, 68-69, 619-631.

Howlett, A.C. (2005) Cannabinoid receptor signaling. Handb Exp Pharmacol, 53-79.

Howlett, A.C., Barth, F., Bonner, T.I., Cabral, G., Casellas, P., Devane, W.A., Felder, C.C., Herkenham, M.,

Mackie, K., Martin, B.R., Mechoulam, R. & Pertwee, R.G. (2002) International Union of Pharmacology.

XXVII. Classification of cannabinoid receptors. Pharmacol Rev, 54, 161-202.

Howlett, A.C., Qualy, J.M. & Khachatrian, L.L. (1986) Involvement of Gi in the inhibition of adenylate cyclase

by cannabimimetic drugs. Mol Pharmacol, 29, 307-313.

Huang, S.M., Bisogno, T., Trevisani, M., Al-Hayani, A., De Petrocellis, L., Fezza, F., Tognetto, M., Petros, T.J.,

Krey, J.F., Chu, C.J., Miller, J.D., Davies, S.N., Geppetti, P., Walker, J.M. & Di Marzo, V. (2002) An

endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors.


Hüfner, K., Binetti, C., Hamilton, D.A., Stephan, T., Flanagin, V.L., Linn, J., Labudda, K., Markowitsch, H.,

Glasauer, S., Jahn, K., Strupp, M. & Brandt, T. (2010) Structural and functional plasticity of the

hippocampal formation in professional dancers and slackliners. Hippocampus.

Hüfner, K., Hamilton, D.A., Kalla, R., Stephan, T., Glasauer, S., Ma, J., Bruning, R., Markowitsch, H.J.,

Labudda, K., Schichor, C., Strupp, M. & Brandt, T. (2007) Spatial memory and hippocampal volume in

humans with unilateral vestibular deafferentation. Hippocampus, 17, 471-485.

Hüfner, K., Stephan, T., Hamilton, D.A., Kalla, R., Glasauer, S., Strupp, M. & Brandt, T. (2009) Gray-matter

atrophy after chronic complete unilateral vestibular deafferentation. Ann N Y Acad Sci, 1164, 383-385.

Igarashi, M. & Guitierrez, O. (1983) Analysis of righting reflex in cats with unilateral and bilateral

labyrinthectomy. ORL J Otorhinolaryngol Relat Spec, 45, 279-289.

Igarashi, M., Ishikawa, K., Ishii, M. & Yamane, H. (1988) Physical exercise and balance compensation after total

ablation of vestibular organs. Prog Brain Res, 76, 395-401.

Inada, H., Maejima, T., Nakahata, Y., Yamaguchi, J., Nabekura, J. & Ishibashi, H. (2010) Endocannabinoids

contribute to metabotropic glutamate receptor-mediated inhibition of GABA release onto hippocampal

CA3 pyramidal neurons in an isolated neuron/bouton preparation. Neuroscience, 165, 1377-1389.

Iordanova, M.D., Burnett, D.J., Aggleton, J.P., Good, M. & Honey, R.C. (2009) The role of the hippocampus in

mnemonic integration and retrieval: complementary evidence from lesion and inactivation studies. Eur J

Neurosci, 30, 2177-2189.

Israel, I., Bronstein, A.M., Kanayama, R., Faldon, M. & Gresty, M.A. (1996) Visual and vestibular factors

influencing vestibular "navigation". Exp Brain Res, 112, 411-419.

Jansen, E.M., Haycock, D.A., Ward, S.J. & Seybold, V.S. (1992) Distribution of cannabinoid receptors in rat

brain determined with aminoalkylindoles. Brain Res, 575, 93-102.

Järbe, T.U., Ross, T., DiPatrizio, N.V., Pandarinathan, L. & Makriyannis, A. (2006) Effects of the CB1R agonist

WIN-55,212-2 and the CB1R antagonists SR-141716 and AM-1387: open-field examination in rats.

Pharmacol Biochem Behav, 85, 243-252.

Jarrard, L.E. (1993) On the role of the hippocampus in learning and memory in the rat. Behav Neural Biol, 60, 9-

26.

Jauregui-Renaud, K., Sang, F.Y., Gresty, M.A., Green, D.A. & Bronstein, A.M. (2008)

Depersonalisation/derealisation symptoms and updating orientation in patients with vestibular disease. J

References 182

Neurol Neurosurg Psychiatry, 79, 276-283.

Jelsing, J., Larsen, P.J. & Vrang, N. (2008) Identification of cannabinoid type 1 receptor expressing cocaine

amphetamine-regulated transcript neurons in the rat hypothalamus and brainstem using in situ

hybridization and immunohistochemistry. Neuroscience, 154, 641-652.

Jensen, D.W. (1983) Survival of function in the deafferentated vestibular nerve. Brain Res, 273, 175-178.

Jhaveri, M.D., Elmes, S.J., Richardson, D., Barrett, D.A., Kendall, D.A., Mason, R. & Chapman, V. (2008)

Evidence for a novel functional role of cannabinoid CB(2) receptors in the thalamus of neuropathic rats.

Eur J Neurosci, 27, 1722-1730.

Jin, K., Xie, L., Kim, S.H., Parmentier-Batteur, S., Sun, Y., Mao, X.O., Childs, J. & Greenberg, D.A. (2004)

Defective adult neurogenesis in CB1 cannabinoid receptor knockout mice. Mol Pharmacol, 66, 204-208.

Jones, G., Pertwee, R.G., Gill, E.W., Paton, W.D., Nilsson, I.M., Widman, M. & Agurell, S. (1974) Relative

pharmacological potency in mice of optical isomers of delta 1-tetrahydrocannabinol. Biochem Pharmacol,

23, 439-446.

Jones, R. (1977) Human effects. NIDA Res Monogr, 128-178.

Kaiser, A., Fedrowitz, M., Ebert, U., Zimmermann, E., Hedrich, H.J., Wedekind, D. & Loscher, W. (2001)

Auditory and vestibular defects in the circling (ci2) rat mutant. Eur J Neurosci, 14, 1129-1142.

Kalueff, A.V., Ishikawa, K. & Griffith, A.J. (2008) Anxiety and otovestibular disorders: linking behavioral

phenotypes in men and mice. Behav Brain Res, 186, 1-11.

Kaminski, N.E., Abood, M.E., Kessler, F.K., Martin, B.R. & Schatz, A.R. (1992) Identification of a functionally

relevant cannabinoid receptor on mouse spleen cells that is involved in cannabinoid-mediated immune

modulation. Mol Pharmacol, 42, 736-742.

Kano, M., Ohno-Shosaku, T., Hashimotodani, Y., Uchigashima, M. & Watanabe, M. (2009) Endocannabinoid-

mediated control of synaptic transmission. Physiol Rev, 89, 309-380.

Karnath, H.O. & Dieterich, M. (2006) Spatial neglect--a vestibular disorder? Brain, 129, 293-305.

Kathmann, M., Bauer, U., Schlicker, E. & Gothert, M. (1999) Cannabinoid CB1 receptor-mediated inhibition of

NMDA- and kainate-stimulated noradrenaline and dopamine release in the brain. Naunyn Schmiedebergs

Arch Pharmacol, 359, 466-470.

Katona, I., Rancz, E.A., Acsady, L., Ledent, C., Mackie, K., Hajos, N. & Freund, T.F. (2001) Distribution of CB1

cannabinoid receptors in the amygdala and their role in the control of GABAergic transmission. J Neurosci,

21, 9506-9518.

Katona, I., Sperlagh, B., Magloczky, Z., Santha, E., Kofalvi, A., Czirjak, S., Mackie, K., Vizi, E.S. & Freund, T.F.

(2000) GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus.

Neuroscience, 100, 797-804.

Katona, I., Sperlagh, B., Sik, A., Kafalvi, A., Vizi, E.S., Mackie, K. & Freund, T.F. (1999) Presynaptically

located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal

interneurons. J Neurosci, 19, 4544-4558.

Katona, I., Urban, G.M., Wallace, M., Ledent, C., Jung, K.M., Piomelli, D., Mackie, K. & Freund, T.F. (2006)

Molecular composition of the endocannabinoid system at glutamatergic synapses. J Neurosci, 26, 5628-

5637.

Kawamura, Y., Fukaya, M., Maejima, T., Yoshida, T., Miura, E., Watanabe, M., Ohno-Shosaku, T. & Kano, M.

References 183

(2006) The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in

the hippocampus and cerebellum. J Neurosci, 26, 2991-3001.

Kearn, C.S., Greenberg, M.J., DiCamelli, R., Kurzawa, K. & Hillard, C.J. (1999) Relationships between ligand

affinities for the cerebellar cannabinoid receptor CB1 and the induction of GDP/GTP exchange. J

Neurochem, 72, 2379-2387.

Keshner, E.A. & Cohen, H. (1989) Current concepts of the vestibular system reviewed: 1. The role of the

vestibulospinal system in postural control. Am J Occup Ther, 43, 320-330.

Kesner, R.P. (2007) Behavioral functions of the CA3 subregion of the hippocampus. Learn Mem, 14, 771-781.

Kessels, R.P., de Haan, E.H., Kappelle, L.J. & Postma, A. (2001) Varieties of human spatial memory: a meta-

analysis on the effects of hippocampal lesions. Brain Res Brain Res Rev, 35, 295-303.

Keuker, J.I., Vollmann-Honsdorf, G.K. & Fuchs, E. (2001) How to use the optical fractionator: an example based

on the estimation of neurons in the hippocampal CA1 and CA3 regions of tree shrews. Brain Res Brain Res

Protoc, 7, 211-221.

Kim, J. & Alger, B.E. (2010) Reduction in endocannabinoid tone is a homeostatic mechanism for specific

inhibitory synapses. Nat Neurosci, 13, 592-600.

Kim, J., Isokawa, M., Ledent, C. & Alger, B.E. (2002) Activation of muscarinic acetylcholine receptors enhances

the release of endogenous cannabinoids in the hippocampus. J Neurosci, 22, 10182-10191.

Kim, M.S., Jin, B.K., Chun, S.W., Lee, M.Y., Lee, S.H., Kim, J.H. & Park, B.R. (1997) Effect of MK801 on

cFos-like protein expression in the medial vestibular nucleus at early stage of vestibular compensation in

uvulonodullectomized rats. Neurosci Lett, 231, 147-150.

King, J., Zheng, Y., Liu, P., Darlington, C.L. & Smith, P.F. (2002) NMDA and AMPA receptor subunit protein

expression in the rat vestibular nucleus following unilateral labyrinthectomy. Neuroreport, 13, 1541-1545.

Klein, T.W., Newton, C.A. & Friedman, H. (2001) Cannabinoids and the immune system. Pain Res Manag, 6,

95-101.

Knierim, J.J., Kudrimoti, H.S. & McNaughton, B.L. (1995) Place cells, head direction cells, and the learning of

landmark stability. J Neurosci, 15, 1648-1659.

Koethe, D., Llenos, I.C., Dulay, J.R., Hoyer, C., Torrey, E.F., Leweke, F.M. & Weis, S. (2007) Expression of

CB1 cannabinoid receptor in the anterior cingulate cortex in schizophrenia, bipolar disorder, and major

depression. J Neural Transm, 114, 1055-1063.

Kondrachuk, A.V. (2001) Simulation and interpretation of experiments with otoliths. J Gravit Physiol, 8, P101-

104.

Kosiorek, P., Hryniewicz, A., Bialuk, I., Zawadzka, A. & Winnicka, M.M. (2003) Cannabinoids alter recognition

memory in rats. Pol J Pharmacol, 55, 903-910.

Kreitzer, A.C. & Regehr, W.G. (2001a) Cerebellar depolarization-induced suppression of inhibition is mediated

by endogenous cannabinoids. J Neurosci, 21, RC174.

Kreitzer, A.C. & Regehr, W.G. (2001b) Retrograde inhibition of presynaptic calcium influx by endogenous

cannabinoids at excitatory synapses onto Purkinje cells. Neuron, 29, 717-727.

Kubie, J.L., Muller, R.U. & Bostock, E. (1990) Spatial firing properties of hippocampal theta cells. J Neurosci,

10, 1110-1123.

Kutner, M.H., Nachtsheim, C.J., Neter, J. & Li, W. (2005) Applied Linear Statistical Models. McGraw-Hill Irwin,

References 184

Boston.

Lacour, M. & Tighilet, B. (2010) Plastic events in the vestibular nuclei during vestibular compensation: the brain

orchestration of a "deafferentation" code. Restor Neurol Neurosci, 28, 19-35.

Lafourcade, C.A. (2009) Presynaptic mechanisms of endocannabinoid-mediated long-term depression in the

hippocampus. J Neurophysiol, 102, 2009-2012.

Landsman, R.S., Burkey, T.H., Consroe, P., Roeske, W.R. & Yamamura, H.I. (1997) SR141716A is an inverse

agonist at the human cannabinoid CB1 receptor. Eur J Pharmacol, 334, R1-2.

Lane, J.I., Witte, R.J., Bolster, B., Bernstein, M.A., Johnson, K. & Morris, J. (2008) State of the art: 3T imaging

of the membranous labyrinth. AJNR Am J Neuroradiol, 29, 1436-1440.

Lawston, J., Borella, A., Robinson, J.K. & Whitaker-Azmitia, P.M. (2000) Changes in hippocampal morphology

following chronic treatment with the synthetic cannabinoid WIN 55,212-2. Brain Res, 877, 407-410.

Lecuit, T. & Lenne, P.F. (2007) Cell surface mechanics and the control of cell shape, tissue patterns and

morphogenesis. Nat Rev Mol Cell Biol, 8, 633-644.

Ledent, C., Valverde, O., Cossu, G., Petitet, F., Aubert, J.F., Beslot, F., Bohme, G.A., Imperato, A., Pedrazzini, T.,

Roques, B.P., Vassart, G., Fratta, W. & Parmentier, M. (1999) Unresponsiveness to cannabinoids and

reduced addictive effects of opiates in CB1 receptor knockout mice. Science, 283, 401-404.

Lee, S.H., Foldy, C. & Soltesz, I. (2010) Distinct endocannabinoid control of GABA release at perisomatic and

dendritic synapses in the hippocampus. J Neurosci, 30, 7993-8000.

Leenaars, M. & Hendriksen, C.F. (2005) Critical steps in the production of polyclonal and monoclonal antibodies:

evaluation and recommendations. ILAR J, 46, 269-279.

Lees, G. & Dougalis, A. (2004) Differential effects of the sleep-inducing lipid oleamide and cannabinoids on the

induction of long-term potentiation in the CA1 neurons of the rat hippocampus in vitro. Brain Res, 997, 1-

14.

Levenes, C., Daniel, H., Soubrie, P. & Crepel, F. (1998) Cannabinoids decrease excitatory synaptic transmission

and impair long-term depression in rat cerebellar Purkinje cells. J Physiol, 510 ( Pt 3), 867-879.

Li, H., Godfrey, D.A. & Rubin, A.M. (1997) Quantitative autoradiography of 5-[3H]6-cyano-7-nitro-

quinoxaline-2,3-dione and (+)-3-[3H]dizocilpine maleate binding in rat vestibular nuclear complex after

unilateral deafferentation, with comparison to cochlear nucleus. Neuroscience, 77, 473-484.

Li, H., Godfrey, T.G., Godfrey, D.A. & Rubin, A.M. (1996) Immunohistochemical study on the distributions of

AMPA receptor subtypes in rat vestibular nuclear complex after unilateral deafferentation. Ann N Y Acad

Sci, 781, 653-655.

Lichtman, A.H. & Martin, B.R. (1996) Delta 9-tetrahydrocannabinol impairs spatial memory through a

cannabinoid receptor mechanism. Psychopharmacology (Berl), 126, 125-131.

Lichtman, A.H. (2000) SR 141716A enhances spatial memory as assessed in a radial-arm maze task in rats. Eur

J Pharmacol, 404, 175-179.

Lichtman, A.H., Dimen, K.R. & Martin, B.R. (1995) Systemic or intrahippocampal cannabinoid administration

impairs spatial memory in rats. Psychopharmacology (Berl), 119, 282-290.

Lichtman, A.H., Varvel, S.A. & Martin, B.R. (2002) Endocannabinoids in cognition and dependence.

Prostaglandins Leukot Essent Fatty Acids, 66, 269-285.

Ligresti, A., Morera, E., Van Der Stelt, M., Monory, K., Lutz, B., Ortar, G. & Di Marzo, V. (2004) Further

References 185

evidence for the existence of a specific process for the membrane transport of anandamide. Biochem J, 380,

265-272.

Lindemann, S., Lessenich, A., Ebert, U. & Loscher, W. (2001) Spontaneous paroxysmal circling behavior in the

ci2 rat mutant: epilepsy with rotational seizures or hyperkinetic movement disorder? Exp Neurol, 172, 437-

445.

Lindsay, L., Liu, P., Gliddon, C., Zheng, Y., Smith, P.F. & Darlington, C.L. (2005) Cytosolic glucocorticoid

receptor expression in the rat vestibular nucleus and hippocampus following unilateral vestibular

deafferentation. Exp Brain Res, 162, 309-314.

Little, P.J., Compton, D.R., Johnson, M.R., Melvin, L.S. & Martin, B.R. (1988) Pharmacology and

stereoselectivity of structurally novel cannabinoids in mice. J Pharmacol Exp Ther, 247, 1046-1051.

Liu, P., Bilkey, D.K., Darlington, C.L. & Smith, P.F. (2003a) Cannabinoid CB1 receptor protein expression in the

rat hippocampus and entorhinal, perirhinal, postrhinal and temporal cortices: regional variations and age-

related changes. Brain Res, 979, 235-239.

Liu, P., Gliddon, C.M., Lindsay, L., Darlington, C.L. & Smith, P.F. (2004) Nitric oxide synthase and arginase

expression changes in the rat perirhinal and entorhinal cortices following unilateral vestibular damage: a

link to deficits in object recognition? J Vestib Res, 14, 411-417.

Liu, P., Zheng, Y., King, J., Darlington, C.L. & Smith, P.F. (2003b) Long-term changes in hippocampal n-methyl-

D-aspartate receptor subunits following unilateral vestibular damage in rat. Neuroscience, 117, 965-970.

Liu, P., Zheng, Y., King, J., Darlington, C.L. & Smith, P.F. (2003c) Nitric oxide synthase and arginase expression

in the vestibular nucleus and hippocampus following unilateral vestibular deafferentation in the rat. Brain

Res, 966, 19-25.

Llano, I., Leresche, N. & Marty, A. (1991) Calcium entry increases the sensitivity of cerebellar Purkinje cells to

applied GABA and decreases inhibitory synaptic currents. Neuron, 6, 565-574.

Loewenstein, O.E. (1974) Comparative morphology and physiology. In Kornhuber, H.H. (ed) Handbook of

sensory physiology. Springer-Verlag, New York, pp. 75-122.

Lombard, C., Nagarkatti, M. & Nagarkatti, P. (2007) CB2 cannabinoid receptor agonist, JWH-015, triggers

apoptosis in immune cells: potential role for CB2-selective ligands as immunosuppressive agents. Clin

Immunol, 122, 259-270.

Lopez, C., Lacour, M., Ahmadi, A.E., Magnan, J. & Borel, L. (2007) Changes of visual vertical perception: a

long-term sign of unilateral and bilateral vestibular loss. Neuropsychologia, 45, 2025-2037.

Lorincz, A. & Nusser, Z. (2008) Specificity of immunoreactions: the importance of testing specificity in each

method. J Neurosci, 28, 9083-9086.

Ludanyi, A., Eross, L., Czirjak, S., Vajda, J., Halasz, P., Watanabe, M., Palkovits, M., Magloczky, Z., Freund, T.F.

& Katona, I. (2008) Downregulation of the CB1 cannabinoid receptor and related molecular elements of

the endocannabinoid system in epileptic human hippocampus. J Neurosci, 28, 2976-2990.

Lutz, B. & Marsicano, G. (2009) Endocannabinoid role in synaptic plasticity and learning. In Squire, L.R. (ed)

Encyclopedia of Neuroscience. Academic Press, pp. 963-975.

Lynn, A.B. & Herkenham, M. (1994) Localization of cannabinoid receptors and nonsaturable high-density

cannabinoid binding sites in peripheral tissues of the rat: implications for receptor-mediated immune

modulation by cannabinoids. J Pharmacol Exp Ther, 268, 1612-1623.

References 186

Maaswinkel, H. & Whishaw, I.Q. (1999) Homing with locale, taxon, and dead reckoning strategies by foraging

rats: sensory hierarchy in spatial navigation. Behav Brain Res, 99, 143-152.

Maaswinkel, H., Jarrard, L.E. & Whishaw, I.Q. (1999) Hippocampectomized rats are impaired in homing by path

integration. Hippocampus, 9, 553-561.

MacKay, W.A. & Murphy, J.T. (1979) Cerebellar modulation of reflex gain. Prog Neurobiol, 13, 361-417.

Mackie, K. & Hille, B. (1992) Cannabinoids inhibit N-type calcium channels in neuroblastoma-glioma cells.


Mackie, K. (2005) Distribution of cannabinoid receptors in the central and peripheral nervous system. Handb

Exp Pharmacol, 299-325.

Mackie, K. (2008) Signaling via CNS cannabinoid receptors. Mol Cell Endocrinol, 286, S60-65.

Mackie, K., Lai, Y., Westenbroek, R. & Mitchell, R. (1995) Cannabinoids activate an inwardly rectifying

potassium conductance and inhibit Q-type calcium currents in AtT20 cells transfected with rat brain

cannabinoid receptor. J Neurosci, 15, 6552-6561.

Maejima, T., Hashimoto, K., Yoshida, T., Aiba, A. & Kano, M. (2001) Presynaptic inhibition caused by

retrograde signal from metabotropic glutamate to cannabinoid receptors. Neuron, 31, 463-475.

Maguire, E.A., Gadian, D.G., Johnsrude, I.S., Good, C.D., Ashburner, J., Frackowiak, R.S. & Frith, C.D. (2000)

Navigation-related structural change in the hippocampi of taxi drivers. Proc Natl Acad Sci U S A, 97, 4398-

4403.

Maguire, E.A., Nannery, R. & Spiers, H.J. (2006) Navigation around London by a taxi driver with bilateral

hippocampal lesions. Brain, 129, 2894-2907.

Mailleux, P. & Vanderhaeghen, J.J. (1992) Distribution of neuronal cannabinoid receptor in the adult rat brain: a

comparative receptor binding radioautography and in situ hybridization histochemistry. Neuroscience, 48,

655-668.

Mallet, P.E. & Beninger, R.J. (1998) The cannabinoid CB1 receptor antagonist SR141716A attenuates the

memory impairment produced by delta9-tetrahydrocannabinol or anandamide. Psychopharmacology (Berl),

140, 11-19.

Malone, D.T., Kearn, C.S., Chongue, L., Mackie, K. & Taylor, D.A. (2008) Effect of social isolation on CB1 and

D2 receptor and fatty acid amide hydrolase expression in rats. Neuroscience, 152, 265-272.

Mandarim-de-Lacerda, C.A., Fernandes-Santos, C. & Aguila, M.B. Image analysis and quantitative morphology.

Methods Mol Biol, 611, 211-225.

Maneuf, Y.P. & Brotchie, J.M. (1997) Paradoxical action of the cannabinoid WIN 55,212-2 in stimulated and

basal cyclic AMP accumulation in rat globus pallidus slices. Br J Pharmacol, 120, 1397-1398.

Manton, K.G., Stallard, E. & Tolley, H. D. (1991) Limits to human life expectancy: Evidence, prospects, and

implications. Population and Development Review, 17, 603-637.

Marchalant, Y., Brothers, H.M. & Wenk, G.L. (2009) New neuron production can be increased in the

hippocampus of aged rats following cannabinoid treatment. Mol Psychiatry, 14, 1067, 1068-1069.

Marchalant, Y., Cerbai, F., Brothers, H.M. & Wenk, G.L. (2008) Cannabinoid receptor stimulation is anti-

inflammatory and improves memory in old rats. Neurobiol Aging, 29, 1894-1901.

Maren, S. & Baudry, M. (1995) Properties and mechanisms of long-term synaptic plasticity in the mammalian

brain: relationships to learning and memory. Neurobiol Learn Mem, 63, 1-18.

References 187

Maresz, K., Carrier, E.J., Ponomarev, E.D., Hillard, C.J. & Dittel, B.N. (2005) Modulation of the cannabinoid

CB2 receptor in microglial cells in response to inflammatory stimuli. J Neurochem, 95, 437-445.

Markowska, A.L. (1999) Sex dimorphisms in the rate of age-related decline in spatial memory: relevance to

alterations in the estrous cycle. J Neurosci, 19, 8122-8133.

Marsicano, G., Goodenough, S., Monory, K., Hermann, H., Eder, M., Cannich, A., Azad, S.C., Cascio, M.G.,

Gutierrez, S.O., van der Stelt, M., Lopez-Rodriguez, M.L., Casanova, E., Schutz, G., Zieglgansberger, W.,

Di Marzo, V., Behl, C. & Lutz, B. (2003) CB1 cannabinoid receptors and on-demand defense against

excitotoxicity. Science, 302, 84-88.

Marsicano, G., Wotjak, C.T., Azad, S.C., Bisogno, T., Rammes, G., Cascio, M.G., Hermann, H., Tang, J.,

Hofmann, C., Zieglgansberger, W., Di Marzo, V. & Lutz, B. (2002) The endogenous cannabinoid system

controls extinction of aversive memories. Nature, 418, 530-534.

Martin, S.J., Grimwood, P.D. & Morris, R.G. (2000) Synaptic plasticity and memory: an evaluation of the

hypothesis. Annu Rev Neurosci, 23, 649-711.

Mato, S. & Pazos, A. (2004) Influence of age, postmortem delay and freezing storage period on cannabinoid

receptor density and functionality in human brain. Neuropharmacology, 46, 716-726.

Mato, S., Chevaleyre, V., Robbe, D., Pazos, A., Castillo, P.E. & Manzoni, O.J. (2004) A single in-vivo exposure

to delta 9THC blocks endocannabinoid-mediated synaptic plasticity. Nat Neurosci, 7, 585-586.

Mato, S., Lafourcade, M., Robbe, D., Bakiri, Y. & Manzoni, O.J. (2008) Role of the cyclic-AMP/PKA cascade

and of P/Q-type Ca++ channels in endocannabinoid-mediated long-term depression in the nucleus

accumbens. Neuropharmacology, 54, 87-94.

Matsuda, L.A., Bonner, T.I. & Lolait, S.J. (1993) Localization of cannabinoid receptor mRNA in rat brain. J

Comp Neurol, 327, 535-550.

Matsuda, L.A., Lolait, S.J., Brownstein, M.J., Young, A.C. & Bonner, T.I. (1990) Structure of a cannabinoid

receptor and functional expression of the cloned cDNA. Nature, 346, 561-564.

Matthews, B.L., Ryu, J.H. & Bockaneck, C. (1989) Vestibular contribution to spatial orientation. Evidence of

vestibular navigation in an animal model. Acta Otolaryngol Suppl, 468, 149-154.

Maurer, C., Mergner, T., Bolha, B. & Hlavacka, F. (2000) Vestibular, visual, and somatosensory contributions to

human control of upright stance. Neurosci Lett, 281, 99-102.

Maykut, M.O. (1985) Health consequences of acute and chronic marihuana use. Prog Neuropsychopharmacol

Biol Psychiatry, 9, 209-238.

Mbongo, F., Qu'hen, C., Vidal, P.P., Tran Ba Huy, P. & de Waele, C. (2009) Role of vestibular input in triggering

and modulating postural responses in unilateral and bilateral vestibular loss patients. Audiol Neurootol, 14,

130-138.

McFarland, M.J. & Barker, E.L. (2004) Anandamide transport. Pharmacol Ther, 104, 117-135.

McFarland, M.J., Porter, A.C., Rakhshan, F.R., Rawat, D.S., Gibbs, R.A. & Barker, E.L. (2004) A role for

caveolae/lipid rafts in the uptake and recycling of the endogenous cannabinoid anandamide. J Biol Chem,

279, 41991-41997.

McGauran, A.M., O'Mara, S.M. & Commins, S. (2005) Vestibular influence on water maze retention: transient

whole body rotations improve the accuracy of the cue-based retention strategy. Behav Brain Res, 158, 183-

187.

References 188

McGregor, I.S., Issakidis, C.N. & Prior, G. (1996) Aversive effects of the synthetic cannabinoid CP 55,940 in

rats. Pharmacol Biochem Behav, 53, 657-664.

McLaughlin, P.J., Winston, K., Swezey, L., Wisniecki, A., Aberman, J., Tardif, D.J., Betz, A.J., Ishiwari, K.,

Makriyannis, A. & Salamone, J.D. (2003) The cannabinoid CB1 antagonists SR 141716A and AM 251

suppress food intake and food-reinforced behavior in a variety of tasks in rats. Behav Pharmacol, 14, 583-

588.

McNaughton, B.L., Battaglia, F.P., Jensen, O., Moser, E.I. & Moser, M.B. (2006) Path integration and the neural

basis of the 'cognitive map'. Nat Rev Neurosci, 7, 663-678.

McPartland, J.M., Glass, M. & Pertwee, R.G. (2007) Meta-analysis of cannabinoid ligand binding affinity and

receptor distribution: interspecies differences. Br J Pharmacol, 152, 583-593.

Means, L.W., Alexander, S.R. & O'Neal, M.F. (1992) Those cheating rats: male and female rats use odor trails in

a water-escape "working memory" task. Behav Neural Biol, 58, 144-151.

Means, L.W., Hardy, W.T., Gabriel, M. & Uphold, J.D. (1971) Utilization of odor trails by rats in maze learning.

J Comp Physiol Psychol, 76, 160-164.

Mechoulam, R. & Gaoni, Y. (1967) The absolute configuration of delta-1-tetrahydrocannabinol, the major active

constituent of hashish. Tetrahedron Lett, 12, 1109-1111.

Mechoulam, R. (1970) Marijuana chemistry. Science, 168, 1159-1166.

Mechoulam, R. (1986) Cannabis as therapeutic agent. CRC Press, Boca Raton, FL.

Mechoulam, R., Ben-Shabat, S., Hanus, L., Ligumsky, M., Kaminski, N.E., Schatz, A.R., Gopher, A., Almog, S.,

Martin, B.R., Compton, D.R. & et al. (1995) Identification of an endogenous 2-monoglyceride, present in

canine gut, that binds to cannabinoid receptors. Biochem Pharmacol, 50, 83-90.

Mechoulam, R., Shani, A., Edery, H. & Grunfeld, Y. (1970) Chemical basis of hashish activity. Science, 169,

611-612.

Mergner, T., Schweigart, G., Fennell, L. & Maurer, C. (2009) Posture control in vestibular-loss patients. Ann N Y

Acad Sci, 1164, 206-215.

Meunier, M., Bachevalier, J., Mishkin, M. & Murray, E.A. (1993) Effects on visual recognition of combined and

separate ablations of the entorhinal and perirhinal cortex in rhesus monkeys. J Neurosci, 13, 5418-5432.

Meyers, J.R., Hu, Z., Lu, Z. & Corwin, J. T. (2009) Hair cell regeneration. In Squire, L.R. (ed) Encyclopedia of

Neuroscience. Academic Press, pp. 1005-1013.

Miettinen, R.A., Kalesnykas, G. & Koivisto, E.H. (2002) Estimation of the total number of cholinergic neurons

containing estrogen receptor-alpha in the rat basal forebrain. J Histochem Cytochem, 50, 891-902.

Miller, L.L. & Branconnier, R.J. (1983) Cannabis: effects on memory and the cholinergic limbic system. Psychol

Bull, 93, 441-456.

Mishkin, M. (1978) Memory in monkeys severely impaired by combined but not by separate removal of

amygdala and hippocampus. Nature, 273, 297-298.

Misner, D.L. & Sullivan, J.M. (1999) Mechanism of cannabinoid effects on long-term potentiation and

depression in hippocampal CA1 neurons. J Neurosci, 19, 6795-6805.

Mitrirattanakul, S., Lopez-Valdes, H.E., Liang, J., Matsuka, Y., Mackie, K., Faull, K.F. & Spigelman, I. (2007)

Bidirectional alterations of hippocampal cannabinoid 1 receptors and their endogenous ligands in a rat

model of alcohol withdrawal and dependence. Alcohol Clin Exp Res, 31, 855-867.

References 189

Mochizuki, T., Okakura-Mochizuki, K., Horii, A., Yamamoto, Y. & Yamatodani, A. (1994) Histaminergic

modulation of hippocampal acetylcholine release in vivo. J Neurochem, 62, 2275-2282.

Moldrich, G. & Wenger, T. (2000) Localization of the CB1 cannabinoid receptor in the rat brain. An

immunohistochemical study. Peptides, 21, 1735-1742.

Molina-Holgado, F., Rubio-Araiz, A., Garcia-Ovejero, D., Williams, R.J., Moore, J.D., Arevalo-Martin, A.,

Gomez-Torres, O. & Molina-Holgado, E. (2007) CB2 cannabinoid receptors promote mouse neural stem

cell proliferation. Eur J Neurosci, 25, 629-634.

Morgan, N.H., Stanford, I.M. & Woodhall, G.L. (2009) Functional CB2 type cannabinoid receptors at CNS

synapses. Neuropharmacology, 57, 356-368.

Morris, R.G., Garrud, P., Rawlins, J.N. & O'Keefe, J. (1982) Place navigation impaired in rats with hippocampal

lesions. Nature, 297, 681-683.

Moser, M.B. (1999) Making more synapses: a way to store information? Cell Mol Life Sci, 55, 593-600.

Motulsky, H. (1995) The GraphPad guide to analyzing radioligand binding data www.graphpad.com. GraphPad

Software, Inc, pp. 1-18.

Mumby, D., Pinel, J. P. J. & Wood, E. R. (1990) Nonrecurring-items delayed nonmatching-to-sample in rats: A

new paradigm for testing nonspatial working memory. Psychobiology, 18, 321-326.

Mumby, D.G. (2001) Perspectives on object-recognition memory following hippocampal damage: lessons from

studies in rats. Behav Brain Res, 127, 159-181.

Munro, S., Thomas, K.L. & Abu-Shaar, M. (1993) Molecular characterization of a peripheral receptor for

cannabinoids. Nature, 365, 61-65.

Murray, E.A. & Mishkin, M. (1986) Visual recognition in monkeys following rhinal cortical ablations combined

with either amygdalectomy or hippocampectomy. J Neurosci, 6, 1991-2003.

Murray, J.B. (1986) Marijuana's effects on human cognitive functions, psychomotor functions, and personality. J

Gen Psychol, 113, 23-55.

Nakamura-Palacios, E.M., Winsauer, P.J. & Moerschbaecher, J.M. (2000) Effects of the cannabinoid ligand SR

141716A alone or in combination with delta9-tetrahydrocannabinol or scopolamine on learning in squirrel

monkeys. Behav Pharmacol, 11, 377-386.

Nakamura, E.M., da Silva, E.A., Concilio, G.V., Wilkinson, D.A. & Masur, J. (1991) Reversible effects of acute

and long-term administration of delta-9-tetrahydrocannabinol (THC) on memory in the rat. Drug Alcohol

Depend, 28, 167-175.

Nardini, M., Jones, P., Bedford, R. & Braddick, O. (2008) Development of cue integration in human navigation.

Curr Biol, 18, 689-693.

Nashner, L.M., Black, F.O. & Wall, C., 3rd (1982) Adaptation to altered support and visual conditions during

stance: patients with vestibular deficits. J Neurosci, 2, 536-544.

Nava, F., Carta, G., Battasi, A.M. & Gessa, G.L. (2000) D(2) dopamine receptors enable delta(9)-

tetrahydrocannabinol induced memory impairment and reduction of hippocampal extracellular

acetylcholine concentration. Br J Pharmacol, 130, 1201-1210.

Nava, F., Carta, G., Colombo, G. & Gessa, G.L. (2001) Effects of chronic Delta(9)-tetrahydrocannabinol

treatment on hippocampal extracellular acetylcholine concentration and alternation performance in the T-

maze. Neuropharmacology, 41, 392-399.

References 190

Nawata, Y., Hiranita, T. & Yamamoto, T. A cannabinoid CB(1) receptor antagonist ameliorates impairment of

recognition memory on withdrawal from MDMA (Ecstasy). Neuropsychopharmacology, 35, 515-520.

Neitz, J. & Jacobs, G.H. (1986) Reexamination of spectral mechanisms in the rat (Rattus norvegicus). J Comp

Psychol, 100, 21-29.

Nestor, L., Roberts, G., Garavan, H. & Hester, R. (2008) Deficits in learning and memory: parahippocampal

hyperactivity and frontocortical hypoactivity in cannabis users. Neuroimage, 40, 1328-1339.

Neu, A., Foldy, C. & Soltesz, I. (2007) Postsynaptic origin of CB1-dependent tonic inhibition of GABA release

at cholecystokinin-positive basket cell to pyramidal cell synapses in the CA1 region of the rat hippocampus.

J Physiol, 578, 233-247.

Newlands, S.D., Dara, S. & Kaufman, G.D. (2005) Relationship of static and dynamic mechanisms in

vestibuloocular reflex compensation. Laryngoscope, 115, 191-204.

Newlands, S.D., Perachio, A. A. (1990) Compensation of horizontal canal related activity in the medial

vestibular nucleus following unilateral labyrinth ablation in the decerebrate gerbil. Experimental Brain

Research, 82, 359-372.

Ni, X., Geller, E.B., Eppihimer, M.J., Eisenstein, T.K., Adler, M.W. & Tuma, R.F. (2004) Win 55212-2, a

cannabinoid receptor agonist, attenuates leukocyte/endothelial interactions in an experimental autoimmune

encephalomyelitis model. Mult Scler, 10, 158-164.

Norre, M.E., Forrez, G. & Stevens, M. (1984) Posturography and vestibular compensation. Acta

Otorhinolaryngol Belg, 38, 619-631.

Núñez, E., Benito, C., Pazos, M.R., Barbachano, A., Fajardo, O., Gonzalez, S., Tolon, R.M. & Romero, J. (2004)

Cannabinoid CB2 receptors are expressed by perivascular microglial cells in the human brain: an

immunohistochemical study. Synapse, 53, 208-213.

Núñez, E., Benito, C., Tolon, R.M., Hillard, C.J., Griffin, W.S. & Romero, J. (2008) Glial expression of

cannabinoid CB(2) receptors and fatty acid amide hydrolase are beta amyloid-linked events in Down's

syndrome. Neuroscience, 151, 104-110.

Nyiri, G., Cserep, C., Szabadits, E., Mackie, K. & Freund, T.F. (2005) CB1 cannabinoid receptors are enriched in

the perisynaptic annulus and on preterminal segments of hippocampal GABAergic axons. Neuroscience,

136, 811-822.

O'Keefe, J. & Dostrovsky, J. (1971) The hippocampus as a spatial map. Preliminary evidence from unit activity

in the freely-moving rat. Brain Res, 34, 171-175.

O'Keefe, J. & Nadel, L. (1978) The hippocampus as a cognitive map. Clarendon Press, Oxford.

O'Keefe, J. (1979) A review of the hippocampal place cells. Prog Neurobiol, 13, 419-439.

O'Mara, S.M., Rolls, E.T., Berthoz, A. & Kesner, R.P. (1994) Neurons responding to whole-body motion in the

primate hippocampus. J Neurosci, 14, 6511-6523.

O'Shea, M. & Mallet, P.E. (2005) Impaired learning in adulthood following neonatal delta9-THC exposure.

Behav Pharmacol, 16, 455-461.

O'Shea, M., McGregor, I.S. & Mallet, P.E. (2006) Repeated cannabinoid exposure during perinatal, adolescent or

early adult ages produces similar longlasting deficits in object recognition and reduced social interaction in

rats. J Psychopharmacol, 20, 611-621.

O'Shea, M., Singh, M.E., McGregor, I.S. & Mallet, P.E. (2004) Chronic cannabinoid exposure produces lasting

References 191

memory impairment and increased anxiety in adolescent but not adult rats. J Psychopharmacol, 18, 502-

508.

Ohno-Shosaku, T., Maejima, T. & Kano, M. (2001) Endogenous cannabinoids mediate retrograde signals from

depolarized postsynaptic neurons to presynaptic terminals. Neuron, 29, 729-738.

Ohno-Shosaku, T., Matsui, M., Fukudome, Y., Shosaku, J., Tsubokawa, H., Taketo, M.M., Manabe, T. & Kano,

M. (2003) Postsynaptic M1 and M3 receptors are responsible for the muscarinic enhancement of retrograde

endocannabinoid signalling in the hippocampus. Eur J Neurosci, 18, 109-116.

Ohno-Shosaku, T., Shosaku, J., Tsubokawa, H. & Kano, M. (2002) Cooperative endocannabinoid production by

neuronal depolarization and group I metabotropic glutamate receptor activation. Eur J Neurosci, 15, 953-

961.

Olabi, B., Bergquist, F. & Dutia, M.B. (2009) Rebalancing the commissural system: mechanisms of vestibular

compensation. J Vestib Res, 19, 201-207.

Olshansky, S.J., Carnes, B.A. & Cassel, C. (1990) In search of Methuselah: estimating the upper limits to human

longevity. Science, 250, 634-640.

Olton, D.S. & Papas, B.C. (1979) Spatial memory and hippocampal function. Neuropsychologia, 17, 669-682.

Olton, D.S. & Werz, M.A. (1978) Hippocampal function and behavior: spatial discrimination and response

inhibition. Physiol Behav, 20, 597-605.

Onaivi, E.S. (2006) Neuropsychobiological evidence for the functional presence and expression of cannabinoid

CB2 receptors in the brain. Neuropsychobiology, 54, 231-246.

Onaivi, E.S., Ishiguro, H., Gong, J.P., Patel, S., Perchuk, A., Meozzi, P.A., Myers, L., Mora, Z., Tagliaferro, P.,

Gardner, E., Brusco, A., Akinshola, B.E., Liu, Q.R., Hope, B., Iwasaki, S., Arinami, T., Teasenfitz, L. &

Uhl, G.R. (2006) Discovery of the presence and functional expression of cannabinoid CB2 receptors in

brain. Ann N Y Acad Sci, 1074, 514-536.

Ong, W.Y. & Mackie, K. (1999) A light and electron microscopic study of the CB1 cannabinoid receptor in

primate brain. Neuroscience, 92, 1177-1191.

Ossenkopp, K.P. & Hargreaves, E.L. (1993) Spatial learning in an enclosed eight-arm radial maze in rats with

sodium arsanilate-induced labyrinthectomies. Behav Neural Biol, 59, 253-257.

Ossenkopp, K.P., Prkacin, A. & Hargreaves, E.L. (1990) Sodium arsanilate-induced vestibular dysfunction in

rats: effects on open-field behavior and spontaneous activity in the automated digiscan monitoring system.

Pharmacol Biochem Behav, 36, 875-881.

Pacheco, M.A., Ward, S.J. & Childers, S.R. (1993) Identification of cannabinoid receptors in cultures of rat

cerebellar granule cells. Brain Res, 603, 102-110.

Palazuelos, J., Aguado, T., Egia, A., Mechoulam, R., Guzman, M. & Galve-Roperh, I. (2006) Non-psychoactive

CB2 cannabinoid agonists stimulate neural progenitor proliferation. FASEB J, 20, 2405-2407.

Pamplona, F.A., Bitencourt, R.M. & Takahashi, R.N. (2008) Short- and long-term effects of cannabinoids on the

extinction of contextual fear memory in rats. Neurobiol Learn Mem, 90, 290-293.

Pan, X., Ikeda, S.R. & Lewis, D.L. (1996) Rat brain cannabinoid receptor modulates N-type Ca2+ channels in a

neuronal expression system. Mol Pharmacol, 49, 707-714.

Panikashvili, D., Simeonidou, C., Ben-Shabat, S., Hanus, L., Breuer, A., Mechoulam, R. & Shohami, E. (2001)

An endogenous cannabinoid (2-AG) is neuroprotective after brain injury. Nature, 413, 527-531.

References 192

Parietti-Winkler, C., Gauchard, G.C., Simon, C. & Perrin, P.P. (2010) Long-term effects of vestibular

compensation on balance control and sensory organisation after unilateral deafferentation due to vestibular

schwannoma surgery. J Neurol Neurosurg Psychiatry, 81, 934-936.

Paxinos, G., Watson, C. (1998) The rat brain in stereotaxic coordinates. Academic Press, San Diego.

Pazos, M.R., Sagredo, O. & Fernandez-Ruiz, J. (2008) The endocannabinoid system in Huntington's disease.

Curr Pharm Des, 14, 2317-2325.

Pertwee, R.G. & Ross, R.A. (2002) Cannabinoid receptors and their ligands. Prostaglandins Leukot Essent Fatty

Acids, 66, 101-121.

Pertwee, R.G. (1997) Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol Ther, 74, 129-180.

Pertwee, R.G. (1999) Pharmacology of cannabinoid receptor ligands. Curr Med Chem, 6, 635-664.

Pertwee, R.G. (2005a) Inverse agonism and neutral antagonism at cannabinoid CB1 receptors. Life Sci, 76, 1307-

1324.

Pertwee, R.G. (2005b) Pharmacological actions of cannabinoids. Handb Exp Pharmacol, 1-51.

Pertwee, R.G. (2006) The pharmacology of cannabinoid receptors and their ligands: an overview. Int J Obes

(Lond), 30 Suppl 1, S13-18.

Peruch, P., Borel, L., Gaunet, F., Thinus-Blanc, G., Magnan, J. & Lacour, M. (1999) Spatial performance of

unilateral vestibular defective patients in nonvisual versus visual navigation. J Vestib Res, 9, 37-47.

Peterson, B.W. & Richmond, F.J. (1988) Control of Head Movement. Oxford University Press., New York.

Petitet, F., Jeantaud, B., Capet, M. & Doble, A. (1997) Interaction of brain cannabinoid receptors with guanine

nucleotide binding protein: a radioligand binding study. Biochem Pharmacol, 54, 1267-1270.

Pettit, D.A., Harrison, M.P., Olson, J.M., Spencer, R.F. & Cabral, G.A. (1998) Immunohistochemical localization

of the neural cannabinoid receptor in rat brain. J Neurosci Res, 51, 391-402.

Piomelli, D. (2003) The molecular logic of endocannabinoid signalling. Nat Rev Neurosci, 4, 873-884.

Piomelli, D., Beltramo, M., Glasnapp, S., Lin, S.Y., Goutopoulos, A., Xie, X.Q. & Makriyannis, A. (1999)

Structural determinants for recognition and translocation by the anandamide transporter. Proc Natl Acad

Sci U S A, 96, 5802-5807.

Pistis, M., Muntoni, A.L., Pillolla, G. & Gessa, G.L. (2002) Cannabinoids inhibit excitatory inputs to neurons in

the shell of the nucleus accumbens: an in vivo electrophysiological study. Eur J Neurosci, 15, 1795-1802.

Pitler, T.A. & Alger, B.E. (1992) Postsynaptic spike firing reduces synaptic GABAA responses in hippocampal

pyramidal cells. J Neurosci, 12, 4122-4132.

Polissidis, A., Chouliara, O., Galanopoulos, A., Marselos, M., Papadopoulou-Daifoti, Z. & Antoniou, K. (2009)

Behavioural and dopaminergic alterations induced by a low dose of WIN 55,212-2 in a conditioned place

preference procedure. Life Sci, 85, 248-254.

Pope, H.G., Jr. & Yurgelun-Todd, D. (1996) The residual cognitive effects of heavy marijuana use in college

students. JAMA, 275, 521-527.

Porter, A.C., Sauer, J.M., Knierman, M.D., Becker, G.W., Berna, M.J., Bao, J., Nomikos, G.G., Carter, P.,

Bymaster, F.P., Leese, A.B. & Felder, C.C. (2002) Characterization of a novel endocannabinoid,

virodhamine, with antagonist activity at the CB1 receptor. J Pharmacol Exp Ther, 301, 1020-1024.

Porter, J.D., Pellis, S.M. & Meyer, M.E. (1990) An open-field activity analysis of labyrinthectomized rats.

Physiol Behav, 48, 27-30.

References 193

Pozzo, T., Berthoz, A., Lefort, L. & Vitte, E. (1991) Head stabilization during various locomotor tasks in humans.

II. Patients with bilateral peripheral vestibular deficits. Exp Brain Res, 85, 208-217.

Prather, P.L., Martin, N.A., Breivogel, C.S. & Childers, S.R. (2000) Activation of cannabinoid receptors in rat

brain by WIN 55212-2 produces coupling to multiple G protein alpha-subunits with different potencies.

Mol Pharmacol, 57, 1000-1010.

Price, D.A., Martinez, A.A., Seillier, A., Koek, W., Acosta, Y., Fernandez, E., Strong, R., Lutz, B., Marsicano, G.,

Roberts, J.L. & Giuffrida, A. (2009) WIN55,212-2, a cannabinoid receptor agonist, protects against

nigrostriatal cell loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's

disease. Eur J Neurosci, 29, 2177-2186.

Pruessner, J.C., Kirschbaum, C., Meinlschmid, G. & Hellhammer, D.H. (2003) Two formulas for computation of

the area under the curve represent measures of total hormone concentration versus time-dependent change.

Psychoneuroendocrinology, 28, 916-931.

Quinn, H.R., Matsumoto, I., Callaghan, P.D., Long, L.E., Arnold, J.C., Gunasekaran, N., Thompson, M.R.,

Dawson, B., Mallet, P.E., Kashem, M.A., Matsuda-Matsumoto, H., Iwazaki, T. & McGregor, I.S. (2008)

Adolescent rats find repeated Delta(9)-THC less aversive than adult rats but display greater residual

cognitive deficits and changes in hippocampal protein expression following exposure.


Quirk, G.J., Muller, R.U. & Kubie, J.L. (1990) The firing of hippocampal place cells in the dark depends on the

rat's recent experience. J Neurosci, 10, 2008-2017.

Qureshi, J., Saady, M., Cardounel, A. & Kalimi, M. (1998) Identification and characterization of a novel

synthetic cannabinoid CP 55,940 binder in rat brain cytosol. Mol Cell Biochem, 181, 21-27.

Ramirez, B.G., Blazquez, C., Gomez del Pulgar, T., Guzman, M. & de Ceballos, M.L. (2005) Prevention of

Alzheimer's disease pathology by cannabinoids: neuroprotection mediated by blockade of microglial

activation. J Neurosci, 25, 1904-1913.

Ranganathan, M. & D'Souza, D.C. (2006) The acute effects of cannabinoids on memory in humans: a review.

Psychopharmacology (Berl), 188, 425-444.

Redfern, M.S., Talkowski, M.E., Jennings, J.R. & Furman, J.M. (2004) Cognitive influences in postural control

of patients with unilateral vestibular loss. Gait Posture, 19, 105-114.

Reibaud, M., Obinu, M.C., Ledent, C., Parmentier, M., Bohme, G.A. & Imperato, A. (1999) Enhancement of

memory in cannabinoid CB1 receptor knock-out mice. Eur J Pharmacol, 379, R1-2.

Rhodes, K.J. & Trimmer, J.S. (2006) Antibodies as valuable neuroscience research tools versus reagents of mass

distraction. J Neurosci, 26, 8017-8020.

Rice, J. (2007) Mathematical statistics and data analysis. Duxbury Press, Belmont, California.

Richfield, E.K. & Herkenham, M. (1994) Selective vulnerability in Huntington's disease: preferential loss of

cannabinoid receptors in lateral globus pallidus. Ann Neurol, 36, 577-584.

Rinaldi-Carmona, M., Barth, F., Heaulme, M., Shire, D., Calandra, B., Congy, C., Martinez, S., Maruani, J.,

Neliat, G., Caput, D. & et al. (1994) SR141716A, a potent and selective antagonist of the brain

cannabinoid receptor. FEBS Lett, 350, 240-244.

Ris, L. & Godaux, E. (1998) Neuronal activity in the vestibular nuclei after contralateral or bilateral

labyrinthectomy in the alert guinea pig. J Neurophysiol, 80, 2352-2367.

References 194

Risey, J. & Briner, W. (1990) Dyscalculia in patients with vertigo. J Vestib Res, 1, 31-37.

Rivers, J.R. & Ashton, J.C. (2010) The development of cannabinoid CBII receptor agonists for the treatment of

central neuropathies. Cent Nerv Syst Agents Med Chem, 10, 47-64.

Robbe, D. & Buzsaki, G. (2009) Alteration of theta timescale dynamics of hippocampal place cells by a

cannabinoid is associated with memory impairment. J Neurosci, 29, 12597-12605.

Robbe, D., Alonso, G. & Manzoni, O.J. (2003) Exogenous and endogenous cannabinoids control synaptic

transmission in mice nucleus accumbens. Ann N Y Acad Sci, 1003, 212-225.

Robbe, D., Kopf, M., Remaury, A., Bockaert, J. & Manzoni, O.J. (2002) Endogenous cannabinoids mediate

long-term synaptic depression in the nucleus accumbens. Proc Natl Acad Sci U S A, 99, 8384-8388.

Robbe, D., Montgomery, S.M., Thome, A., Rueda-Orozco, P.E., McNaughton, B.L. & Buzsaki, G. (2006)

Cannabinoids reveal importance of spike timing coordination in hippocampal function. Nat Neurosci, 9,

1526-1533.

Robinson, L., Goonawardena, A.V., Pertwee, R.G., Hampson, R.E. & Riedel, G. (2007) The synthetic

cannabinoid HU210 induces spatial memory deficits and suppresses hippocampal firing rate in rats. Br J

Pharmacol, 151, 688-700.

Robinson, L., Hinder, L., Pertwee, R.G. & Riedel, G. (2003) Effects of delta9-THC and WIN-55,212-2 on place

preference in the water maze in rats. Psychopharmacology (Berl), 166, 40-50.

Rodgers, R.J., Evans, P.M. & Murphy, A. (2005) Anxiogenic profile of AM-251, a selective cannabinoid CB1

receptor antagonist, in plus-maze-naive and plus-maze-experienced mice. Behav Pharmacol, 16, 405-413.

Rodriguez de Fonseca, F., Del Arco, I., Bermudez-Silva, F.J., Bilbao, A., Cippitelli, A. & Navarro, M. (2005)

The endocannabinoid system: physiology and pharmacology. Alcohol Alcohol, 40, 2-14.

Rodriguez de Fonseca, F., Del Arco, I., Martin-Calderon, J.L., Gorriti, M.A. & Navarro, M. (1998) Role of the

endogenous cannabinoid system in the regulation of motor activity. Neurobiol Dis, 5, 483-501.

Rodriguez de Fonseca, F., Gorriti, M.A., Fernandez-Ruiz, J.J., Palomo, T. & Ramos, J.A. (1994) Downregulation

of rat brain cannabinoid binding sites after chronic delta 9-tetrahydrocannabinol treatment. Pharmacol

Biochem Behav, 47, 33-40.

Rolls, E.T. (1996) A theory of hippocampal function in memory. Hippocampus, 6, 601-620.

Roloff, A.M. & Thayer, S.A. (2009) Modulation of excitatory synaptic transmission by Delta 9-

tetrahydrocannabinol switches from agonist to antagonist depending on firing rate. Mol Pharmacol, 75,

892-900.

Romero, J., Berrendero, F., Manzanares, J., Perez, A., Corchero, J., Fuentes, J.A., Fernandez-Ruiz, J.J. & Ramos,

J.A. (1998) Time-course of the cannabinoid receptor down-regulation in the adult rat brain caused by

repeated exposure to delta9-tetrahydrocannabinol. Synapse, 30, 298-308.

Romero, J., Garcia, L., Fernandez-Ruiz, J.J., Cebeira, M. & Ramos, J.A. (1995) Changes in rat brain cannabinoid

binding sites after acute or chronic exposure to their endogenous agonist, anandamide, or to delta 9-

tetrahydrocannabinol. Pharmacol Biochem Behav, 51, 731-737.

Romero, J., Lastres-Becker, I., de Miguel, R., Berrendero, F., Ramos, J.A. & Fernandez-Ruiz, J. (2002) The

endogenous cannabinoid system and the basal ganglia. biochemical, pharmacological, and therapeutic

aspects. Pharmacol Ther, 95, 137-152.

Ronesi, J., Gerdeman, G.L. & Lovinger, D.M. (2004) Disruption of endocannabinoid release and striatal long-

References 195

term depression by postsynaptic blockade of endocannabinoid membrane transport. J Neurosci, 24, 1673-

1679.

Roozendaal, B., Phillips, R.G., Power, A.E., Brooke, S.M., Sapolsky, R.M. & McGaugh, J.L. (2001) Memory

retrieval impairment induced by hippocampal CA3 lesions is blocked by adrenocortical suppression. Nat

Neurosci, 4, 1169-1171.

Rossier, J., Haeberli, C. & Schenk, F. (2000) Auditory cues support place navigation in rats when associated with

a visual cue. Behav Brain Res, 117, 209-214.

Rothblat, L.A. & Hayes, L.L. (1987) Short-term object recognition memory in the rat: nonmatching with trial-

unique junk stimuli. Behav Neurosci, 101, 587-590.

Routtenberg, A. (1984) The CA3 pyramidal cell in the hippocampus: Site of intrinsic expression and extrinsic

control of memory formation. In Squire, L.R. & Butters, N. (ed) Neuropsychology of Memory. Guilford

Press, New York, pp. 536-546.

Rubino, T., Realini, N., Braida, D., Alberio, T., Capurro, V., Vigano, D., Guidali, C., Sala, M., Fasano, M. &

Parolaro, D. (2009a) The depressive phenotype induced in adult female rats by adolescent exposure to

THC is associated with cognitive impairment and altered neuroplasticity in the prefrontal cortex. Neurotox

Res, 15, 291-302.

Rubino, T., Realini, N., Braida, D., Guidi, S., Capurro, V., Vigano, D., Guidali, C., Pinter, M., Sala, M.,

Bartesaghi, R. & Parolaro, D. (2009b) Changes in hippocampal morphology and neuroplasticity induced

by adolescent THC treatment are associated with cognitive impairment in adulthood. Hippocampus, 19,

763-772.

Rubino, T., Vigano, D., Massi, P. & Parolaro, D. (2000a) Changes in the cannabinoid receptor binding, G protein

coupling, and cyclic AMP cascade in the CNS of rats tolerant to and dependent on the synthetic

cannabinoid compound CP55,940. J Neurochem, 75, 2080-2086.

Rubino, T., Vigano, D., Massi, P., Spinello, M., Zagato, E., Giagnoni, G. & Parolaro, D. (2000b) Chronic delta-9-

tetrahydrocannabinol treatment increases cAMP levels and cAMP-dependent protein kinase activity in

some rat brain regions. Neuropharmacology, 39, 1331-1336.

Rueda-Orozco, P.E., Soria-Gomez, E., Montes-Rodriguez, C.J., Martinez-Vargas, M., Galicia, O., Navarro, L. &

Prospero-Garcia, O. (2008) A potential function of endocannabinoids in the selection of a navigation

strategy by rats. Psychopharmacology (Berl), 198, 565-576.

Russell, N.A., Horii, A., Smith, P.F., Darlington, C.L. & Bilkey, D.K. (2003a) Bilateral peripheral vestibular

lesions produce long-term changes in spatial learning in the rat. J Vestib Res, 13, 9-16.

Russell, N.A., Horii, A., Smith, P.F., Darlington, C.L. & Bilkey, D.K. (2003b) Long-term effects of permanent

vestibular lesions on hippocampal spatial firing. J Neurosci, 23, 6490-6498.

Russell, N.A., Horii, A., Smith, P.F., Darlington, C.L. & Bilkey, D.K. (2006) Lesions of the vestibular system

disrupt hippocampal theta rhythm in the rat. J Neurophysiol, 96, 4-14.

Ryberg, E., Vu, H.K., Larsson, N., Groblewski, T., Hjorth, S., Elebring, T., Sjogren, S. & Greasley, P.J. (2005)

Identification and characterisation of a novel splice variant of the human CB1 receptor. FEBS Lett, 579,

259-264.

Ryu, J.H., McCabe, B. F. (1976) Central vestibular compensation. Effect of bilateral labyrinthectomy on neural

activity in the medial vestibular nucleus. Archives of Otolaryngology, 102, 71-76.

References 196

Sakurai, Y. (1990) Hippocampal cells have behavioral correlates during the performance of an auditory working

memory task in the rat. Behav Neurosci, 104, 253-263.

Sakurai, Y. (1994) Involvement of auditory cortical and hippocampal neurons in auditory working memory and

reference memory in the rat. J Neurosci, 14, 2606-2623.

Sakurai, Y. (2002) Coding of auditory temporal and pitch information by hippocampal individual cells and cell

assemblies in the rat. Neuroscience, 115, 1153-1163.

Saman, Y., Bamiou, D.E. & Gleeson, M. (2009) A contemporary review of balance dysfunction following

vestibular schwannoma surgery. Laryngoscope, 119, 2085-2093.

Sang, F.Y., Jauregui-Renaud, K., Green, D.A., Bronstein, A.M. & Gresty, M.A. (2006)

Depersonalisation/derealisation symptoms in vestibular disease. J Neurol Neurosurg Psychiatry, 77, 760-

766.

Sanudo-Pena, M.C. & Walker, J.M. (1998) A novel neurotransmitter system involved in the control of motor

behavior by the basal ganglia. Ann N Y Acad Sci, 860, 475-479.

Sanudo-Pena, M.C., Patrick, S.L., Patrick, R.L. & Walker, J.M. (1996) Effects of intranigral cannabinoids on

rotational behavior in rats: interactions with the dopaminergic system. Neurosci Lett, 206, 21-24.

Saper, C.B. & Sawchenko, P.E. (2003) Magic peptides, magic antibodies: guidelines for appropriate controls for

immunohistochemistry. J Comp Neurol, 465, 161-163.

Saunders, R.C., Murray, E.A. & Mishkin, M. (1984) Further evidence that amygdala and hippocampus

contribute equally to recognition memory. Neuropsychologia, 22, 785-796.

Save, E., Cressant, A., Thinus-Blanc, C. & Poucet, B. (1998) Spatial firing of hippocampal place cells in blind

rats. J Neurosci, 18, 1818-1826.

Sawant, P.M., Holland, P.T., Mountfort, D.O. & Kerr, D.S. (2008) In vivo seizure induction and pharmacological

preconditioning by domoic acid and isodomoic acids A, B and C. Neuropharmacology, 55, 1412-1418.

Saxon, D.W., Anderson, J.H. & Beitz, A.J. (2001) Transtympanic tetrodotoxin alters the VOR and Fos labeling in

the vestibular complex. Neuroreport, 12, 3051-3055.

Schaeppi, U., Krinke, G., FitzGerald, R.E. & Classen, W. (1991) Impaired tunnel-maze behavior in rats with

sensory lesions: vestibular and auditory systems. Neurotoxicology, 12, 445-454.

Schatz, A.R., Lee, M., Condie, R.B., Pulaski, J.T. & Kaminski, N.E. (1997) Cannabinoid receptors CB1 and CB2:

a characterization of expression and adenylate cyclase modulation within the immune system. Toxicol Appl

Pharmacol, 142, 278-287.

Schautzer, F., Hamilton, D., Kalla, R., Strupp, M. & Brandt, T. (2003) Spatial memory deficits in patients with

chronic bilateral vestibular failure. Ann N Y Acad Sci, 1004, 316-324.

Schirmer, M., Kaiser, A., Lessenich, A., Lindemann, S., Fedrowitz, M., Gernert, M. & Loscher, W. (2007c)

Auditory and vestibular defects and behavioral alterations after neonatal administration of streptomycin to

Lewis rats: Similarities and differences to the circling (ci2/ci2) Lewis rat mutant. Brain Res, 1155, 179-195.

Schirmer, M., Lessenich, A., Lindemann, S. & Loscher, W. (2007a) Marked differences in response to dopamine

receptor antagonism in two rat mutants, ci2 and ci3, with lateralized rotational behavior. Behav Brain Res,

180, 218-225.

Schirmer, M., Nobrega, J.N., Harrison, S.J. & Loscher, W. (2007b) Alterations in dopamine D3 receptors in the

circling (ci3) rat mutant. Neuroscience, 144, 1462-1469.

References 197

Schlicker, E. & Kathmann, M. (2001) Modulation of transmitter release via presynaptic cannabinoid receptors.

Trends Pharmacol Sci, 22, 565-572.

Schlicker, E., Timm, J., Zentner, J. & Gothert, M. (1997) Cannabinoid CB1 receptor-mediated inhibition of

noradrenaline release in the human and guinea-pig hippocampus. Naunyn Schmiedebergs Arch Pharmacol,

356, 583-589.

Schmidt-Koenig, K. (1965) Current problems in bird orientation. Adv Study Behav, 1, 217-278.

Schneider, M. & Koch, M. (2002) The cannabinoid agonist WIN 55,212-2 reduces sensorimotor gating and

recognition memory in rats. Behav Pharmacol, 13, 29-37.

Schneider, M. & Koch, M. (2003) Chronic pubertal, but not adult chronic cannabinoid treatment impairs

sensorimotor gating, recognition memory, and the performance in a progressive ratio task in adult rats.


Schneider, M. & Koch, M. (2005) Deficient social and play behavior in juvenile and adult rats after neonatal

cortical lesion: effects of chronic pubertal cannabinoid treatment. Neuropsychopharmacology, 30, 944-957.

Schneider, M. & Koch, M. (2007) The effect of chronic peripubertal cannabinoid treatment on deficient object

recognition memory in rats after neonatal mPFC lesion. Eur Neuropsychopharmacol, 17, 180-186.

Schneider, M., Schomig, E. & Leweke, F.M. (2008) Acute and chronic cannabinoid treatment differentially

affects recognition memory and social behavior in pubertal and adult rats. Addict Biol, 13, 345-357.

Scoville, W.B. & Milner, B. (1957) Loss of recent memory after bilateral hippocampal lesions. J Neurol

Neurosurg Psychiatry, 20, 11-21.

Selley, D.E., Stark, S., Sim, L.J. & Childers, S.R. (1996) Cannabinoid receptor stimulation of guanosine-5'-O-(3-

[35S]thio)triphosphate binding in rat brain membranes. Life Sci, 59, 659-668.

Serpa, A., Ribeiro, J.A. & Sebastiao, A.M. (2009) Cannabinoid CB(1) and adenosine A(1) receptors

independently inhibit hippocampal synaptic transmission. Eur J Pharmacol, 623, 41-46.

Sharp, P.E., Blair, H.T., Etkin, D. & Tzanetos, D.B. (1995) Influences of vestibular and visual motion

information on the spatial firing patterns of hippocampal place cells. J Neurosci, 15, 173-189.

Shen, M. & Thayer, S.A. (1998) The cannabinoid agonist Win55,212-2 inhibits calcium channels by receptor-

mediated and direct pathways in cultured rat hippocampal neurons. Brain Res, 783, 77-84.

Shen, M., Piser, T.M., Seybold, V.S. & Thayer, S.A. (1996) Cannabinoid receptor agonists inhibit glutamatergic

synaptic transmission in rat hippocampal cultures. J Neurosci, 16, 4322-4334.

Shire, D., Carillon, C., Kaghad, M., Calandra, B., Rinaldi-Carmona, M., Le Fur, G., Caput, D. & Ferrara, P.

(1995) An amino-terminal variant of the central cannabinoid receptor resulting from alternative splicing. J

Biol Chem, 270, 3726-3731.

Shoaib, M. (2008) The cannabinoid antagonist AM251 attenuates nicotine self-administration and nicotine-

seeking behaviour in rats. Neuropharmacology, 54, 438-444.

Sim-Selley, L.J., Brunk, L.K. & Selley, D.E. (2001) Inhibitory effects of SR141716A on G-protein activation in

rat brain. Eur J Pharmacol, 414, 135-143.

Sim, L.J., Hampson, R.E., Deadwyler, S.A. & Childers, S.R. (1996) Effects of chronic treatment with delta9-

tetrahydrocannabinol on cannabinoid-stimulated [35S]GTPgammaS autoradiography in rat brain. J

Neurosci, 16, 8057-8066.

Sim, L.J., Selley, D.E. & Childers, S.R. (1995) In vitro autoradiography of receptor-activated G proteins in rat

References 198

brain by agonist-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate binding. Proc Natl Acad Sci U S A,

92, 7242-7246.

Simantov, R., Crispino, M., Hoe, W., Broutman, G., Tocco, G., Rothstein, J.D. & Baudry, M. (1999) Changes in

expression of neuronal and glial glutamate transporters in rat hippocampus following kainate-induced

seizure activity. Brain Res Mol Brain Res, 65, 112-123.

Sink, K.S., McLaughlin, P.J., Wood, J.A., Brown, C., Fan, P., Vemuri, V.K., Peng, Y., Olszewska, T., Thakur,

G.A., Makriyannis, A., Parker, L.A. & Salamone, J.D. (2008) The novel cannabinoid CB1 receptor neutral

antagonist AM4113 suppresses food intake and food-reinforced behavior but does not induce signs of

nausea in rats. Neuropsychopharmacology, 33, 946-955.

Sirkin, D.W., Precht, W. & Courjon, J.H. (1984) Initial, rapid phase of recovery from unilateral vestibular lesion

in rat not dependent on survival of central portion of vestibular nerve. Brain Res, 302, 245-256.

Sjostrom, P.J., Turrigiano, G.G. & Nelson, S.B. (2003) Neocortical LTD via coincident activation of presynaptic

NMDA and cannabinoid receptors. Neuron, 39, 641-654.

Skaggs, W.E. (1993) An information-theoretic approach to deciphering the hippocampal code. In Hanson, S.J.,

Cowan, J. D. & Giles, C. L. (ed) Advances in neural information processing systems. Morgan Kaufmann,

San Mateo, CA, pp. P 1030-1037.

Skaper, S.D., Buriani, A., Dal Toso, R., Petrelli, L., Romanello, S., Facci, L. & Leon, A. (1996) The ALIAmide

palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate

paradigm of excitotoxic death in cerebellar granule neurons. Proc Natl Acad Sci U S A, 93, 3984-3989.

Smith, P.F. & Curthoys, I.S. (1988a) Neuronal activity in the contralateral medial vestibular nucleus of the

guinea pig following unilateral labyrinthectomy. Brain Res, 444, 295-307.

Smith, P.F. & Curthoys, I.S. (1988b) Neuronal activity in the ipsilateral medial vestibular nucleus of the guinea

pig following unilateral labyrinthectomy. Brain Res, 444, 308-319.

Smith, P.F. & Curthoys, I.S. (1989) Mechanisms of recovery following unilateral labyrinthectomy: a review.

Brain Res Brain Res Rev, 14, 155-180.

Smith, P.F. (1997) Vestibular-hippocampal interactions. Hippocampus, 7, 465-471.

Smith, P.F., Brandt, T., Strupp, M., Darlington, C.L. & Zheng, Y. (2009) Balance before reason in rats and

humans. Ann N Y Acad Sci, 1164, 127-133.

Smith, P.F., Darlington, C.L. & Curthoys, I.S. (1986) The effect of visual deprivation on vestibular compensation

in the guinea pig. Brain Res, 364, 195-198.

Smith, P.F., Darlington, C.L. & Zheng, Y. (2010) Move it or lose it--is stimulation of the vestibular system

necessary for normal spatial memory? Hippocampus, 20, 36-43.

Smith, P.F., Geddes, L.H., Baek, J.-H., Darlington, C.L., and Zheng, Y. (2010, in press) Modulation of memory

in humans by vestibular lesions and galvanic vestibular stimulation. Front Neur.

Smith, P.F., Horii, A., Russell, N., Bilkey, D.K., Zheng, Y., Liu, P., Kerr, D.S. & Darlington, C.L. (2005a) The

effects of vestibular lesions on hippocampal function in rats. Prog Neurobiol, 75, 391-405.

Smith, P.F., Zheng, Y., Goddard, M. & Darlington, C. L. (2007) Cognitive Disorders Research Trends. Nova

Science Publishers.

Smith, P.F., Zheng, Y., Horii, A. & Darlington, C.L. (2005b) Does vestibular damage cause cognitive dysfunction

in humans? J Vestib Res, 15, 1-9.

References 199

Solinas, M. & Goldberg, S.R. (2005) Motivational effects of cannabinoids and opioids on food reinforcement

depend on simultaneous activation of cannabinoid and opioid systems. Neuropsychopharmacology, 30,

2035-2045.

Solowij, N. (1995) Do cognitive impairments recover following cessation of cannabis use? Life Sci, 56, 2119-

2126.

Solowij, N., Stephens, R.S., Roffman, R.A., Babor, T., Kadden, R., Miller, M., Christiansen, K., McRee, B. &

Vendetti, J. (2002) Cognitive functioning of long-term heavy cannabis users seeking treatment. JAMA, 287,

1123-1131.

Song, C. & Howlett, A.C. (1995) Rat brain cannabinoid receptors are N-linked glycosylated proteins. Life Sci, 56,

1983-1989.

Spiers, H.J., Burgess, N., Maguire, E.A., Baxendale, S.A., Hartley, T., Thompson, P.J. & O'Keefe, J. (2001)

Unilateral temporal lobectomy patients show lateralized topographical and episodic memory deficits in a

virtual town. Brain, 124, 2476-2489.

Squire, L.R. (1992) Memory and the hippocampus: a synthesis from findings with rats, monkeys, and humans.

Psychol Rev, 99, 195-231.

Squire, L.R. & Zola-Morgan, S. (1991) The medial temporal lobe memory system. Science, 253, 1380-1386.

Squire, L.R. & Zola, S.M. (1996) Structure and function of declarative and nondeclarative memory systems.


Squire, L.R., Stark, C.E. & Clark, R.E. (2004) The medial temporal lobe. Annu Rev Neurosci, 27, 279-306.

Staab, J.P. & Ruckenstein, M.J. (2003) Which comes first? Psychogenic dizziness versus otogenic anxiety.

Laryngoscope, 113, 1714-1718.

Stackman, R.W. & Herbert, A.M. (2002) Rats with lesions of the vestibular system require a visual landmark for

spatial navigation. Behav Brain Res, 128, 27-40.

Stackman, R.W. & Taube, J.S. (1997) Firing properties of head direction cells in the rat anterior thalamic nucleus:

dependence on vestibular input. J Neurosci, 17, 4349-4358.

Stackman, R.W., Clark, A.S. & Taube, J.S. (2002) Hippocampal spatial representations require vestibular input.


Steffenach, H.A., Sloviter, R.S., Moser, E.I. & Moser, M.B. (2002) Impaired retention of spatial memory after

transection of longitudinally oriented axons of hippocampal CA3 pyramidal cells. Proc Natl Acad Sci U S

A, 99, 3194-3198.

Stella, N., Schweitzer, P. & Piomelli, D. (1997) A second endogenous cannabinoid that modulates long-term

potentiation. Nature, 388, 773-778.

Stubley-Weatherly, L., Harding, J.W. & Wright, J.W. (1996) Effects of discrete kainic acid-induced hippocampal

lesions on spatial and contextual learning and memory in rats. Brain Res, 716, 29-38.

Stupien, G., Florian, C. & Roullet, P. (2003) Involvement of the hippocampal CA3-region in acquisition and in

memory consolidation of spatial but not in object information in mice. Neurobiol Learn Mem, 80, 32-41.

Suàrez, J., Bermudez-Silva, F.J., Mackie, K., Ledent, C., Zimmer, A., Cravatt, B.F. & de Fonseca, F.R. (2008)

Immunohistochemical description of the endogenous cannabinoid system in the rat cerebellum and

functionally related nuclei. J Comp Neurol, 509, 400-421.

Suàrez, J., Llorente, R., Romero-Zerbo, S.Y., Mateos, B., Bermudez-Silva, F.J., de Fonseca, F.R. & Viveros, M.P.

References 200

(2009) Early maternal deprivation induces gender-dependent changes on the expression of hippocampal

CB(1) and CB(2) cannabinoid receptors of neonatal rats. Hippocampus, 19, 623-632.

Suenaga, T. & Ichitani, Y. (2008) Effects of hippocampal administration of a cannabinoid receptor agonist WIN

55,212-2 on spontaneous object and place recognition in rats. Behav Brain Res, 190, 248-252.

Suenaga, T., Kaku, M. & Ichitani, Y. (2008) Effects of intrahippocampal cannabinoid receptor agonist and

antagonist on radial maze and T-maze delayed alternation performance in rats. Pharmacol Biochem Behav,

91, 91-96.

Sugiura, T. & Waku, K. (2002) Cannabinoid receptors and their endogenous ligands. J Biochem, 132, 7-12.

Sugiura, T. (2009) Physiological roles of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand.

Biofactors, 35, 88-97.

Sugiura, T., Kishimoto, S., Oka, S. & Gokoh, M. (2006) Biochemistry, pharmacology and physiology of 2-

arachidonoylglycerol, an endogenous cannabinoid receptor ligand. Prog Lipid Res, 45, 405-446.

Sugiura, T., Kobayashi, Y., Oka, S. & Waku, K. (2002) Biosynthesis and degradation of anandamide and 2-

arachidonoylglycerol and their possible physiological significance. Prostaglandins Leukot Essent Fatty

Acids, 66, 173-192.

Sugiura, T., Kondo, S., Kishimoto, S., Miyashita, T., Nakane, S., Kodaka, T., Suhara, Y., Takayama, H. & Waku,

K. (2000) Evidence that 2-arachidonoylglycerol but not N-palmitoylethanolamine or anandamide is the

physiological ligand for the cannabinoid CB2 receptor. Comparison of the agonistic activities of various

cannabinoid receptor ligands in HL-60 cells. J Biol Chem, 275, 605-612.

Sugiura, T., Kondo, S., Sukagawa, A., Nakane, S., Shinoda, A., Itoh, K., Yamashita, A. & Waku, K. (1995) 2-

Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res

Commun, 215, 89-97.

Sutherland, B.A., Rahman, R.M., Clarkson, A.N., Shaw, O.M., Nair, S.M. & Appleton, I. (2009) Cerebral heme

oxygenase 1 and 2 spatial distribution is modulated following injury from hypoxia-ischemia and middle

cerebral artery occlusion in rats. Neurosci Res, 65, 326-334.

Sutherland, R.J. & Dyck, R. H. (1984) Place navigation by rats in a swimming pool. Can J Pschol, 38, 322-347.

Suzuki, M., Kitano, H., Ito, R., Kitanishi, T., Yazawa, Y., Ogawa, T., Shiino, A. & Kitajima, K. (2001) Cortical

and subcortical vestibular response to caloric stimulation detected by functional magnetic resonance

imaging. Brain Res Cogn Brain Res, 12, 441-449.

Szabo, B., Siemes, S. & Wallmichrath, I. (2002) Inhibition of GABAergic neurotransmission in the ventral

tegmental area by cannabinoids. Eur J Neurosci, 15, 2057-2061.

Szabo, B., Wallmichrath, I., Mathonia, P. & Pfreundtner, C. (2000) Cannabinoids inhibit excitatory

neurotransmission in the substantia nigra pars reticulata. Neuroscience, 97, 89-97.

Tagliaferro, P., Javier Ramos, A., Onaivi, E.S., Evrard, S.G., Lujilde, J. & Brusco, A. (2006) Neuronal

cytoskeleton and synaptic densities are altered after a chronic treatment with the cannabinoid receptor

agonist WIN 55,212-2. Brain Res, 1085, 163-176.

Takahashi, K.A. & Castillo, P.E. (2006) The CB1 cannabinoid receptor mediates glutamatergic synaptic

suppression in the hippocampus. Neuroscience, 139, 795-802.

Takahashi, R.N., Pamplona, F.A. & Fernandes, M.S. (2005) The cannabinoid antagonist SR141716A facilitates

memory acquisition and consolidation in the mouse elevated T-maze. Neurosci Lett, 380, 270-275.

References 201

Talpos, J.C., Dias, R., Bussey, T.J. & Saksida, L.M. (2008) Hippocampal lesions in rats impair learning and

memory for locations on a touch-sensitive computer screen: the "ASAT" task. Behav Brain Res, 192, 216-

225.

Taylor, H.G. (1998) Analysis of the medical use of marijuana and its societal implications. J Am Pharm Assoc

(Wash), 38, 220-227.

Tchernichovski, O. & Benjamini, Y. (1998) The dynamics of long-term exploration in the rat. Part II. An

analytical model of the kinematic structure of rat exploratory behavior. Biol Cybern, 78, 433-440.

Terranova, J.P., Michaud, J.C., Le Fur, G. & Soubrie, P. (1995) Inhibition of long-term potentiation in rat

hippocampal slices by anandamide and WIN55212-2: reversal by SR141716 A, a selective antagonist of

CB1 cannabinoid receptors. Naunyn Schmiedebergs Arch Pharmacol, 352, 576-579.

Terranova, J.P., Storme, J.J., Lafon, N., Perio, A., Rinaldi-Carmona, M., Le Fur, G. & Soubrie, P. (1996)

Improvement of memory in rodents by the selective CB1 cannabinoid receptor antagonist, SR 141716.


Terrazas, A., Krause, M., Lipa, P., Gothard, K.M., Barnes, C.A. & McNaughton, B.L. (2005) Self-motion and the

hippocampal spatial metric. J Neurosci, 25, 8085-8096.

Thomas, B.F., Gilliam, A.F., Burch, D.F., Roche, M.J. & Seltzman, H.H. (1998) Comparative receptor binding

analyses of cannabinoid agonists and antagonists. J Pharmacol Exp Ther, 285, 285-292.

Tsou, K., Brown, S., Sanudo-Pena, M.C., Mackie, K. & Walker, J.M. (1998) Immunohistochemical distribution

of cannabinoid CB1 receptors in the rat central nervous system. Neuroscience, 83, 393-411.

Tsou, K., Mackie, K., Sanudo-Pena, M.C. & Walker, J.M. (1999) Cannabinoid CB1 receptors are localized

primarily on cholecystokinin-containing GABAergic interneurons in the rat hippocampal formation.


Turrigiano, G.G. (2008) The self-tuning neuron: synaptic scaling of excitatory synapses. Cell, 135, 422-435.

Turu, G. & Hunyady, L. (2010) Signal transduction of the CB1 cannabinoid receptor. J Mol Endocrinol, 44, 75-

85.

Valverde, O., Karsak, M. & Zimmer, A. (2005) Analysis of the endocannabinoid system by using CB1

cannabinoid receptor knockout mice. Handb Exp Pharmacol, 117-145.

van der Meer, M.A., Knierim, J.J., Yoganarasimha, D., Wood, E.R. & van Rossum, M.C. (2007) Anticipation in

the rodent head direction system can be explained by an interaction of head movements and vestibular

firing properties. J Neurophysiol, 98, 1883-1897.

van der Stelt, M., Mazzola, C., Esposito, G., Matias, I., Petrosino, S., De Filippis, D., Micale, V., Steardo, L.,

Drago, F., Iuvone, T. & Di Marzo, V. (2006) Endocannabinoids and beta-amyloid-induced neurotoxicity in

vivo: effect of pharmacological elevation of endocannabinoid levels. Cell Mol Life Sci, 63, 1410-1424.

Van Sickle, M.D., Duncan, M., Kingsley, P.J., Mouihate, A., Urbani, P., Mackie, K., Stella, N., Makriyannis, A.,

Piomelli, D., Davison, J.S., Marnett, L.J., Di Marzo, V., Pittman, Q.J., Patel, K.D. & Sharkey, K.A. (2005)

Identification and functional characterization of brainstem cannabinoid CB2 receptors. Science, 310, 329-

332.

Vander Wall, S.B. (1982) An experimental analysis of cache recovery in Clark's nutcracker. Anim Behav, 30, 84-

94.

Varma, N., Carlson, G.C., Ledent, C. & Alger, B.E. (2001) Metabotropic glutamate receptors drive the

References 202

endocannabinoid system in hippocampus. J Neurosci, 21, RC188.

Varvel, S.A. & Lichtman, A.H. (2002) Evaluation of CB1 receptor knockout mice in the Morris water maze. J

Pharmacol Exp Ther, 301, 915-924.

Varvel, S.A., Anum, E.A. & Lichtman, A.H. (2005) Disruption of CB(1) receptor signaling impairs extinction of

spatial memory in mice. Psychopharmacology (Berl), 179, 863-872.

Vinod, K.Y., Arango, V., Xie, S., Kassir, S.A., Mann, J.J., Cooper, T.B. & Hungund, B.L. (2005) Elevated levels

of endocannabinoids and CB1 receptor-mediated G-protein signaling in the prefrontal cortex of alcoholic

suicide victims. Biol Psychiatry, 57, 480-486.

Vitte, E., Derosier, C., Caritu, Y., Berthoz, A., Hasboun, D. & Soulie, D. (1996) Activation of the hippocampal

formation by vestibular stimulation: a functional magnetic resonance imaging study. Exp Brain Res, 112,

523-526.

Waespe, W., Schwarz, U., Wolfenburger, M. (1992) Firing characteristics of vestibular nuclei neurons in the alert

monkey after bilateral vestibular neurectomy. Experimental Brain Research, 89, 311-322.

Wallace, D.G. & Whishaw, I.Q. (2003) NMDA lesions of Ammon's horn and the dentate gyrus disrupt the direct

and temporally paced homing displayed by rats exploring a novel environment: evidence for a role of the

hippocampus in dead reckoning. Eur J Neurosci, 18, 513-523.

Wallace, M.J., Blair, R.E., Falenski, K.W., Martin, B.R. & DeLorenzo, R.J. (2003) The endogenous cannabinoid

system regulates seizure frequency and duration in a model of temporal lobe epilepsy. J Pharmacol Exp

Ther, 307, 129-137.

Wallace, D.G., Gorny, B. & Whishaw, I.Q. (2002a) Rats can track odors, other rats, and themselves: implications

for the study of spatial behavior. Behav Brain Res, 131, 185-192.

Wallace, D.G., Hines, D.J. & Whishaw, I.Q. (2002b) Quantification of a single exploratory trip reveals

hippocampal formation mediated dead reckoning. J Neurosci Methods, 113, 131-145.

Wallace, D.G., Hines, D.J., Pellis, S.M. & Whishaw, I.Q. (2002c) Vestibular information is required for dead

reckoning in the rat. J Neurosci, 22, 10009-10017.

Wallmichrath, I. & Szabo, B. (2002) Analysis of the effect of cannabinoids on GABAergic neurotransmission in

the substantia nigra pars reticulata. Naunyn Schmiedebergs Arch Pharmacol, 365, 326-334.

Walsh, S., Mnich, K., Mackie, K., Gorman, A.M., Finn, D.P. & Dowd, E. (2010) Loss of cannabinoid CB1

receptor expression in the 6-hydroxydopamine-induced nigrostriatal terminal lesion model of Parkinson's

disease in the rat. Brain Res Bull, 81, 543-548.

Wang, S.J. (2003) Cannabinoid CB1 receptor-mediated inhibition of glutamate release from rat hippocampal

synaptosomes. Eur J Pharmacol, 469, 47-55.

Watanabe, S. & Yoshida, M. (2007) Auditory cued spatial learning in mice. Physiol Behav, 92, 906-910.

Wegener, N., Kuhnert, S., Thuns, A., Roese, R. & Koch, M. (2008) Effects of acute systemic and intra-cerebral

stimulation of cannabinoid receptors on sensorimotor gating, locomotion and spatial memory in rats.


Weinstein, A., Brickner, O., Lerman, H., Greemland, M., Bloch, M., Lester, H., Chisin, R., Sarne, Y., Mechoulam,

R., Bar-Hamburger, R., Freedman, N. & Even-Sapir, E. (2008) A study investigating the acute dose-

response effects of 13 mg and 17 mg Delta 9- tetrahydrocannabinol on cognitive-motor skills, subjective

and autonomic measures in regular users of marijuana. J Psychopharmacol, 22, 441-451.

References 203

West, M.J. & Gundersen, H.J. (1990) Unbiased stereological estimation of the number of neurons in the human

hippocampus. J Comp Neurol, 296, 1-22.

West, M.J., Slomianka, L. & Gundersen, H.J. (1991) Unbiased stereological estimation of the total number of

neurons in thesubdivisions of the rat hippocampus using the optical fractionator. Anat Rec, 231, 482-497.

Whishaw, I.Q. & Brooks, B.L. (1999) Calibrating space: exploration is important for allothetic and idiothetic

navigation. Hippocampus, 9, 659-667.

Whishaw, I.Q. & Gorny, B. (1999) Path integration absent in scent-tracking fimbria-fornix rats: evidence for

hippocampal involvement in "sense of direction" and "sense of distance" using self-movement cues. J

Neurosci, 19, 4662-4673.

Whishaw, I.Q. & Maaswinkel, H. (1998) Rats with fimbria-fornix lesions are impaired in path integration: a role

for the hippocampus in "sense of direction". J Neurosci, 18, 3050-3058.

Whishaw, I.Q. & Tomie, J.A. (1997) Piloting and dead reckoning dissociated by fimbria-fornix lesions in a rat

food carrying task. Behav Brain Res, 89, 87-97.

Whishaw, I.Q. (1985) Formation of a place learning-set by the rat: a new paradigm for neurobehavioral studies.

Physiol Behav, 35, 139-143.

Whishaw, I.Q. (1998) Spatial mapping takes time. Hippocampus, 8, 122-130.

Whishaw, I.Q., Coles, B.L. & Bellerive, C.H. (1995) Food carrying: a new method for naturalistic studies of

spontaneous and forced alternation. J Neurosci Methods, 61, 139-143.

Whishaw, I.Q., Hines, D.J. & Wallace, D.G. (2001) Dead reckoning (path integration) requires the hippocampal

formation: evidence from spontaneous exploration and spatial learning tasks in light (allothetic) and dark

(idiothetic) tests. Behav Brain Res, 127, 49-69.

Whishaw, I.Q., Oddie, S.D., McNamara, R.K., Harris, T.L. & Perry, B.S. (1990) Psychophysical methods for

study of sensory-motor behavior using a food-carrying (hoarding) task in rodents. J Neurosci Methods, 32,

123-133.

Wiener, S.I., Korshunov, V.A., Garcia, R. & Berthoz, A. (1995) Inertial, substratal and landmark cue control of

hippocampal CA1 place cell activity. Eur J Neurosci, 7, 2206-2219.

Wilkinson, D., Nicholls, S., Pattenden, C., Kilduff, P. & Milberg, W. (2008) Galvanic vestibular stimulation

speeds visual memory recall. Exp Brain Res, 189, 243–248.

Wilkinson, D., Zubko, O., DeGutis, J., Milberg, W. & Potter, J. (2010) Improvement of a figure copying deficit

during subsensory galvanic vestibular stimulation. J. Neuropsychol, 4, 107-118.

Williams, S.M., Bryan-Lluka, L.J. & Pow, D.V. (2005) Quantitative analysis of immunolabeling for serotonin

and for glutamate transporters after administration of imipramine and citalopram. Brain Res, 1042, 224-

232.

Wilson, R.I. & Nicoll, R.A. (2001) Endogenous cannabinoids mediate retrograde signalling at hippocampal

synapses. Nature, 410, 588-592.

Wilson, V.J. & Melvill Jones, G. (1979) Mammalian vestibular physiology. Plenum Press, New York and London.

Winer, B.J., Brown, D. R. & Michels, K. M. (ed) (1991) Statistical Principles in Experimental Design. McGraw

Hill, New York.

Winsauer, P.J., Lambert, P. & Moerschbaecher, J.M. (1999) Cannabinoid ligands and their effects on learning and

performance in rhesus monkeys. Behav Pharmacol, 10, 497-511.

References 204

Winters, B.D., Forwood, S.E., Cowell, R.A., Saksida, L.M. & Bussey, T.J. (2004) Double dissociation between

the effects of peri-postrhinal cortex and hippocampal lesions on tests of object recognition and spatial

memory: heterogeneity of function within the temporal lobe. J Neurosci, 24, 5901-5908.

Winters, B.D., Saksida, L.M. & Bussey, T.J. (2008) Object recognition memory: neurobiological mechanisms of

encoding, consolidation and retrieval. Neurosci Biobehav Rev, 32, 1055-1070.

Wise, L.E., Thorpe, A.J. & Lichtman, A.H. (2009) Hippocampal CB(1) receptors mediate the memory impairing

effects of Delta(9)-tetrahydrocannabinol. Neuropsychopharmacology, 34, 2072-2080.

Wolf, S.A., Bick-Sander, A., Fabel, K., Leal-Galicia, P., Tauber, S., Ramirez-Rodriguez, G., Muller, A., Melnik,

A., Waltinger, T.P., Ullrich, O. & Kempermann, G. (2010) Cannabinoid receptor CB1 mediates baseline

and activity-induced survival of new neurons in adult hippocampal neurogenesis. Cell Commun Signal, 8,

12.

Wolff, M.C. & Leander, J.D. (2003) SR141716A, a cannabinoid CB1 receptor antagonist, improves memory in a

delayed radial maze task. Eur J Pharmacol, 477, 213-217.

Wong, D.F., Kuwabara, H., Horti, A.G., Raymont, V., Brasic, J., Guevara, M., Ye, W., Dannals, R.F., Ravert, H.T.,

Nandi, A., Rahmim, A., Ming, J.E., Grachev, I., Roy, C. & Cascella, N. (2010) Quantification of cerebral

cannabinoid receptors subtype 1 (CB1) in healthy subjects and schizophrenia by the novel PET radioligand

[(11)C]OMAR. Neuroimage.

Yakushin, S.B., Raphan, T., Buttner-Ennever, J.A., Suzuki, J. & Cohen, B. (2005) Spatial properties of central

vestibular neurons of monkeys after bilateral lateral canal nerve section. J Neurophysiol, 94, 3860-3871.

Yamanaka, T., Him, A., Cameron, S.A. & Dutia, M.B. (2000) Rapid compensatory changes in GABA receptor

efficacy in rat vestibular neurones after unilateral labyrinthectomy. J Physiol, 523 Pt 2, 413-424.

Yim, T.T., Hong, N.S., Ejaredar, M., McKenna, J.E. & McDonald, R.J. (2008) Post-training CB1 cannabinoid

receptor agonist activation disrupts long-term consolidation of spatial memories in the hippocampus.


Yoshida, T., Hashimoto, K., Zimmer, A., Maejima, T., Araishi, K. & Kano, M. (2002) The cannabinoid CB1

receptor mediates retrograde signals for depolarization-induced suppression of inhibition in cerebellar

Purkinje cells. J Neurosci, 22, 1690-1697.

Zajonc, T.P. & Roland, P.S. (2005) Vertigo and motion sickness. Part I: vestibular anatomy and physiology. Ear

Nose Throat J, 84, 581-584.

Zar, J.H. (4th ed) (1999) Biostatistical analysis. Prentice Hall, Upper Saddle River, New Jersey.

Zennou-Azogui, Y., Borel, L., Lacour, M., Ez-Zaher, L. & Ouaknine, M. (1993) Recovery of head postural

control following unilateral vestibular neurectomy in the cat. Neck muscle activity and neuronal correlates

in Deiters' nuclei. Acta Otolaryngol Suppl, 509, 1-19.

Zhang, M., Martin, B.R., Adler, M.W., Razdan, R.K., Ganea, D. & Tuma, R.F. (2008) Modulation of the balance

between cannabinoid CB(1) and CB(2) receptor activation during cerebral ischemic/reperfusion injury.


Zhang, M., Martin, B.R., Adler, M.W., Razdan, R.K., Jallo, J.I. & Tuma, R.F. (2007) Cannabinoid CB(2)

receptor activation decreases cerebral infarction in a mouse focal ischemia/reperfusion model. J Cereb

Blood Flow Metab, 27, 1387-1396.

Zhang, R., Ashton, J., Horii, A., Darlington, C.L. & Smith, P.F. (2005a) Immunocytochemical and stereological

References 205

analysis of GABA(B) receptor subunit expression in the rat vestibular nucleus following unilateral

vestibular deafferentation. Brain Res, 1037, 107-113.

Zhang, R., Smith, P.F. & Darlington, C.L. (2005b) Immunocytochemical and stereological study of

glucocorticoid receptors in rat medial vestibular nucleus neurons and the effects of unilateral vestibular

deafferentation. Acta Otolaryngol, 125, 1258-1264.

Zhao, W., Cavallaro, S., Gusev, P. & Alkon, D.L. (2000) Nonreceptor tyrosine protein kinase pp60c-src in spatial

learning: synapse-specific changes in its gene expression, tyrosine phosphorylation, and protein-protein

interactions. Proc Natl Acad Sci U S A, 97, 8098-8103.

Zheng, Y., Balabhadrapatruni, S., Masumura, C., Munro, O., Darlington, C.L. & Smith, P.F. (2009a) Bilateral

vestibular deafferentation causes deficits in a 5-choice serial reaction time task in rats. Behav Brain Res,

203, 113-117.

Zheng, Y., Darlington, C.L. & Smith, P.F. (2004) Bilateral labyrinthectomy causes long-term deficit in object

recognition in rat. Neuroreport, 15, 1913-1916.

Zheng, Y., Darlington, C.L. & Smith, P.F. (2006) Impairment and recovery on a food foraging task following

unilateral vestibular deafferentation in rats. Hippocampus, 16, 368-378.

Zheng, Y., Goddard, M., Darlington, C.L. & Smith, P.F. (2007) Bilateral vestibular deafferentation impairs

performance in a spatial forced alternation task in rats. Hippocampus, 17, 253-256.

Zheng, Y., Goddard, M., Darlington, C.L. & Smith, P.F. (2008) Effects of bilateral vestibular deafferentation on

anxiety-related behaviours in Wistar rats. Behav Brain Res, 193, 55-62.

Zheng, Y., Goddard, M., Darlington, C.L. & Smith, P.F. (2009b) Long-term deficits on a foraging task after

bilateral vestibular deafferentation in rats. Hippocampus, 19, 480-486.

Zheng, Y., Horii, A., Appleton, I., Darlington, C.L. & Smith, P.F. (2001) Damage to the vestibular inner ear

causes long-term changes in neuronal nitric oxide synthase expression in the rat hippocampus.


Zheng, Y., Kerr, D.S., Darlington, C.L. & Smith, P.F. (2003) Unilateral inner ear damage results in lasting

changes in hippocampal CA1 field potentials in vitro. Hippocampus, 13, 873-878.

Zheng, Y., Mason-Parker, S.E., Logan, B., Darlington, C.L., Smith, P.F. & Abraham, W.C. (2010) Hippocampal

synaptic transmission and LTP in vivo are intact following bilateral vestibular deafferentation in the rat.


Zimmer, A., Zimmer, A.M., Hohmann, A.G., Herkenham, M. & Bonner, T.I. (1999) Increased mortality,

hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice. Proc Natl Acad Sci U S A, 96,

5780-5785.

Zimmerberg, B., Glick, S.D. & Jarvik, M.E. (1971) Impairment of recent memory by marihuana and THC in

rhesus monkeys. Nature, 233, 343-345.

Zingler, V.C., Weintz, E., Jahn, K., Mike, A., Huppert, D., Rettinger, N., Brandt, T. & Strupp, M. (2008) Follow-

up of vestibular function in bilateral vestibulopathy. J Neurol Neurosurg Psychiatry, 79, 284-288.

Zola, S.M., Squire, L.R., Teng, E., Stefanacci, L., Buffalo, E.A. & Clark, R.E. (2000) Impaired recognition

memory in monkeys after damage limited to the hippocampal region. J Neurosci, 20, 451-463.

Zola-Morgan, S. & Squire, L.R. (1985) Medial temporal lesions in monkeys impair memory on a variety of tasks

sensitive to human amnesia. Behav Neurosci, 99, 22-34.

References 206

Zola-Morgan, S. & Squire, L.R. (1986) Memory impairment in monkeys following lesions limited to the

hippocampus. Behav Neurosci, 100, 155-160.

Zola-Morgan, S., Squire, L.R., Clower, R.P. & Rempel, N.L. (1993) Damage to the perirhinal cortex exacerbates

memory impairment following lesions to the hippocampal formation. J Neurosci, 13, 251-265.

zu Eulenburg, P., Stoeter, P. & Dieterich, M. (2010) Voxel-based morphometry depicts central compensation

after vestibular neuritis. Ann Neurol, 68, 241-249.

The New Beginning 206

This unforgettable journey has taught me

Perseverance to endure difficult times,

humility in gaining knowledge,

Diligence to work consistently.

But by the grace of God I am what I am, and His grace to me was not without effect.

<1 Corinthians 15:10>

CHANGES IN THE ENDOCANNABINOID YSTEM

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