Defective dentate nucleus GABA receptors in essential tremor - Brain

BRAINA JOURNAL OF NEUROLOGY

Defective dentate nucleus GABA receptorsin essential tremorSarah Paris-Robidas,1,2,* Elodie Brochu,1,2,* Marion Sintes,1,2 Vincent Emond,1,2

Melanie Bousquet,1,2 Milene Vandal,1,2 Mireille Pilote,1,2 Cyntia Tremblay,1,2

Therese Di Paolo,1,2 Ali H. Rajput,3 Alex Rajput3 and Frederic Calon1,2

1 Faculty of Pharmacy, Laval University, Quebec, QC G1V 0A6, Canada

2 Centre Hospitalier de l’Universite Laval (CHUL) Research Centre, Quebec, QC G1V 4G2, Canada

3 Division of Neurology, Royal University Hospital, University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada

*These authors contributed equally to this work.

Correspondence to: Dr Frederic Calon,

Centre Hospitalier de l’Universite Laval (CHUL) Research Centre,

Room T205, 2705 Laurier Blvd,

Quebec, QC G1V 4G2, Canada

E-mail: [email protected]

The development of new treatments for essential tremor, the most frequent movement disorder, is limited by a poor under-

standing of its pathophysiology and the relative paucity of clinicopathological studies. Here, we report a post-mortem decrease

in GABAA (35% reduction) and GABAB (22–31% reduction) receptors in the dentate nucleus of the cerebellum from individuals

with essential tremor, compared with controls or individuals with Parkinson’s disease, as assessed by receptor-binding auto-

radiography. Concentrations of GABAB receptors in the dentate nucleus were inversely correlated with the duration of essential

tremor symptoms (r2 = 0.44, P5 0.05), suggesting that the loss of GABAB receptors follows the progression of the disease. In

situ hybridization experiments also revealed a diminution of GABAB(1a + b) receptor messenger RNA in essential tremor (#27%).

In contrast, no significant changes of GABAA and GABAB receptors (protein and messenger RNA), GluN2B receptors, cytochrome

oxidase-1 or GABA concentrations were detected in molecular or granular layers of the cerebellar cortex. It is proposed that a

decrease in GABA receptors in the dentate nucleus results in disinhibition of cerebellar pacemaker output activity, propagating

along the cerebello-thalamo-cortical pathways to generate tremors. Correction of such defective cerebellar GABAergic drive

could have a therapeutic effect in essential tremor.

Keywords: essential tremor; cerebellum; GABA receptors; dentate nucleus; deep cerebellar nuclei

Abbreviations: GABA = �-aminobutyric acid

IntroductionMore than 10 million Americans suffer from essential tremor,

making it the most prevalent adult-onset movement disorder

(Moghal et al., 1994; Louis and Ferreira, 2010). Essential tremor

is characterized by a bilateral action tremor affecting predomin-

antly the arms, the head and/or the voice (Rajput et al., 2004;

Louis, 2005). Formerly regarded as benign, the symptoms of

essential tremor evolve gradually and can become very disturbing

for patients. Recent studies have emphasized the presence of

doi:10.1093/brain/awr301 Brain 2012: 135; 105–116 | 105

Received May 16, 2011. Revised September 2, 2011. Accepted September 12, 2011. Advance Access publication November 26, 2011

� The Author (2011). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.

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abnormalities within �-aminobutyric acid (GABA)-ergic Purkinje

cells in a significant proportion of patients with essential tremor

(Louis et al., 2007, 2009, 2010; Axelrad et al., 2008; Shill et al.,

2008). Several lines of evidence dating back to the early 1970s

suggest that cerebellar dysfunctions reverberating through the

cerebello-thalamo-cortical pathway play a key role in essential

tremor (Deuschl et al., 2000; McAuley and Marsden, 2000;

Pinto et al., 2003; Elble and Deuschl, 2009; Schnitzler et al.,

2009).

The standard pharmaceutical care of essential tremor has essen-

tially not changed in the last 40 years and still relies on primidone

and propranolol, which display moderate efficacy to decrease the

amplitude of tremor in less than half of patients (Lyons et al.,

2003; Louis et al., 2010; Deuschl et al., 2011). The small

number of post-mortem studies performed on essential tremor

subjects is likely linked to the limited therapeutic arsenal available

today to treat essential tremor (Louis, 2005, 2009).

The fact that drugs acting on GABAA receptors, such as primi-

done, benzodiazepines or ethanol, decrease tremor amplitude sug-

gests that altered GABAergic neurotransmission is involved in

essential tremor. Although the exact location of this impaired

GABAergic current is unknown, GABA dysfunction in the cerebel-

lum has been suggested as an aetiological factor in essential tremor

(Deuschl et al., 2000; Kralic et al., 2005; Louis, 2005; Elble and

Deuschl, 2009). More specifically, post-mortem studies indicate

that cerebellar GABAergic Purkinje cells show increased numbers

of axonal swellings and a reduction in numbers in a significant pro-

portion of subjects with essential tremor (Louis et al., 2007, 2009;

Axelrad et al., 2008; Shill et al., 2008). Low levels of GABA have

been reported in the CSF of patients with essential tremor com-

pared with controls (Mally and Baranyi, 1994; Mally et al., 1996).

Moreover, toxins such as aflatrem, penitrem A or harmaline have

been proposed to induce tremor in rodents by interacting with

GABA receptors (Cavanagh et al., 1998; Miwa, 2007). In addition,

GABAA receptor �1 subunit knockout mice exhibit postural and

kinetic tremors, partly reproducing some features of essential

tremor (Kralic et al., 2005). Finally, a recent PET study revealed

decreased binding of 11C-flumazenil at the benzodiazepine recep-

tor site of the GABAA receptor in several brain regions, supporting

the possibility of a general GABAergic dysfunction in essential

tremor (Boecker et al., 2010).

To characterize GABAergic neurotransmission within the cere-

bellum of patients with essential tremor, we have conducted post-

mortem investigations of GABAA and GABAB receptors by auto-

radiography and in situ hybridization on horizontal cerebellar

sections. Since the major GABAergic tract within the cerebellum

connects Purkinje cells to deep cerebellar nuclei and all outgoing

information leaving the cerebellum is funnelled through the deep

cerebellar nuclei, it was essential to measure GABA receptor in this

structure. However, deep cerebellar nuclei are both very small and

irregularly shaped, and it is therefore technically challenging to

measure their protein or messenger RNA content with available

neuroimaging approaches or techniques requiring tissue homogen-

ates. Autoradiography and in situ hybridization were thus selected

because these techniques allow the 2D resolution necessary to

quantify the expression of these receptors in the deep cerebellar

nuclei, notably the dentate nucleus—the largest and one of the

most important subparts of the deep cerebellar nuclei. A group of

patients with essential tremor (n = 10) was compared with control

(n = 16) and patients with Parkinson’s disease (n = 10), taking ad-

vantage of a clinicopathological study in which detailed clinical

variables have been prospectively recorded by the same neurolo-

gists (A.H.R. and A.R.).

Materials and methods

Clinicopathological assessment ofpatientsPatients with essential tremor and Parkinson’s disease controls were all

followed at the Movement Disorders Clinic Saskatchewan at 6- or

12-month intervals (Rajput et al., 2004, 2009). The Movement

Disorders Clinic Saskatchewan has operated uninterrupted since

1968. Autopsy was restricted to those patients who have been as-

sessed by the Movement Disorders Clinic Saskatchewan neurologists

(A.H.R. and A.R.). The clinical diagnosis of essential tremor was made

as described previously (Rajput et al., 2004). All cases with essential

tremor had postural and/or kinetic tremor for several years. All cases

with Parkinson’s disease had resting tremor: two were tremor-

dominant cases and eight were classical (mixed) with approximately

equal severity of bradykinesia, rigidity and tremor (Rajput et al., 2009).

For this study, three main criteria were used to select subjects with

essential tremor: the absence of neuropathologically confirmed

Parkinson’s disease; the presence of dentate nucleus in the tissue block;

and age and gender matching with the two other groups. The severity

of tremor was recorded at each visit and was based on the visual

assessment of tremor amplitude at any site [Unified Parkinson’s

Disease Rating Scale (UPDRS III)] and the history of its impact on

daily activities (Rajput et al., 2004). As essential tremor is often mis-

diagnosed as parkinsonian tremor (Rajput et al., 2004; Jain et al.,

2006; Shahed and Jankovic, 2007; Minen and Louis, 2008;

Benito-Leon et al., 2009; Adler et al., 2011), tissue from patients with

Parkinson’s disease were included to identify changes that are specific

for essential tremor (i.e. kinetic tremor versus resting tremor). Clinical

data including the age and mode of onset, severity of the disease,

duration of disease, response to treatment and adverse effects of

treatment, were recorded prospectively after each clinical assessment.

General information such as other diagnosis, sex, cause and age of

death were also documented. All autopsies were done within 25 h of

death. Table 1 shows a summary of relevant information available

regarding the subjects involved in the study and more detailed infor-

mation is provided in Supplementary Table 1. The maximal severity of

tremor scores is given in Table 1 and Supplementary Table 1.

Tissue handling and processingDetails of autopsy procedures have been described previously (Rajput

et al., 2004, 2009). One half of the brain was histologically examined

by a neuropathologist using representative sections including: the cere-

bral cortex, hippocampus, caudate, lentiform nucleus, thalamus,

mid-brain, pons, medulla and cerebellum with dentate nucleus.

Standard stains including silver stain and �-synuclein, ubiquitin and

tau immunostaining were used routinely as they became commercially

available (Rajput et al., 2009). Neuropathologists produced detailed

reports, which were provided to the family and discussed where

appropriate. In some cases with essential tremor, cerebellar Purkinje

106 | Brain 2012: 135; 105–116 S. Paris-Robidas et al.

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cells counts were performed by a neuropathologist as reported previ-

ously (Rajput et al., 2011). The other half of the brain was frozen at

�80�C and was cut by hand in the frontal plane into 2–3-mm thick

slices. Coronal slices containing molecular layer of the cerebellar

cortex, granular layer of the cerebellar cortex and dentate nucleus

were cryostat-sectioned (20 mm), thaw-mounted onto SuperFrostPlus

75 � 50-mm slides (Brain Research Laboratories), desiccated overnight

at 4�C, and stored at �80�C until assayed. For homogenate-based

studies such as western immunoblotting or high-performance liquid

chromatography, cerebellar cortex extracts (�100 mg) were homoge-

nized and processed to generate a Tris-buffered saline-soluble fraction

containing extracellular and cytosolic proteins, a detergent-soluble frac-

tion containing membrane-bound proteins and a formic acid-soluble

fraction containing detergent-insoluble proteins, as described in detail

previously (Tremblay et al., 2007; Julien et al., 2008). Cerebellar pH

was measured in 10 mg of tissue diluted into 10 volumes of unbuf-

fered, deionized, distilled water, as described previously (Kingsbury

et al., 1995; Calon et al., 2003a; Julien et al., 2008). Indeed, tissue

pH has been identified as one of the best markers to assess the degree

of preservation of post-mortem brain tissue, as it is particularly sensi-

tive to ante-mortem agonal phase and is a good marker of messenger

RNA degradation (Kingsbury et al., 1995; Li et al., 2004; Catts et al.,

2005; Lipska et al., 2006; Mexal et al., 2006; Vawter et al., 2006; Atz

et al., 2007; Weis et al., 2007).

Receptor binding autoradiographyGABAA receptor density was assessed with 3H-flunitrazepam (GE-

Healthcare; 87 Ci/mmol) allosteric binding to the �1- and �2-interface

(benzodiazepine site) of the GABAA receptor on cerebellum sections,

using a previously reported autoradiography technique (Calon et al.,

2003a). Non-specific binding was determined in presence of 1-mM

clonazepam. GABAB receptor density was evaluated with 3H-CGP

54626 {3-N [1-(S)-(3,4-dichlorophenyl)-ethylamino]-2-(S)-hydroxypro-

pyl-P-(cyclo-hexylmethyl)-phosphinic acid; ANAWA; 40 Ci/mmol}

binding to the antagonist sites of the GABAB receptor using previously

published procedures (Calon et al., 2001, 2003a). Non-specific binding

was measured with 500mM (� )-baclofen (Tocris Bioscience).

N-methy-D-aspartic acid receptors containing GluN2B subunits

(GluR"2/NR2B) were quantified using a high-affinity selective antag-

onist 3H-Ro 25-6981 (F. Hoffmann-La Roche Ltd; 27.6 Ci/mmol;

Mutel et al., 1998; Calon et al., 2003b). Ro 25-6981 is an ifenprodil

derivative that has a high affinity for the GluN2B subunit (Mutel et al.,

1998; Calon et al., 2003b). Non-specific binding was determined by

adding 10 mM Ro 04-5595 hydrochloride (F. Hoffmann–La Roche Ltd)

to the incubation buffer. Slide-mounted tissue sections were exposed

to Kodak Biomax MR film (Carestream Health) along with standards

(3H-micro-scales, GE Healthcare) during 2, 6 and 12 weeks for3H-flunitrazepam, 3H-CGP 54626 and 3H-Ro 25-6981, respectively.

Macroscopic quantification of all autoradiograms was performed

on a KODAK Image Station 4000 MM Digital Imaging System

(Molecular Imaging Software version 5.0.0.90). Optical densities were

measured in the molecular layer of the cerebellar cortex, the granular

layer of the cerebellar cortex and the dentate nucleus of the deep

cerebellar nuclei.

Western immunoblottingFor western immunoblotting, protein concentration was determined

using bicinchoninic acid assays (Pierce). Equal amounts of protein per

sample (20mg of total protein per lane) were added to Laemmli’s

loading buffer, heated to 95�C for 5 min before loading, and subjected

to sodium dodecyl sulphate–polyacrylamide gel electrophoresis.

Proteins were electroblotted onto polyvinylidene fluoride membranes

(Immobilon, Millipore) before blocking in 5% non-fat dry milk and

0.5% bovine serum albumin (Invitrogen) in phosphate-buffered

saline containing 0.1% Tween for 1 h. Membranes were immuno-

blotted with appropriate primary and secondary antibodies followed

by chemiluminescence reagents (Lumiglo reserve, KPL Inc.). Optical

densities of bands were directly quantified using a KODAK Image

Station 4000 MM Digital Imaging System. The following antibodies

were used in this study for western immunoblotting: anti-GABAB

(NeuroMab), anti-cytochrome oxidase-1 (Santa Cruz Biotechnology)

and anti-actin (Sigma).

Table 1 Selected characteristics of study volunteers and comparison of tissue quality between groups

Characteristics Study group Statistical comparison

Control Parkinson’sdisease

Essentialtremor

Test Result

n 16 10 10 – –

Men, ratio 6/16 2/10 2/10 Contingency 0.51

Age at death, mean (SD) (years) 76 (6) 82 (8) 84 (11) ANOVA 0.08

Age of onset, mean (SD) (years) – 63 (12) 54 (20) – –

Disease duration, mean (SD) (years) – 19 (8) 30 (18) – –

Brain pH mean (SD) 6.14 (0.31) 6.46 (0.19) 6.36 (0.17) ANOVA 0.06

Post-mortem interval, mean(SD) (h) 19 (6) 18 (6) 15 (8) ANOVA 0.32

Brain weight, mean (SD) (g) 1249 (49) 1248 (150) 1178 (172) ANOVA 0.49

Hoehn and Yahr scale, mean (SD) – 3.5 (0.2) – – –

Tremor severity, UPDRS, mean (SD) – – 2.0 (0.3) – –

CO-1 messenger RNA DN-DCN, mean (SD) 525 (103) 562 (134) 584 (191) ANOVA 0.68

CO-1 messenger RNA MolCtx, mean (SD) 433 (114) 437 (122) 490 (102) ANOVA 0.51

CO-1 messenger RNA GraCtx, mean (SD) 872 (206) 931 (191) 1013 (159) ANOVA 0.31

Poly-T messenger RNA MolCtx, mean (SD) 124 (119) 86 (33) 105 (72) ANOVA 0.59

CO-1 = cytochrome oxydase-1; DN-DCN = dentate nucleus of the deep cerebellar nuclei; GraCtx = granular layer of the cerebellar cortex; MolCtx = molecular layer of the

cerebellar cortex; UPDRS = Unified Parkinson’s Disease Rating Scale.

Dentate nucleus GABA receptors in essential tremor Brain 2012: 135; 105–116 | 107

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In situ hybridizationThe general methodology for in situ hybridization has been fully de-

scribed previously (Wisden and Morris, 1994; Calon et al., 2001; Julien

et al., 2008, 2009). GABAA receptors contain an intrinsic ligand-gated

chloride channel, formed by the pentameric assembly of multiple sub-

units. In situ hybridization, histochemistry and quantitative real-time

polymerase chain reaction data show that deep cerebellar nuclei neu-

rons mainly express the �1, �2, b2 and �2 subunit messenger RNAs

(Gambarana et al., 1993; Rotter et al., 2000; Linnemann et al., 2006).

Oligonucleotide probes for the human GABAA receptor subunits �1,

�2, b2 and �2 have thus been synthesized according to previous pub-

lications (Nicholson and Faull, 1996; Petri et al., 2002) and are sum-

marized in Supplementary Table 2.

Metabotropic GABAB receptors are heterodimers consisting of

GABAB1 and GABAB2 subunits (Jones et al., 1998). In situ hybridization

has been performed using oligonucleotide probes for GABAB(1a + b) and

GABAB(2) as described previously (Calon et al., 2001). Oligonucleotide

sequences were specific for the coding region of the GABAB(1a + b) re-

ceptor messenger RNA (NM_001470.2, 1807–1763 and 2933–2884),

whereas those specific to the GABAB(2) receptor (NM_005458.6,

557–513 and 1315–1271), as described (Calon et al., 2001;

Supplementary Table 2).

Subunit I of cytochrome c oxidase messenger RNA is encoded by

the mitochondrial genome and is a classical marker of neuronal activity

(Wong-Riley, 1989; Hirsch et al., 2000). Subunit I of cytochrome c

oxidase oligonucleotide probes were chosen for their coding sequence

within the human mitochondrial genome (NC_005089.1, 6268–6221,

6578–6534, 6854–6805 and 7441–7392) and are fully described in

Supplementary Table 2. To further assess total messenger RNA con-

tent, in situ hybridization using a poly-T probe was performed to label

poly-A tails, which are present on the vast majority of eukaryotic mes-

senger RNAs (Julien et al., 2009).

Oligonucleotides were labelled with 33P-dATP (PerkinElmer) using a

3-terminal deoxynucleotidyltransferase enzyme kit (New England

Biolabs). The reaction was carried out at 37�C for 60 min, and labelled

oligonucleotides were purified using a QIAquick� Nucleotide Removal

Kit (Qiagen). The purified probes were kept at �20�C until assayed

the next day. Pre-hybridization and hybridization conditions were

exactly as described (Julien et al., 2009). Slides were exposed to

Kodak Biomax MR film for 28, 28, 7 and 2 days for GABAA, GABAB,

poly-T and subunit I of cytochrome c oxidase probes, respectively,

and macroscopic optical density quantification was performed using

the KODAK Image Station. The hybridization signal value from a

single section was obtained after subtracting the labelling from

the white matter quantified in the same section. The final data

from each individual were from the mean of four to eight tissue sec-

tions. Non-specific hybridization to tissue sections was found to be neg-

ligible, as determined by adding a 100-fold excess of unlabelled probes.

Amine quantification byhigh-performance liquidchromatographyGABA was measured by high-performance liquid chromatography

(HPLC) coupled with UV detection. Supernatants from Tris-buffered

saline fraction extracts of cerebellum were directly derivated with the

reagent dansyl chloride (Sigma-Aldrich) based on previously published

methods with slight modifications (Saller and Czupryna, 1989; Calon

et al., 1999). Briefly, 50ml of dansyl chloride (1.2 mg/ml) and 50 ml of

sample (Tris-buffered saline extracts) or standard solution, also in

Tris-buffered saline, were mixed and then incubated for 30 min at

90�C. After a 10-min centrifugation at 9000 rpm (4�C), the super-

natant was immediately injected into the chromatograph consisting

of a Waters 717 plus autosampler automatic injector set at 6�C, a

Waters 1525 binary pump equipped with an Atlantis dC18 (3 ml;

3.9 � 150 mm) column, and a Waters 2487 Dual l Absorbance detect-

or (Waters limited). Absorbance was set at 337 nm and the sensitivity

at 0.5 absorbance units full scale. The mobile phase consisted of a

water-acetonitrile mixture (88.5–11.35% v/v) containing 0.15%

(v/v) of phosphoric acid and was delivered at a rate of 0.8 ml/min.

Samples were run in duplicates and peaks were identified and quanti-

fied using the Breeze software (Waters limited). HPLC concentration

values were normalized to tissue weight.

Data and statistical analysesStatistical comparisons of means between groups were performed

using ANOVA when the homogeneity of variance was confirmed

(P4 0.05 using Bartlett’s test). Log transformations of the data were

used when needed to equalize variance and to provide more normally

distributed measures. Post hoc tests were used to determine significant

difference between groups (ANOVA: Newman–Keuls). Adjustments

for age of death or cerebellar pH were performed using analysis of

covariance (ANCOVA), when needed. Using the least squares method,

coefficients of correlation and significance of the linear relationship

between parameters were determined with a simple regression

model. Adjustment for additional variables (age of death or cerebellar

pH) was performed using partial correlation analyses. Likelihood ratio

analysis of contingency tables using the Pearson’s method was used in

the investigation of categorical data such as sex and apoE "4 allele

status, the statistical result being distributed as chi-squared. All statis-

tical analyses were performed using JMP Statistical Analysis Software

(version 8.0.2) and P 5 0.05 were considered to be statistically

significant.

ResultsPrior to experiments, assessment of tissue quality was performed

by measuring tissue pH, cytochrome oxidase (subunit I of cyto-

chrome c oxidase) messenger RNA and total messenger RNA con-

tent in the cerebellum from our sample series. First, cerebellar pH

was comparable between groups suggesting equivalent tissue

quality between groups, although there was a trend for reduced

brain pH in controls (Table 1). Secondly, strong subunit I of cyto-

chrome c oxidase messenger RNA signal was detected in the deep

cerebellar nuclei and cerebellar cortex, showing relative distribu-

tion identical to a previous report in the monkey (Hevner and

Wong-Riley, 1991). No major change between groups was

detected indicating that groups were comparable in terms

of metabolic activity (Table 1). Thirdly, in situ hybridization of

poly-T oligonucleotide showed that total messenger RNA content

did not differ between experimental groups (Table 1). Overall,

these data confirm relative equivalent tissue quality between

groups.

Autoradiography of 3H-flunitrazepam to GABAA and 3H-CGP

54626 to GABAB receptors in the human cerebellum are displayed

in Figs 1 and 2. The general distribution of GABAA receptors

within the cerebellum was in agreement with earlier analyses in

human, monkey and rodent tissue sections (Bowery et al., 1987;


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Dennis et al., 1988). Binding of 3H-CGP 54626 to GABAB recep-

tors was readily detectable in the dentate nucleus (Fig. 2), but

stronger in the cerebellar cortex, particularly in the molecular

layer, conforming with previous autoradiographic studies in

rodents, monkeys and humans (Bowery et al., 1987; Turgeon

and Albin, 1993; Billinton et al., 1999; Calon et al., 2000).

These patterns were also consistent with earlier immunohisto-

chemical evaluation of GABAB receptors in human and rodent

Figure 1 Decreased GABAA receptors in the dentate nucleus of individuals with essential tremor. (A) Representative autoradiograms of

human cerebellum sections showing 3H-flunitrazepam to the benzodiazepine site of GABAA receptors in the dentate nucleus and the

cerebellar cortex of individuals with essential tremor (ET) or Parkinson’s disease (PD), compared with controls (Ctrl). Specific binding of3H-flunitrazepam in the dentate nucleus (B), molecular layer (D) and granular layer (E) of the cerebellar cortex. Correlation analyses

between GABAA receptors and the duration of essential tremor symptoms showed a trend towards inverse relationship between the

duration of essential tremor and GABAA receptors in the dentate nucleus (C). Each point represents an individual and the horizontal bar is

the average (n = 9–15 per group). Statistical comparisons were performed using an ANOVA followed by Newman–Keuls post hoc test or

linear regression.


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cerebellum (Margeta-Mitrovic et al., 1999; Billinton et al., 2000;

Kulik et al., 2002).

The quantification of the specific binding of 3H-flunitrazepam

and 3H-CGP 54 626 revealed a significant decrease of GABAA

(35% versus controls and 35% versus Parkinson’s disease) and

GABAB (#22% versus controls and #31% versus Parkinson’s dis-

ease) receptors in the dentate nucleus of patients with essential

tremor, compared with the two other groups (Figs 1 and 2). The

effects remained statistically significant after adjustment for cov-

ariates age of death and cerebellar pH using ANCOVA.

Figure 2 Decreased GABAB receptors in the dentate nucleus of individuals with essential tremor. (A) Representative autoradiograms of

human cerebellum sections showing 3H-CGP 54626 to GABAB receptors in the dentate nucleus and the cerebellar cortex of individuals

with essential tremor (ET) or Parkinson’s disease (PD), compared with controls (Ctrl). Specific binding of 3H-CGP 54626 to GABAB

receptors in the dentate nucleus (B), molecular layer (D) and granular layer (E) of the cerebellar cortex. Correlation analyses between

GABAA receptors and the duration of essential tremor symptoms showed an inverse relationship between the duration of essential tremor

and GABAB receptors in the dentate nucleus (C). Each point represents an individual and the horizontal bar is the average (n = 9–15

per group). Statistical comparisons were performed using an ANOVA followed by Newman–Keuls post hoc test or linear regression.


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Interestingly, the concentrations of GABAB receptors in the den-

tate nucleus were inversely correlated [Pearson correlation coeffi-

cient (pairwise detection method) = 0.66, r2 = 0.44, P = 0.0365]

with the duration of essential tremor symptoms, indicating that

the longer the clinical expression of the disease, the lower the

levels of GABAB receptors (Fig. 2C). Adjustment for age of

death or cerebellar pH using partial correlations had minimal

effect on the significance of the correlation. Controlling for both

age of death and cerebellar pH, a correlation of �0.757

(P = 0.0402) was obtained, as estimated by restricted maximum

likelihood method. In contrast, no significant differences between

groups were detected in either molecular or granular layers of the

cerebellar cortex (Figs 1D and E and 2D and E). The absence of

change in GABAB receptors in the cerebellar cortex was confirmed

by western immunoblot (Supplementary Fig. 1). No difference in3H-Ro 25-6981 specific binding to GluN2B subunits of N-methy-D-

aspartic acid receptors was detected in any subregion of the cere-

bellum, including the dentate nucleus, confirming that the de-

crease of GABAB receptor was specific (Supplementary Fig. 2).

We next ascertained whether the loss of GABA receptor in the

dentate nucleus was associated with reduced messenger RNA

transcription by performing in situ hybridization of subunits human

GABAA receptor subunit �1, �2, b2 and �2 (Supplementary Figs 3

and 4). Strong hybridization signals for �1-, b2- and �2 subunits

were detected in the cerebellar cortex, mostly in the granular layer

(Supplementary Fig. 3). The �2 subunit displayed a different ex-

pression pattern being almost exclusively expressed in the Purkinje

cells layer (Supplementary Figs 3 and 4). Overall, the distribution

of GABAA receptor subunit messenger RNA is in agreement with

previous work in the rat cerebellum (Laurie et al., 1992;

Gambarana et al., 1993; Rotter et al., 2000; Linnemann et al.,

2006). An increase of GABAA receptor subunit �1 and b2 was

detected in the cerebellar cortex of patients with Parkinson’s dis-

ease (Supplementary Fig. 4). However, the signal in the dentate

nucleus was too weak to allow reliable quantification, except for

�1 (Supplementary Fig. 4), but was not different between groups

(Supplementary Fig. 4).

In situ hybridization experiments targeting GABAB(1a + b) and

GABAB(2) messenger RNA were performed in the same series of

samples. The messenger RNA distribution of GABAB(1a + b) and

GABAB(2) messenger RNA was stronger in the molecular layer of

cerebellar cortex (Fig. 3A and B), as expected from previous stu-

dies in rodents (Billinton et al., 1999; Bischoff et al., 1999; Durkin

et al., 1999; Clark et al., 2000; Liang et al., 2000). However, only

GABAB(1a + b) messenger RNA was detectable in the dentate nu-

cleus (Fig. 3A). When comparing groups of patients, there was a

significant decrease in the density of messenger RNA for

GABAB(1a + b) in the dentate nucleus from individuals with essential

tremor, compared with controls without neurological impairment

(P50.01) and to individuals suffering from Parkinson’s disease

(P50.05; Fig. 3C). ANCOVA revealed that the changes of

GABAB(1a + b) messenger RNA remained significant after adjust-

ment for age of death and cerebellar pH. No difference in

GABAB(1a + b) or GABAB(2) subunit messenger RNA was seen in

the cerebellar cortex (Fig. 3D and E). Inverse relationships between

age of death and GABAB(1a + b) messenger RNA were detected in

the cerebellar cortex (r2 = 0.10, P50.05) and the dentate nucleus

(r2 = 0.15, P50.05).

Finally, to probe whether the changes in receptor were asso-

ciated with obvious changes in the production or release of the

neurotransmitter GABA, we determined the post-mortem concen-

tration of GABA in the cerebellar cortex by HPLC. However, no

significant difference was detected (Fig. 4).

DiscussionTo our knowledge, we report the first evidence of a statistically

significant neurochemical alteration within cerebellar nuclei that

distinguishes patients with essential tremor as a group from

Parkinson’s disease and/or age-matched controls. The decrease

in GABA receptors was present in patients who suffered tremors

for years and as such, may represent a long-term neurochemical

substrate of essential tremor. We propose that the disruption of

the GABAergic input into the deep cerebellar nuclei contributes to

the generation of oscillatory information conveyed to the thalamus

and the motor cortex, expressed clinically as action tremor

(Supplementary Fig. 5).

What may have caused a reduction ofGABA receptors?A first explanation is that the loss of GABA receptors is a conse-

quence of a neurodegenerative process in the dentate nucleus,

consistent with the correlation between the loss of GABAB recep-

tor and with the progression of the disease. However, our data

clearly show that the reduction of GABA receptor is specific: no

alteration of N-methy-D-aspartic acid receptors or subunit I of

cytochrome c oxidase messenger RNA has been observed in the

present sample series. Accordingly, post-mortem studies have not

reported frank evidence of cell death in the deep cerebellar

nuclei (Louis et al., 2007; Elble and Deuschl, 2009), although

degeneration of the deep cerebellar nuclei has been observed in

a subset of patients with essential tremor (Louis et al., 2006,

2007).

Secondly, a downregulation of dentate nucleus GABAA and

GABAB receptors may result from an exaggerated GABAergic

input from Purkinje cells, which project from the cerebellar cortex

to the deep cerebellar nuclei. In classical pharmacology, a decrease

in post-synaptic receptors is often a compensatory reaction to

pre-synaptic overactivity. Thus, the decrease in GABAA and

GABAB receptors could be a post-synaptic consequence of in-

creased GABA input from Purkinje cells. However, although

GABA concentration in the cerebellum rather displayed a trend

towards an increase, it was not statistically significant and thus

does not confirm this viewpoint. Alternatively, the decrease of

GABA receptors in the dentate nucleus could be a consequence

of a loss of GABA receptor located on Purkinje cells axons. This

hypothesis is consistent with reports of Purkinje cell loss, axonal

swellings (torpedoes) and other abnormalities that have been

observed in essential tremor (Louis et al., 2007; Axelrad et al.,

2008; Shill et al., 2008). However, while only GABAB receptors

are located pre-synaptically (see below), both GABAA and GABAB


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receptors were downregulated. In addition, the concomitant loss

of GABAB receptor messenger RNA transcripts rather suggests that

the loss of GABA receptors arises from a transcription decrease in

dentate nucleus cells.

A third possibility is that the GABAA and GABAB receptor reduc-

tion per se plays a causative role in essential tremor by restricting

the post-synaptic action of GABA released from Purkinje cell

axons, thereby disinhibiting deep cerebellar nuclei neurons. The

ensuing overactivity of deep cerebellar nuclei neurons would

spread up through the cerebellar-thalamo-cortical circuit, possibly

contributing to the generation of tremors (Supplementary Fig. 5).

How can a reduction of GABA receptorsin the dentate nucleus play a role inessential tremor?As mentioned in the ‘Introduction’ section, one of the most widely

accepted hypotheses proposes that essential tremor is caused by

central oscillators propagating within the cerebello-thalamo-

cortical network (Deuschl et al., 2000; McAuley and Marsden,

2000; Pinto et al., 2003; Elble and Deuschl, 2009; Schnitzler

et al., 2009). Virtually all output from the cerebellum originates

from deep cerebellar nuclei neurons, which project their axons to

various structures, including the ventral lateral nucleus of the thal-

amus forming a complex circuitry with cortico-thalamic afferents,

thalamo-cortical projection neurons and thalamic GABAergic inter-

neurons (Heck and Sultan, 2002; Ilinsky and Kultas-Ilinsky, 2002).

Given the key position of the deep cerebellar nuclei in this circuit-

ry, it is clear that the neurological information processed in the

cerebellum has to interact with GABA receptors in deep cerebellar

nuclei neurons before leaving the cerebellum. The deep cerebellar

nuclei have been known to play a crucial role in the generation of

tremor and experiments involving the local cooling or ablation of

dentate nucleus cells induces oscillations resembling intention tre-

mors in non-human primates (Growdon et al., 1967; Brooks et al.,

1973; Goldberger and Growdon, 1973; Cooke and Thomas,

1976).

Figure 3 Decreased GABAB receptor subunits messenger RNA in the dentate nucleus of individuals with essential tremor. The expression

of GABAB(1a + b) (A) and GABAB(2) (B) receptor subunits was determined using in situ hybridization and quantified in the dentate nucleus

(C) and the cerebellar cortex (E and F). Correlation analyses show an association between GABAB(1a + b) receptors messenger RNA and the

duration of essential tremor symptoms in the dentate nucleus (D). Each point represents an individual and the horizontal bar is the average

(n = 9–15 per group). Statistical comparisons were performed using an ANOVA followed by Newman–Keuls post hoc test (C, E and F) or

linear regression (D).


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Electrophysiological studies show that all dentate nucleus cells

are sensitive to GABAergic input from Purkinje cells (Uusisaari and

Knopfel, 2008), whereas immunocytochemistry, autoradiography

and in situ hybridization experiments in rodent and human tissue

have detected both GABAA and GABAB receptors in the deep cere-

bellar nuclei (Kingsbury et al., 1980; Bowery et al., 1987; Chu

et al., 1990; Albin et al., 1991; Gambarana et al., 1993;

Turgeon and Albin, 1993). Electrophysiological recording of deep

cerebellar nuclei neurons confirms that both GABAA and GABAB

receptors mediate the inhibitory input coming from Purkinje cells

(Ito et al., 1970; Mouginot and Gahwiler, 1996; Chen et al.,

2005). In addition, GABAB receptors are present on the pre-

synaptic terminals of Purkinje cells into the deep cerebellar nuclei

(Mouginot and Gahwiler, 1996). Interestingly, electrophysiology

data also indicate that deep cerebellar nuclei neurons belong to

the family of single cell oscillators, with a pacemaker-like activity

(Jahnsen, 1986; Llinas, 1988; Llinas and Muhlethaler, 1988;

Mouginot and Gahwiler, 1996). Populations of neurons with an

inherent tendency for spontaneous rhythmic discharge (i.e. pace-

makers) have been identified in vertebrates and invertebrates

(Iles and Pearson, 1969) and have long been suspected to be a

driving force in essential tremor (DeLong, 1978). In the case of

deep cerebellar nuclei neurons, it has been shown that their

pacemaker-like activity is under tonic control by the GABAergic

input from Purkinje cells (Mouginot and Gahwiler, 1996). Even

more compelling is the demonstration that deep cerebellar nuclei

neurons can impose their rhythmicity to their thalamic target

neurons with highly regular patterns of activity (Pinault and

Deschenes, 1992).

Therefore, a decrease of deep cerebellar nuclei GABAA and

GABAB receptors in essential tremor, as reported here, is likely

to have a critical impact on cerebellar output, as schematized in

Supplementary Fig. 5. In this scheme, without the GABAergic in-

hibitory input from Purkinje cells, the pacemaker activity of

dentate nucleus neurons would be the origin of the oscillatory pat-

terns climbing the cerebello-thalamo-cortical pathways in essential

tremor (Supplementary Fig. 5). In the normal setting, GABA

released by axons originating from Purkinje cells interacts with

GABA receptor to restrain the pacemaker activity of deep cerebel-

lar nuclei neurons. It is suggested here that the lack of GABA

receptors in the dentate nucleus leads to incorrect response to

the GABAergic input from Purkinje cells. The ensuing disinhibition

of dentate nucleus neurons would then facilitate the transmission

of oscillatory discharge of information up to target neurons in the

thalamus. Such faulty tremogenic information would then be con-

veyed from the thalamus to the primary motor, pre-motor and

supplemental areas of the brain cortex (Supplementary Fig. 5).

Ensuing oscillation in the 10 Hz range reaching the motor cortex

could modulate descending motor pathways to limb muscle and

thus drive essential tremor (McAuley and Marsden, 2000).

Interestingly, one of the most efficient treatments of essential

tremor is deep brain stimulation of the ventral intermediate nu-

cleus of the thalamus (Zesiewicz et al., 2005; Pahwa et al., 2006;

Stani et al., 2009). The efficacy against tremor of the deep brain

stimulation technique has been attributed to its capacity to disrupt

pathological thalamic oscillatory activity, thereby stopping the

propagation of tremor-inducing signal climbing the cerebello-tha-

lamo-cortical system (Lozano et al., 2002; Kane et al., 2009).

Therefore, a defect of deep cerebellar nuclei GABAergic receptor

could play an upstream role in the generation of tremor.

The present results possibly explain the pharmacological efficacy

of GABAergic drugs in essential tremor, and further support the

hypothesis that GABAergic compounds specifically acting at the

deep cerebellar nuclei level could be even more efficient. There

is good evidence that tremor may be suppressed by substances

that facilitate inhibitory neurotransmission mediated by GABA

(Louis, 1999). For example, intrathecal pump releasing a GABAB

receptor agonist baclofen has already been successfully used to de-

crease tremor in a case report (Weiss et al., 2003). Intrathalamic

injection of GABAA receptor agonist led to tremor reduction

(Pahapill et al., 1999). A tremorlytic action of GABAA or GABAB

receptor agonists has also been reported in animal models (Tariq

et al., 2001; Paterson et al., 2009). Several clinical studies have

been performed with GABA analogues, such as gabapentin, preg-

abalin or benzodiazepines showing some efficacy (Louis, 2005;

Zesiewicz et al., 2007; Ferrara et al., 2009). An additional phase

IV multi-site, prospective, double-blind, randomized, placebo-

controlled, cross-over trial is currently being performed with preg-

abalin (ClinicalTrials.gov Identifier: NCT00584376). However, the

complexity of using GABAergic treatments stems from the diffi-

culty of selectively targeting one small nucleus like the deep cere-

bellar nuclei, which makes adverse effects practically unavoidable

with current therapies. However, the fact that the dentate nucleus

expresses GABAB(1a + b), but not GABAB(2) subunits, suggests that

GABAB receptors in the dentate nucleus may be specific to this

nucleus. Indeed, the vast majority of GABAB receptor heterodimers

contain both a GABAB(1a + b) and a GABAB(2) subunit, coexpressed

by the same cells (Benke et al., 1999; Billinton et al., 1999;

Bischoff et al., 1999; Durkin et al., 1999; Clark et al., 2000;

Liang et al., 2000; Bettler and Tiao, 2006). In structures where

only GABAB(1a + b) can be found, it has been proposed that an

Figure 4 GABA concentrations in the cerebellar cortex com-

paring individuals with essential tremor or Parkinson’s disease

versus controls. GABA concentrations were determined by high

performance liquid chromatography and expressed in ng/mg of

tissue. Each point represents an individual and the horizontal bar

is the average (n = 9–15 per group). Statistical comparisons were

performed using an ANOVA followed by Newman–Keuls post

hoc test.


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unidentified subunit may couple to GABAB(1a + b) to form a func-

tional receptor (Bowery et al., 2002; Bowery, 2010; Marshall and

Foord, 2010). If such is the case within the dentate nucleus, the

GABAB receptor assembly might be unique there, which opens the

door to tissue-specific pharmacological targeting. Then, hypothet-

ically, an agonist specific for GABAB receptor subspecies present in

the dentate nucleus could be used to re-establish the tonic inhib-

ition on dentate nucleus neurons and reduce tremor.

Although we had access to a wide range of clinical data, some

important information such as Braak (Alzheimer’s disease and

Parkinson’s disease) scores, CERAD (Consortium to Establish a

Registry for Alzheimer’s Disease) stages and ante-mortem cogni-

tive performance, were not documented. Therefore, the fact that

we were not able to link our observation on GABA receptors to

Alzheimer’s disease-related neurodegeneration and symptoms is a

limitation of our study. Finally, it is important to point out that the

decrease in GABA receptors correlated with the progression of

essential tremor, as patients who suffered for a longer time had

lower levels of GABA receptors. This suggests that this GABAergic

defect is not an acute phenomenon but is established progressively

during the evolution of the disease.

ConclusionOwing to the small number of clinicopathological studies, our

understanding of the pathophysiology of essential tremor has

lagged behind other CNS diseases. The present investigation is

probably the first to report a neurochemical difference in indi-

viduals with essential tremor, versus controls or patients with

Parkinson’s disease, using human brain tissue. Since GABAergic

input into deep cerebellar nuclei neurons is critical for the regula-

tion of its pacemaker activity extending through cerebello-

thalamo-cortical networks, a reduction of GABA receptors may

play an important role in the generation of tremor. Finally, our

results suggest that GABA receptors within the deep cerebellar

nuclei are potential drug targets in essential tremor.

AcknowledgementsThe authors wish to thank the patients and families who gener-

ously donated brain tissue to our research program; Novartis

(Basel, Switzerland) for the gift of 3H-CGP 54626; and F.

Hoffmann-La Roche Ltd (Basel, Switzerland) for the gift of3H-Ro 25-6981 and Ro 04-5595.

FundingThe International Essential Tremor Foundation (IETF); the Fonds

d’Enseignement et de Recherche of the Faculty of Pharmacy of

Laval University; and the Canada Foundation for Innovation

(10307). F.C. was supported by a New Investigator Award from

Canadian Institutes of Health Research (CAN-76833) and a salary

award from the Fonds de la recherche en sante du Quebec.

Supplementary materialSupplementary material is available at Brain online.

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