How to investigate behavior and cognitive abilities in rodents in a social group slideshare

How to Investigate Behavior and Cognitive Abilities of Individual Rodents in a Social Group

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Today’s Presenters:

Holger Russig, PhDScientific Director,

TSE Systems

David P. Wolfer, MDInstitute of Anatomy and Zurich Center

for Integrative Human Physiology, University of Zurich

Institute of Human Movement Sciences and Sport, ETH Zurich

Ewelina Knapska, PhDNencki Institute of Experimental

BiologyWarsaw, Poland

Please submit questions for our guest speakers during the presentation through the Questions

Window.

Automated Behavioral and Cognitive Screening of Socially Housed Mice

Holger Russig, PhDScientific Director,

TSE Systems

Copyright InsideScientific & TSE Systems. All Rights Reserved.


Hallmarks of intelligent behavioral & cognitive testing

1. Focus on true translational research – test animals within their social environment

2. Remove stress, fear and anxiety – reduce experimenter interference

3. Remove human bias – automatize testing to standardize data acquisition & analysis

4. Allow high-throughput screening – Long-term continuous testing during light & dark phases for days or even weeks

5. Flexibility - multiple animals & paradigms within a single system

Inspiring Design 1. Spacious central compartment with food grid and shelter

2. Four fully automated operant conditioning corners

3. Variety of sensors and actors providing the possibility to program an endless number of testing paradigms

Inspiring DesignSensors:• 1 RFID antenna• 1 presence sensor• 2 light beam

nosepoke sensors• 2 lickometer

Actors:• 2 automated doors• 2 rows of 3 stimulus

LEDs• Air-puff valve

Light beams register START / END of a nose

poke event

Lickometer register the number and duration of

licks

Presence detectors & RFID antennas register START / END

of a corner visit

Nose Poke LicksCorner Visit

Classical & Operant Conditioning 1. Sensors and Actors can be used flexibly to program classical or operant conditioning tasks

2. Software based shaping of animals behavior

3. Modulation of reinforcement

Free programming of many behavioral and cognitive tasks

Spatial and Temporal• Place Learning• Avoidance Learning• Reversal Learning• Alternation• Serial Reversal• Patrolling• Coverage• Drinking Sessions/

Temporal Learning

Social and Others• Competition/Hierarchy Analysis• Differential Synchronization

Spontaneous Behavior• Free Adaptation• Nosepoke Adaptation

Operant Conditioning• Continued Stimulus

(LED Scheme)• Fixed Ratio• Progressive Ratio• Impulsivity & Differential

Reinforcement of Low Responding (DRL)

Memory• Impulsivity &

Delay Discounting• Attentional Shift• Neophobia• Conditioned Aversion

Discrimination Learning & Preferences• Light Discrimination

(LED Scheme)• Taste Aversion• Compound Cue

Software Operation of the IntelliCage is controlled by graphical oriented Software consisting of three parts:

1. Designer

2. Controller

3. Analyzer

Designer:

• Hardware definition

• Provides graphical tools to design and store conditioning tasks

• Handling of animal ID, groups, clusters, and modules for temporal experimental control

• Definition of cognitive test schedule series containing different experiments

Controller:• Runs experiments by

executing the "Experiment file "

edited in the Designer

• Extracts and stores the behavioral events

• Visualizes the basic behavioral parameters during the ongoing

experiment, allowing for online-monitoring

• Alarm function for high animal welfare

Analyzer:

• Explore, extract, and preprocess data produced and stored

by the Controller

• Original data files stay unmodified

• Data representation and export in graphical and text format

• Filter and statistical analysis options

Social Housing – high throughput• Up to 16 animals share one cage

• 8 cages run by 1 controller

• Social behavior and hierarchies can be established and evaluated

• Animals “choose” when to enter an operant corner

…”Following or avoiding other mice in corner visits”…” this is the first method that permits the mapping of the social networks of mice and the effects of those social networks on reward-induced behaviors”…

(Parkitna et al., PLoS One 2014)

Lipp et al. 2005

Reduced Data Variability

• Fully automated

• No human bias

• No stress induced by human handling

• High standardization

10 min open field exploration (video tracking)

10 min exploration (IntelliCage)

Knapska et al. 2006

Automated Experimentation

Transfer of validated behavioral paradigms into an automated setup

Models of Spatial learning and Memory

Automated Experimentation

• Automated detection of individual cognitive deficits or enhancements within social groups

• Comparable to individual testing

Konopka et al. 2010

Automation Saves Time

• Automated replacement of time consuming and labour – intensive behavioral paradigms, such as the Morris water maze

6 trials a day + probe trial7 days intensive work

6 days of experimentation 1 h study preparation

Herrera et al. 2008, Konopka et al. 2010

VS

Summary

• IntelliCage is the next generation behavior & cognition test system

• Animals in social groups, high translational value and animal welfare

• Fully automated, high standardization, efficiency and accuracy

• Long-term High-throughput screening with great experimental flexibility

• IntelliCage reduces lab space, animals and costs

IntelliCage Product Overview Video

click to view video

http://youtu.be/Ta35IpMt5Pg

Behavioral phenotyping in IntelliCage: from spontaneous behavior to cognition

David P. Wolfer, MDInstitute of Anatomy and Zurich Center for

Integrative Human Physiology, University of Zurich

Institute of Human Movement Sciences and Sport, ETH Zurich


What we will cover today...

Adaptation Stages• Prescreening by monitoring

of spontaneous behavior

• Adaptation of activity to scheduled drinking sessions

Cognitive Test Battery• Hippocampus-dependent

spatial learning tasks

• Reaction time task to measure motor impulsivity

Mice familiarize themselves with IntelliCageduring three successive stages of adaptation

J Neurosci Meth 234:26-37, 2014

08:00 20:00

Free Adaptation

Free access to water

with all doors kept open

at all times

08:00 20:00

Nosepoke Adaptation

Nosepoke opens door once per visit for 5s, everywhere

at al times

08:00 20:00

Session Adaptation

Nosepoke opens door everywhere but only during

drinking sessions

Many parameters are measured during adaptationstages to characterize spontaneous behavior

Visits

total visits (x/d)visits dark/light-index (%)visits session-index (%)

visits in 1st half of dark phase (%)visits in 1st third of session (%)

light visits (x/h)active phase visits (x/h)

empty visits (%)pokes only visits (%)drinking visits (%)repeated visits (%)

repeated visit pattern (%)corner preference strength (%)corner preference volatility (%)

cumulative corner preference CV (%)corner preference alternation (%)

median visit duration (s) emptymedian visit duration (s) pokes onlymedian visit duration (s) drinking

Pokes

total pokes (x/d)pokes dark/light-index (%)pokes session-index (%)

pokes in 1st half of dark phase (%)pokes in 1st third of session (%)

active phase pokes (x/h)pokes with drinking (%)

alternating pokes (%)pokes per visit (x)

side preference strength (%)side preference volatility (%)

cumulative side preference CV (%)

median poke duration (s) emptymedian poke duration (s) drinking

median poke latency (s) firstmedian poke latency (s) repeated

median poke latency (s) alternating

Licks

total licks (x/d)licks dark/light-index (%)

licks in 1st half of dark phase (%)licks in 1st third of session (%)

active phase licks (x/h)

licking sessions per visit (x)licks per poke (x)

total contact time (s/d)contact time dark/light-index (%)

licking session duration (s)contact time per poke (s)

lick interval (ms)lick duration (ms)

licking frequency (Hz)

same set of 50 variables used forfree, nosepoke and drinking session adaptation

PCA analysis extracts 11 dimensions of independent variation of spontaneous behavior in IntelliCage


1 strong spontaneous corner preferencesrepetitive & stereotyped visit patternscorner preference

8strong spontaneous side preferencessmall fraction of alternating pokesside preference

4 many visits and pokes per unit timelow fraction of visits and pokes with licksvisits & pokes

3little activity during light phase: mostvisits, pokes and licks during dark phaselight phase inactivity

2little fragmentation of drinking with fewerbut longer drinking pokes with many licksdrinking poke size

5long (non-drinking) visits with slowpoking rhythm during visitsvisit duration

7most visits, pokes and licks occurduring the first half of the dark phase

early dark activity

6overall many licks and longcumulative lick contact timelick number

9high licking frequency during drinkingpokes, long licks with short intervalslicking frequency

10many pokes per visits, poke number elevated relative to visit numberpokes per visit

11large fraction of pokeless visits,few visits with drinkingpokeless visits

27%

21%

14%

20%

1 2 3 4 5 6 7 8 9 10 11

23% 11% 9% 9% 7% 6% 5% 4% 3% 3% 2%

n = 1542

cumulative corner preference CV (%)corner preference strength (%)corner preference volatility (%)repeated visits (%)repeated visit sequences (%)corner preference alternation (%)cumulative side preference CV (%)median poke duration (s) lickless

drinking time per poke (s)licks per poke (x)contact time per poke (s)median poke duration (s) drinking

visits dark/light-index (%)licks dark/light-index (%)pokes dark/light-index (%)contact time dark/light-index (%)light visits (x/h)

dark phase pokes (x/h)total pokes (x/d)total visits (x/d)dark phase visits (x/h)pokes only visits (%)pokes with drinking (%)visits with driking (%)

median visit duration (s) pokes onlymedian visit duration (s) pokelessmedian poke latency (s) alternatingmedian poke latency (s) firstmedian poke latency (s) repeated

total licks (x/d)dark phase licks (x/h)total contact time (s/d)

visits in 1st half of dark phase (%)pokes in 1st half of dark phase (%)licks in 1st half of dark phase (%)

alternating pokes (%)side preference strength (%)side preference volatility (%)

lick interval (ms)licking frequency (Hz)lick duration (ms)

pokes per visit (x)drinking pokes per visit (x)median visit duration (s) drinking

pokeless visits (%)

0.88000.8710

-0.86200.82400.7700

-0.59400.58600.4730

-0.01700.0470

-0.07400.2790

-0.01900.05500.02400.0590

-0.0870

-0.1960-0.1650-0.1640-0.1690-0.27300.38500.1510

0.00900.2240

-0.1260-0.13400.3760

-0.1440-0.1490-0.1480

0.00100.1070

-0.0460

-0.13400.3840

-0.5690

0.1580-0.0400-0.1610

0.07300.32100.2330

0.1370

0.03100.0130

-0.0010-0.0010-0.0280-0.06200.05600.1660

0.91400.80200.74700.7180

-0.0360-0.04000.0020

-0.0330-0.0830

-0.1960-0.1940-0.1290-0.12700.1260

-0.1420-0.1750

-0.0630-0.00100.0880

-0.05700.0290

0.27100.25800.3590

-0.04100.05300.0020

0.0300-0.0220-0.0070

-0.20800.03500.2800

-0.1250-0.22000.2410

0.0940

0.02200.02500.03300.04300.0280

-0.06200.06400.0540

-0.0330-0.01900.0180

-0.0640

0.93800.93100.91900.9140

-0.6330

0.0740-0.0810-0.05800.0820

-0.13500.09400.0840

0.1140-0.01500.1190

-0.07400.0650

-0.15000.0860

-0.1290

0.04500.03200.0170

-0.01500.0350

-0.0340

-0.08800.12300.0460

0.02200.0280

-0.0020

0.0530

-0.3040-0.14000.2130

-0.0550-0.03300.0640

-0.3450-0.2000

-0.0610-0.14700.0190

-0.2910

0.0450-0.0450-0.0300-0.03900.5960

0.85400.85200.82800.81500.7240

-0.6590-0.6440

-0.02900.0280

-0.30000.1820

-0.0690

0.16800.16300.1720

0.0060-0.07900.0170

-0.0730-0.02200.1190

-0.15100.05900.2510

0.1630-0.1320-0.0950

-0.0020

0.07600.0640

-0.03000.08900.11400.0530

-0.02400.3870

-0.06000.1730

-0.13500.0970

0.00300.03800.04000.0410

-0.0890

-0.0230-0.0260-0.1660-0.17000.0680

-0.0780-0.0240

0.73600.72500.63800.46100.4130

-0.0720-0.0660-0.0960

0.01500.0420

-0.0720

-0.12200.09200.0700

-0.00700.0960

-0.1100

0.11600.00900.3930

-0.0540

-0.0920-0.05100.0250

-0.07000.02100.11000.0670

-0.0710

0.17900.20400.12200.0440

0.00200.0220

-0.02200.01100.2130

0.18000.19900.25600.2390

-0.41600.41400.4680

-0.1140-0.06900.0050

-0.38100.0830

0.84200.84200.6740

0.05900.0590

-0.0290

0.0750-0.0170-0.0160

-0.16500.17300.0740

-0.05400.0700

-0.0540

-0.1440

0.01900.0040

-0.0420-0.0060-0.0180-0.04700.0004

-0.0500

0.01300.0260

-0.0300-0.0030

-0.01800.06700.00100.06300.0240

-0.0100-0.00200.02700.0210

-0.08400.08000.0640

-0.0060-0.05900.0820

-0.02500.0030

0.04100.0510

-0.0010

0.93300.91400.8580

-0.03300.01200.0280

0.0080-0.0170-0.0410

-0.01300.0180

-0.0002

0.0160

0.16800.2730

-0.11300.30500.19200.11700.5660

-0.2000

-0.0350-0.0170-0.06200.1060

0.0400-0.00700.0280

-0.0110-0.0900

0.05900.0460

-0.0440-0.0270-0.09100.10200.0190

0.00100.02500.22400.1330

-0.1010

-0.0250-0.0270-0.0390

0.03400.0240

-0.0260

-0.87400.8650

-0.6320

0.0740-0.0360-0.0730

0.13500.09800.1200

0.0920

-0.0860-0.08800.0760

-0.0680-0.0530-0.0090-0.1600-0.3760

0.16400.23800.4610

-0.2950

0.03400.0990

-0.00900.10800.0490

0.10800.10800.09400.09700.1250

-0.2080-0.1030

0.0700-0.0110-0.05400.1710

-0.0730

0.21900.25400.3960

-0.0005-0.07700.0270

-0.0140-0.06600.1390

-0.87700.77200.6950

0.0370-0.1130-0.1550

-0.0120

0.09800.2100

-0.02100.27000.28800.2340

-0.0870-0.0720

-0.0450-0.0600-0.0090-0.0760

-0.05400.03000.01200.0290

-0.1020

0.19100.1970

-0.2030-0.2110-0.0620-0.14900.0760

0.4010-0.09200.09100.21300.2180

-0.0120-0.01800.0450

-0.04400.00500.0320

-0.17400.1110

-0.0400

0.0690-0.10000.0330

0.89300.74100.7040

-0.0300

-0.01800.08400.02300.10200.10700.11100.09500.0920

0.0590-0.01500.1360

-0.0180

0.1030-0.04800.0550

-0.03300.1110

-0.2140-0.22000.28500.3050

-0.16700.0740

-0.4640

-0.21200.0140

-0.01000.10700.0230

-0.0950-0.10400.0003

0.03900.0110

-0.0380

-0.05600.0030

-0.0180

0.0310-0.07500.1420

-0.18400.2300

-0.0660

0.9090

DBA/2

Inbred strains show unique and reproducibleprofiles of spontaneous behavior


1.52

0.93

1.16

0.85

1.71

1.57

1.45

1.38

1.78

1.00

0.86

-0.22

-0.22

-0.42

0.47

0.21

0.43

-0.65

0.52

-0.22

-1.06

0.45

C03 C07

light phase inactivity

early dark activity

-0.57

-0.32

-1.09

-1.17

-0.95

-0.83

-1.07

-1.55

-0.29

-1.28

0.16

0.32

0.32

0.18

0.00

0.06

0.23

0.05

0.32

0.11

0.85

-0.10

0.28

0.52

-0.33

-0.01

0.06

0.01

0.53

-0.03

-0.10

0.09

-0.39

-0.50

-0.42

-0.62

-0.25

-0.54

-0.82

-0.60

-0.38

-0.56

-0.39

-0.28

C04 C05 C10 C11

visit and poke number

visit duration

pokes per visit

empty visits

-0.45

-0.18

-1.22

-0.59

-0.42

-0.81

-0.92

-0.72

-0.67

0.64

-0.26

0.48

0.06

-0.25

0.09

0.44

-0.68

-0.19

0.20

-0.33

-1.05

-0.82

0.14

0.34

0.65

0.42

1.13

0.33

0.55

0.33

0.30

0.52

0.38

C02 C06 C09

drinking poke size

lick number

licking frequency

16

16

16

46

52

16

53

52

23

22

23

-0.45

-0.10

0.13

-0.16

-0.28

-0.41

-0.21

-0.23

-0.13

-0.14

0.10

1.27

1.33

1.37

0.88

0.98

0.51

0.63

0.48

0.77

0.31

0.48

DBA/2 HamburgDBA/2 StockholmDBA/2 RomaDBA/2 Zurich 1DBA/2 Zurich 2DBA/2 Zurich 3DBA/2 Zurich 4DBA/2 Zurich 5

DBA/2 (4 strains)129S2 (4 strains)BALB/c (4 strains)

C01 C08N

corner preference

side preference

Effect profiles

Strain

s vs. C

57B

L/6

C57BL/6

129S2

BALB/c

Lesions and mutations are associated with distinctprofiles of spontaneous behavior


0.08

0.35

1.03

0.27

-0.03

0.55

0.22

-0.44

-0.25

0.82

-0.83

1.08

1.65

-0.31

-0.08

-0.51

0.10

0.38

0.77

-0.20

0.79

-0.09

0.26

0.06

0.16

0.28

-0.26

0.21

C03 C07

light phase inactivity

early dark activity

0.44

0.60

0.60

-0.35

0.37

-0.19

-0.10

-0.21

-0.52

0.67

0.91

-0.19

-0.04

-0.47

-0.27

-0.94

-0.75

0.37

0.03

-0.29

0.02

-0.13

0.15

-0.04

0.25

0.36

-1.34

0.32

-0.23

-0.18

-0.54

-0.05

-0.26

0.20

-0.04

-0.11

0.34

0.36

-0.06

-0.24

-1.58

0.00

1.30

0.48

1.27

0.09

1.10

0.24

-0.28

0.13

0.11

-0.26

-0.11

0.81

-0.11

-0.28

C04 C05 C10 C11

visit and poke number

visit duration

pokes per visit

empty visits

0.29

0.30

0.00

0.41

-0.30

-0.79

-0.36

0.45

0.27

-1.19

-0.28

-0.27

-0.43

1.34

0.41

-0.29

-0.03

0.43

-0.05

-0.82

-0.79

-0.27

-0.24

0.08

0.19

0.22

0.70

1.82

-0.16

0.37

0.00

0.25

0.02

-0.05

-0.44

-0.21

0.09

-0.15

-0.47

-0.83

0.62

-0.79

C02 C06 C09

drinking poke size

lick number

licking frequency

381314

323616161416

6426302140

0.74

2.50

0.84

0.03

0.16

0.02

0.00

0.13

-0.04

0.24

0.38

0.42

1.63

-0.11

-0.34

-1.72

-0.41

-0.11

-0.17

-0.46

0.19

-0.03

-0.64

0.10

-0.19

-0.28

2.64

-0.08

hippocampal lesion 1hippocampal lesion 2hippocampal lesion 3

dorsal striatal lesionmedial habenular lesionSNI x Grn-/- 10mtSNI x Grn-/- 2mtSNI 10mtSNI 2mt

MUNC-18+/-SNAP-25+/-Thy1/5xFAD

PDGF/EPO-tg6ßAPPsα/sα-DM

C01 C08N

corner preference

side preference

Mu

tant vs.

Wild

type

Effect profiles

Lesio

n v

s. C

on

trol

Hipp

mHbdlCP

mutation

• Normal mice efficiently adapt activity to drinking sessions

• Mice with hippocampal lesions are unable to do this

• Impairment is not seen when other brain structures are lesioned

visi

ts (x

/h)

lesion p<.0008bin p<.0001bin x lesion p<.0001lesioned 14sham 14

lesion n.s.bin p<.0001bin x lesion n.s.lesioned 16sham 20

lesion n.s.bin p<.0001bin x lesion p<.0257lesioned 12sham 10

1h bins 7-20h

10

30

50

70

90

10

30

50

70

90

1h bins 7-20h

10

30

50

70

90

1h bins 7-20h

Hipp PFC dlCP

Mice with hippocampal lesions have troubleadapting their activity to a drinking session schedule

• Location of reward is predicted by increasingly difficult rules

• Combination with drinking sessions increases motivation

• Patrolling task reversal is very challenging for normal mice

A battery of hippocampus-dependent spatial learning tasks in IntelliCage

Nosepoke opens doors during session in one of four corners only

08:00 20:00

Rule predicting next reward depends on task

Corner preference task with reversalSame corner rewarded throughout acquisition phase, switched to opposite corner during reversal

Serial reversal taskRewarded corner changes randomly between drinking sessions, remains constant during session

Chaining task with reversalRewarded corner is anti- or clockwise relative to most recently visited corner, direction switched during reversal

Patrolling task with reversalRewarded corner is anti- or clockwise relative to last rewarded corner, direction switched during reversal

1 of 4 rewarded

• Hippocampus is not needed for simple corner preference learning

• Adaptation to changing rules depends on hippocampus

• Hippocampus is needed to learn visit patterns

Complete hippocampal lesions impair learning inIC serial reversal, chaining and patrolling tasks

7 lesion12 sham

-30

-10

10

30

50

70

% c

orre

ct -

cha

nce

acquisition

corner preference

time p<.0001lesion ns

interaction ns

reversal serial


interaction ns

time p<.0001lesion p<.0022interaction ns

14 lesion13 sham

1st, 2nd – 2nd last, last day

chaining

acquisition reversal

time p<.0001lesion p<.0444

int. p<.0001

13 lesion13 sham


int. p<.0001


patrollingacquisition reversal


int. p<.0123

14 lesion13 sham


int. p<.0001


• Test battery discriminates between hippocampal and striatal lesions

• Slightly “better” performance in corner preference acquisition may reflect reduced exploration

Dorsolateral striatal lesions do not interferenegatively with spatial and pattern learning in IC

acquisition

corner preference

reversal serial

chaining



16 lesion15 sham

-30

-10

10

30

50

70

% c

orre

ct -

cha

nce



interaction ns


interaction ns


interaction ns


interaction ns


interaction ns


1st, 2nd – 2nd last, last day 1st, 2nd – 2nd last, last day 1st, 2nd – 2nd last, last day

16 lesion15 sham

16 lesion15 sham

• Profile was hippocampus-like already during adaptation

• Deficit in IntelliCage is confirmed in radial- and T-maze

• Good scores in simplest task correlate with poor exploration in the open field

APPSα-DM show deficits in IC reversal and pattern learning that are reminiscent of hippocampal lesions

acquisition

corner preference

reversal serial

chaining



9 mutant13 control

-30

-10

10

30

50

70

% c

orre

ct -

cha

nce

time p<.0001geno p<.0001interaction ns

time p<.0001geno ns

int. p<.0011



time p<.0001geno ns

interaction ns



9 mutant13 control

time p<.0001genotype nsinteraction ns

time p<.0001geno p<.0011int. p<.0055


9 mutant13 control

EMBO J 30:2266-2280, 2011

• This lesion also impairs learning in conventional “hippocampus-dependent” tasks

• Running batteries of multiple tests is efficient in Intelli-Cage and often necessary to recognize specific brain lesions

Medial habenular lesions impair IC spatial and pattern learning in a way highly similar to hippocampal lesions

acquisition

corner preference

reversal serial

chaining



11 lesion23 control

-30

-10

10

30

50

70

% c

orre

ct -

cha

nce


interaction ns





int. p<.0001



11 lesion23 control


int. p<.0001


int. p<.0001


11 lesion23 control

Front Behav Neurosci 7:17, 2013

• Cognitive function is not limited to hippocampus-dependent learning

• IntelliCage corners can be used as mini-Skinner boxes that work 24/7

• mice make 100-150 spontaneous responses per day

Measuring motor impulsivity usingan IntelliCage reaction time task

08:00 20:00

1st poke starts trial and determines correct door,at any time in any corner

Correct responsePoke at correct door while LED is switched on, latency recorded

Premature responsePoke at any door during delay- no effect during training stage- ends trial and prevents correct response during testing stage

Time errorPoke after correct door has closed

Side errorPoke at incorrect door while correct door is open

Omission errorNo further poke after delay

delay period with both doors closed, randomly 0.5 - 1.5 - 2.5 s

Correct door opens and LEDs go on for 5s at end of delay if no premature

responses occurred


• Same traits of D2 mice are also found by individual testing in the conventionalreaction time task

• Testing impulsivity in conventional operant conditioning chambers is time consuming and labor intensive

D2 make more premature responses than B6 andhave longer reaction times in the IC reaction time task

0

20

40

60

80

100

Premature responses (%)

12 D211 B6

time p<.0024strain ns

interaction ns

day1,2-8,9

delay0.5-1.5-2.5s

delay p<.0001strain ns

interaction ns

training

12 D211 B6

time p<.0001strain p<.0019Int. p<.0161

day1,2-8,9

delay0.5-1.5-2.5s

delay p<.0001strain p<.0019Interaction ns

testing

0

.2

.4

.6

.8

1

1.2

1.4

Correct response latency (s)

12 D211 B6

time p<.0001strain p<.0131interaction ns

day1,2-8,9

delay0.5-1.5-2.5s

delay nsstrain p<.0017interaction ns

testing

• Impulsive responding suggests role of medial habenula in behavioral inhibition

• Response latencyis not affected by the lesion

• Both observations are confirmed in conventionalreaction time test

Medial habenular lesions increase prematureresponses in the IC reaction time task

Premature responses (%) Correct response latency (s)

0

20

40

60

80

100

11 lesion22 control


interaction ns

day1,2-4,5

delay0.5-1.5-2.5s

delay p<.0001lesion ns

interaction ns

time p<.0001lesion p<.0104Interaction ns

day1,2-6,7

delay0.5-1.5-2.5s

delay p<.0001lesion p<.0104

Int. p<.0347

11 lesion22 control

0

.2

.4

.6

.8

1

1.2

1.4

time nslesion ns

interaction ns

day1,2-6,7

delay0.5-1.5-2.5s

delay nslesion ns

interaction ns

11 lesion22 control

training testing testing


Conclusion…

• IntelliCage is a versatile operant conditioning environment permitting to design a virtually unlimited number of different behavioral tests

• Prescreening of mice using spontaneous behavior during adaptation allows to optimize test batteries for subsequent screening

• Long-term 24h observation permits analysis of circadian regulation and temporal adaptation of behavior

• IntelliCage spatial learning tasks are specifically sensitive to hippocampal lesions and mutations affecting hippocampal function

• IntelliCage is equivalent to conventional operant conditioning in detecting motor impulsivity and increased reaction time

Vootele VoikarElisabetta VannoniGiovanni ColaciccoMaria AlvarezInger DrescherClaudia Meyer

Hans-Peter LippSven KrackowAnton Rau

Max Gassmann, Vetsuisse & ZIHP University of ZurichUlrike Müller, University of HeidelbergShigeyoshi Itohara, RIKEN Brain Science InstituteGregor Eichele, MPI Biophysical Chemistry, Göttingen

Acknowledgments:

Multiple Behavioral Measures In The IntelliCage System

Ewelina Knapska, PhD

Nencki Institute of Experimental Biology

Warsaw, Poland


What we will cover today...

1. Goal: Understanding links between behavior and the amygdalaMeasure: Studying appetitively and aversively motivated learning in the IntelliCage system –

a. How to compare different behaviors in well balanced conditions,b. How to design the experiment to collect brain tissue at the certain point of the behavioral training.

2. Goal: Shedding light on autismMeasure: Monitoring different measures of mouse behavior at the same time –

a. How to identify associated symptoms separately,b. How behaviors can be modulated by circadian rhytm, c. How we can standardize the behavioral measures.

Classical Behavioral Tests Intellicage System

stress due to the human contact minimized human contact

isolation anxiety no isolation anxiety

extreme novelty of the experimental environment

experiment is performed in a familiar environment

problems with results’ replication highly replicable results

incidential measures enables long time testing

variability between laboratories comparable results

Classical Behavioral Testing VS. IntelliCage System

Appetitively And Aversively Motivated Learning In The IntelliCage System –

comparing different behaviors in well balanced conditions

In the place preference training mice learned to associate sweetened water with one of the corners, whereas in the place avoidance training visiting one of the corners was punished with an air-puff.

The mice acquired both responses very quickly.

Place Preference vs. Place Avoidance – Behavioral Characteristics

sweetened water

water

air-puff

PLACE PREFERENCE TRAINING PLACE AVOIDANCE TRAINING%

OF

VISI

TS IN

RE

WAR

DED

/PU

NIS

HED

CO

RNER

BEFORE AFTER 2H AFTER 5 DAYS

BEFORE AFTER 2H AFTER 5 DAYS

Knapska E, Walasek G, Nikolaev E, Neuhäusser-Wespy F, Lipp HP, Kaczmarek L, Werka T. (2006) Differential involvement of the central amygdala in appetitive versus aversive learning. Learn Mem, 13: 192-200

We used the place preference and place avoidance protocols to investigate activation of the amygdala measured with c-Fos expression. c-Fos is a marker of activated neurons, with the highest expression level ~ 90 min after stimulation.

To harvest brains for immunohistochemistry we applied shaping procedure during which animals learned that water access is limited to a fixed 2-hour period daily.

Place Preference vs. Place Avoidance – Shaping Procedure


wd – water deprivation

P-Pref – place preference

P-Av – place avoidance

Brains from the trained mice were taken and c-Fos expression in the amygdala was assessed.

We found higher c-Fos expression in the central amygdala after place preference than place avoidance training.

Place Preference vs. Place Avoidance – Patterns of Amygdala Activation


P-PREF

P-AV

Place Preference

Place Avoidance

MMP-9, a protein involved in synaptic plasticity, learning and memory is regulated by c-Fos.

Thus, we investigated the effects of its deletion on learning in place preference and place avoidance paradigms.

Place Preference vs. Place Avoidance In Matrix Metaloproteinase 9 Mice – Behavioral Characteristics

Place preference learning was impaired in MMP-9 knock-out mice when one of the corners was associated with either sweetened water (A) or tap water (B).

In contrast, place avoidance learning was intact (C). Knapska E, Lioudyno V, Kiryk A,

Mikosz M, Gorkiewicz T, Michaluk P, Gawlak M, Chaturvedi M, Mochol G, Balcerzyk M, Wojcik DK, Wilczynski GM, Kaczmarek L. (2013) Reward learning requires activity of matrix metalloproteinase-9 in the central amygdala. Journal of Neuroscience, 33(36):14591–14600.

To better balance conditions of appetitively and aversively motivated trainings we used the same modality of the reward and punishment. The mice learned to discriminate between two bottles in one corner, one containing sweetened water and another with tap water (appetitively motivated training, A) or one containing water with quinine solution (bitter) and another with tap water (aversively motivated training, D).

Place Preference vs. Place Avoidance In Matrix Metaloproteinase 9 Mice – Behavioral Characteristics

MMP-9 knock-outs were impaired during appetitively motivated discrimination learning (B, C), but not in aversively motivated discrimination learning (E, F).

We also examined control measures, such as amount of liquids drunk by mice (I, J)

Knapska E, Lioudyno V, Kiryk A, Mikosz M, Gorkiewicz T, Michaluk P, Gawlak M, Chaturvedi M, Mochol G, Balcerzyk M, Wojcik DK, Wilczynski GM, Kaczmarek L. (2013) Reward learning requires activity of matrix metalloproteinase-9 in the central amygdala. Journal of Neuroscience, 33(36):14591–14600.

To confirm the functional role of MMP-9 in appetitively motivated learning in the central amygdala we blocked its activity by specific inhibitor, TIMP-9.

We stereotactically injected nanoparticles that gradually released TIMP-9 in the central amygdala of trained mice. The behavioral effects were identical as observed in knock-out animals.

Place Preference vs. Place Avoidance In Matrix Metaloproteinase 9 Mice – Functional Role of MMP-9

Knapska E, Lioudyno V, Kiryk A, Mikosz M, Gorkiewicz T, Michaluk P, Gawlak M, Chaturvedi M, Mochol G, Balcerzyk M, Wojcik DK, Wilczynski GM, Kaczmarek L. (2013) Reward learning requires activity of matrix metalloproteinase-9 in the central amygdala. Journal of Neuroscience, 33(36):14591–14600.

Injection Sites

Appetitively Motivated Discrimination Learning

Aversively Motivated Discrimination Learning

Monitoring Different Measures Of Mouse Behavior At The Same Time

Autism – Disorder of Neural Development

http://en.wikipedia.org/

DSM-V, American Psychiatric Association, 2013

Communication Difficulties

Stereotyped BehaviorsSocial Deficits

Different Severity Of Symptoms

Prenatal exposure to valproic acid – a mouse model of autism

Evoked by the toxic environmental factors– e.g., prenatal exposure to valproic acid (VPA)

VPA

pregnant female

offspring with behavioral impairments characteristic for autism

Balb/c and C57BL/6

Assessment of cognitive rigidity and learning abilities

(DSM-V, American Psychiatric Association, 2013)

To understand mechanisms that underlie the observed deficits we need tests that identify symptoms separately

• repetitive behaviors, perseveration - insistence on sameness, inability to break habits or change behavioral patterns

• autism is also diagnosed with accompanying intellectual impairment, with cognitive deficits unrelated to repetitive or restricted behaviors

To assess cognitive performance and perseveration simultaneously we used place preference paradigm followed by reversal learning (position of the reward was changed to opposite corner).

We measured learning in place preference and place reversal paradigms, and at the same time, frequency of visits to the corner that was no longer rewarded (perseveration).

Assessment of cognitive rigidity and learning abilities – experimental scheme

Adaptation phase Testing phase

Phase Simple (SA) and

nose-poke adaptation (NPA)

Place preference learning (PPL)

Reversal learning(RL)

Schematic drawing – IntelliCage overview

corner with10% sucrose

no access corner

previouslyrewarded corner

corner withwater

Puscian A, Leski S, Gorkiewicz T, Meyza K, Lipp HP, Knapska E. (2014) A novel automated behavioral test battery assessing cognitive rigidity in two genetic mouse models of autism. Front Behav Neurosci., 8:140.

We observed impaired place reversal learning in C57BL/6 mice (E), and the impairment was associated with perseveration (H).

In contrast, in Balb/c valproate-treated mice we observed impaired both place and reversal learning (C, F), the impairment not associated with perseveration (I).

Assessment of cognitive rigidity and learning abilities – Results

Place Learning

Place Re-Learning

PerserverationPuscian A, Leski S, Gorkiewicz T, Meyza K, Lipp HP, Knapska E. (2014) A novel automated behavioral test battery assessing cognitive rigidity in two genetic mouse models of autism. Front Behav Neurosci., 8:140.

One of our aims was standardization of the tests.

To this end we compared performance of several cohorts of mice (n=12 per cohort).

For further use we choose the most reliable measures.

The Chosen Measures Are Very Replicable…


We standardized a battery of automated, highly replicable tests that allowed for testing of cognitive abilities along with preservative behaviors in group-housed mice.

Assessment of cognitive rigidity and learning abilities - Summary


C57BL/6valproate-treated

BALB/cvalproate-treated

impaired place reversal learning?

related toperseveration?

modulated by the circadian rhythm (present only in the light phase of the day)

Summary...

IntelliCage allows for:

• comparing appetitivley and aversively motivated learning in well balanced conditions

• simultaneous assessment of cognitive abilities and perseveration

The tests:

• have fine resolution: allow for verifying mouse models symptom by symptom to find the underlying neuronal mechanisms

• are efficient and highly replicable

• allow for taking brain tissue for further analyses

Thank You!

For additional information on high-through put behavior phenotyping and tools for studying behavior and cognition in both mice and rat research applications please visit:

www.tse-systems.com



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