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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
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
Copyright InsideScientific & TSE Systems. All Rights Reserved.
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
J Neurosci Meth 234:26-37, 2014
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
J Neurosci Meth 234:26-37, 2014
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
J Neurosci Meth 234:26-37, 2014
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
time p<.0001lesion ns
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
time p<.0001lesion p<.0002
int. p<.0001
1st, 2nd – 2nd last, last day
patrollingacquisition reversal
time p<.0001lesion p<.0014
int. p<.0123
14 lesion13 sham
time p<.0001lesion p<.0001
int. p<.0001
1st, 2nd – 2nd last, last day
• 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
acquisition reversal
patrollingacquisition reversal
16 lesion15 sham
-30
-10
10
30
50
70
% c
orre
ct -
cha
nce
time p<.0001lesion p<.0035interaction ns
time p<.0001lesion ns
interaction ns
time p<.0001lesion ns
interaction ns
time p<.0001lesion ns
interaction ns
time p<.0001lesion ns
interaction ns
time p<.0001lesion ns
interaction ns
time p<.0001lesion p<.0471interaction 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
acquisition reversal
patrollingacquisition reversal
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 p<.0022interaction ns
1st, 2nd – 2nd last, last day
time p<.0001geno ns
interaction ns
time p<.0001geno p<.0244interaction ns
1st, 2nd – 2nd last, last day
9 mutant13 control
time p<.0001genotype nsinteraction ns
time p<.0001geno p<.0011int. p<.0055
1st, 2nd – 2nd last, last day
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
acquisition reversal
patrollingacquisition reversal
11 lesion23 control
-30
-10
10
30
50
70
% c
orre
ct -
cha
nce
time p<.0001lesion ns
interaction ns
time p<.0001lesion p<.0289interaction ns
time p<.0001lesion p<.0022interaction ns
1st, 2nd – 2nd last, last day
time p<.0001lesion ns
int. p<.0001
time p<.0001lesion p<.0002interaction ns
1st, 2nd – 2nd last, last day
11 lesion23 control
time p<.0001lesion p<.0128
int. p<.0001
time p<.0001lesion p<.0053
int. p<.0001
1st, 2nd – 2nd last, last day
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
Front Behav Neurosci 7:17, 2013
• 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
time p<.0001lesion ns
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
Front Behav Neurosci 7:17, 2013
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
Copyright InsideScientific & TSE Systems. All Rights Reserved.
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
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
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
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
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…
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 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
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.
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!
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