The document describes a study that investigated the effects of an enriched environment on rat behavior and response to pharmacological interventions. Rats were housed in either a standard environment or an enriched one involving more space, objects, and stimulation. They underwent tests for anxiety, learning, and memory, including an open field test, Morris water maze, and object recognition task. Results showed enriched rats had less anxiety-like behavior, better spatial learning and memory, and a stronger preference for novel objects after a delay. However, housing had little effect on response to the cognitive impairing drug scopolamine. The study suggests an enriched environment can improve rat behavior but may not alter response to all drugs.

1. John Birrane
Monique Peeters

2. Contents
Introduction
Methods
Results
Discussion

4. Introduction
 Rats frequently used as an animal model in
psychopharmacological research
 How comparable are they? (Patterson-Kane, Hunt & Harper, 1999).
 People: everyday novel objects
 Standard rats: always same environment
Enriched rats (with novel objects) better?

5. Enriched environment?
 Larger group of animals (Hamm, Temple, O’Dell, Pike &
Lyeth, 1996; Wainwright et al., 1993)
 More room to move arround (Arai & Feig, 2010; Hamm et
al., 1996; Wainwright et al., 1993).
 Lots of different object, stimulating all senses (Bennet et
al., 1964; Hamm et al., 1996)

6. Advantages enriched
environment
 More dendritic arborisation, neurogenesis, glial cells and
improved learning (Kempermann, Kuhn, & Gage, 1997).
 More synaptic plasticity (Rampon et al., 2000).
The best way to investigate these advantages is the
performance on different tasks (Cao, Huang, and Ruan,
2008).
 Effect on pharmacological interventions?

7. Open Field test (Hall, 1934)
 Measures anxiety and exploratory behaviour
(Wainwright et al., 1993).
 The field is divided into different areas: wall, corner and
centre
 Measures: behaviour and time spend in different areas
open field.
 Anxiety: more time in corners and near wall, low distance
moved, more boli (Walsh & Cummins, 1976)
 Exploratory behaviour: Number of rears and leans, and
high distance moved.

8. Morris water maze (Morris, 1981)
 Measures spatial learning and memory (Morris, 1981)
 Circular tank, filled with water, with a platform placed
beneath water surface (Buccafusco, 2009).
 Rats will find the platform faster everyday regardless of
starting position.
 Measures: Swimming speed, distance moved, time in the
zone of the platform.
 Probe trials: no platform, check whether rats still know
the location of the platform.

9. Object Recognition task
 AKA Novel object preference task (Mumby, Gaskin, Glenn,
Schramek & Lehmann, 2002)
 Two trails: one trial with two similar objects and one trial
with an old and a new object (Mumby et al., 2002)
 Rats have preference for novel object (Dix & Aggleton,
1998; Ennaceur & Delacour, 1988)
 Looking at novel object correlates with memory of the
familiar objects (Ennaceur & Delacour, 1988)

10. Scopolamine
 Cognitive impairing drug, blocks muscarinic receptors
 Animal model of Alzheimer’s disease
How will the environment influence the effect of
scopolamine on memory?
 Hypothesis: For enriched rats more scopolamine is
needed to achieve a memory impairing effect.

12. Methods
36 Male Wistar
Rats
18 Standard 18 Enriched
condition Condition

13.  From six weeks of age placed in
enviroment
 Tests started with 12 weeks
 Used to human contact
 Reversed light-dark cycle: lights on from
19:00-7:00
 Unlimited access to food and water
 21 C 1 C, humidity 45%-55%

14. Standard
 Three rats per cage  six cages
 40cm long x 20cm high x 25cm wide
 Objects
 Cardboard tube
 Wooden stick
 Sawdust bedding

15. Enriched
Picture by Martina

16. Enriched
 Two large cages with nine rats each
 150cm long x 80cm high x 90cm wide
 Environment changed and cleaned every week
 Novel stimuli
 Objects: PVC pipes, treadmills, brick stones, metal grid,
rope, wood sticks, different types of nesting material.

17. Tests we used:
 Open field test
 Anxiety behaviour
 Morris water maze
 Learning
 Object Recognition task (novel object preference task)
 Memory

18. Open field
 Square box: 1m x 1m, grey floor
 Tested four days in a row, day 4 had novel object
 Five minutes exploration time
Measurements
 By computer programme (EthoVision)
 Distance moved, time near walls, time in centre, time in
corner
 By human
 Rears, leans, boli

19. Morris water maze
 Large water tank: 122cm ø, 80cm height
 Small platform placed 40cm from wall and 1cm below
water surface
 Four different starting positions
 Acquistion trial: 4 days in a row, 4 trials/day
 Probe trial: day 3 (trial nr 5) and day 5 (trial nr 1)
 Reversal trials (other platform location): day 5 and 6, 4
trials/day
 EthoVision is used for the measurements

20. Object Recognition task
 Rats placed in round box, 1 transparent side
 3 minutes exploration time each trial
 Trial1: two similar objects
 Trial2: one familiar object (like in trial1) and one new object.
 Mug, jar, small bottle, cone
 No medication
 One hour delay
 Twenty-four hour delay
 With medication
 One hour delay  cognitive impairing drugs

21. Object recognition task (2)
Scopolamine
 Three doses: injected 1ml/kg
 0.00mg/ml (saline=control)  0.00 mg/kg
 0.03mg/ml  0.03 mg/kg
 0.30mg/ml  0.30mg/kg
 Subcutaneous
 30 minutes before first trial
 Double blind

22. Object Recognition task (3)
Short Movie (or at least some pictures)

23. Statistical Analysis
Open field
 Day 1-3  split-plot
 Day 4  independent samples t-test
Morris water maze
 Acquisition  split-plot
 Probe  independent samples t-test
 Reversal  split-plot
Object recognition task
 No medication:  split-plot
 Scopolamine:  univariate BS ANOVA

25. Open Field:
Days 1-3
Independent Measure Day Group Interaction
variable
Distance travelled Anxiety/exploratory F(2,68)= 23.617, F(1,34)= 30.500 F(2,68)= 7.490,
behaviour p<0.001 p<0.001 p=0.001
Time in centre of Anxiety n.s. n.s. F(2,68)= 3.553, p
field <0.05
Time in corners Anxiety n.s. n.s. n.s.
Thigmotaxis/Time at Anxiety n.s. n.s. n.s.
wall
Leaning Exploratory n.s. F(1,34) = 10.400, n.s.
behaviour p<0.05
Rearing Exploratory F(2,68)= 4.181, n.s. n.s.
behaviour p=0.019
Boli Anxiety F(2,68) = 8.108, n.s. n.s
p=0.001

26. Distance Moved Leaning
Time in Centre Boli

27. Rears
There was only a group
effect between leaning
and distance travelled.
The standard rats had a
higher distance moved
and number of leans.

28. Analysis of the open-field (day 4)
Independent Measure Group Standard Enriched
variable
Distance travelled Anxiety/exploratory t= 5.03, df= 34, p < M= 1675.41, SD= M= 1218.90, SD=
behaviour .01 156.86, N=18 351.74, N= 18
Time in centre of Anxiety t= 2.12, df= 34, p < M= 50.60, SD= 13.07, M= 35.44, SD= 27.32,
field .05 N= 18 N= 18
Time in corners Anxiety n.s. M= 31.98, SD= 7.71, M= 54.51, SD= 26.19,
N=18 N= 18
Thigmotaxis/Time at Anxiety n.s. M= 60.81, SD= 10.37, M= 54.07, SD= 17.59,
wall N=18 N=18
Time spent at novel Exploratory n.s. M=6.61, SD= 5.45, N= M= 5.98, SD= 5.73,
object behaviour 18 N=18
Leaning Exploratory n.s. M= 19.42, SD= 5.72, M= 16.94, SD= 4.35,
behaviour N=18 N= 18
Rearing Exploratory n.s. M= 14.49, SD= 6.87, M= 16.37, SD= 5.71,
behaviour N= 18 N= 18
Boli Anxiety n.s. M= 3.61, SD= 3.26, M= 2.28, SD= 2.80,
N= 18 N= 18

29. Distance Moved
There was only a group
effect in the distance
moved and time spend in
centre. The standard rats
had again a higher
distance moved, and they
Time in Centre have spend more time in
the centre of the open
field.

30. Morris water maze
Acquisition trial
Acquisition trial Morris water maze (MWM)
Independent Measure Day Group Interaction
variable
Distance moved F(5,102) = 98.189, F(1,34) = 5.036, n.s.
p<0.001 p=0.031
Escape latency Learning F(3,102) = 78.374, n.s. n.s.
p<0.001
Mean velocity F(5,102) =7.868, n.s. n.s.
p<0.001

31. Distance moved Mean Velocity
Time in zone

32. Probe trial
Independent samples t-tests
Zone 0-60 seconds 0-30 seconds 30-60 seconds
North n.s n.s. n.s.
East n.s n.s n.s.
South n.s. n.s. n.s.
West t(34) = 2.565, p<.05 t(34) = 3.044 , p<.005 n.s.
Platform zone t= -.2.24, df= 34, p < t= 2.37, df= 34, p < .05 n.s.
.05
Random zone n.s. n.s. n.s.

33. Time in zone platform vs time in random zone
Time in zone West

34. Reversal trial (new platform location)
Reversal trials
Independent variable Measure Day Group
Distance Moved F(1,34) = 17.939, n.s.
p<0.001
Mean Velocity F(1,34) = 13.680, n.s.
p=0.001
Escape latency Learning F(1,34) = 24.644, n.s.
(reversal) p<0.001

35. Distance moved Time in zone
Mean velocity

36. Object recognition task
No medication: • Effect of delay:
F(1,33) =24.348, P<0.001
• Group effect:
F(1, 33) = 5.133, P<0.05
D2 value is time spend at novel
object minus the time spend at No effects on total
old object corrected for exploration time or
exploration time preference for the left
or right object

37. Scopolamine
 D2 Value
No effect on group:
F (1, 99) = 0.189, p=0.665
Dose effect:
F (2, 99) = 13.961, p<0.01

38. Exploration time trial1 (E1):
 Group (n.s.):
F(1,98)= 1.720, p=0.193
 Dose effect:
F(2,98)=3.111, p=0.049
Exploration time trial2 (E2):
 Group (n.s.):
F(1,99) = 1.715. p=0.193
 Dose:
F(2,99)= 6.602, p=0.002

39. Conclusions
 Open field:
• Standard rats moved more on all four days
• Enriched rats spent more time in corners of open
field
• Leans and rears increase over time
• Number of defeciations decline

40.  Morris water maze:
• Standard rats moved more
• Decline in distance moved over the days on both
groups
• Enriched rats spent more time in the west zone and
the zone of the platform during probe trials

41.  Novel object preference task:
• Enriched rats have a stronger preference for the
novel object after 24 hours delay (no medication)
• With scopolamine there was a strong effect of the
dose, but no effect between groups on the
exploration time and the preference for the novel
object.

43. Open field results: Habituation the key?
Crusio and Schwegler (1987) and Crusio (2001)
negatively correlate increased intra and infra
hippocampal mossy fibre (IIHMF) projections with
exploration.
Denenberg (1969) carried out a factor analysis of open
field behaviour and found that increased ambulation
was linked to anxious behaviour on only the first day.
Indicative of the opposite for subsequent days.

44. Problems with this interpretation...
1. Some of these experiments conducted on mice...
Wishaw and Tomie (1996) claim that rats and mice
are comparable for a range of behavioural tests (as
long as they don't involve water!). Neurobiological
differences may exist though...
2. Meshi et al. (2006) demonstrated that behavioural
differences caused by EE were not due to
neurogenesis observed in van Praag's experiments.

45. The importance of exercise for
enrichment
(a,f) = controls
(b,g) = learner
(c,h) = swimmer
(d,i) = runner
(e,j) = enrichment
In this experiment, enrichment
had no effect on proliferation,
but exercise did.
Enrichment did affect the
survival of the newborn cells
positively (85% vs. 56%).

46. Types of enrichment and standard conditions (Simpson & Kelly,
2011).
Social or physical
enrichment? Both?
Dissociable effects?
Standard housing conditions of
our rats did feature some physical
enrichment (stick, cardboard
tubes) along with moderate social
enrichment (two litter-mates).
Different combinations in the literature

47. Perhaps standard rats were too enriched...
Lots of research in the literature features singly housed rats
as controls. This deprivation state not a suitable control.
However, our results were not entirely anomalous. MWM
performance reflected previous research in that enriched
housing led to faster acquisition and more time spent in PZ
during probe trial.

48. Performance in the ORT
No significant difference was observed between STD and EE rats
in d2. There was a significant effect for dose at the lower dose,
however.
Has scopolamine outlived its usefulness? Klinkenberg and
Blokland (2010) affirm that scopolamine is an effective amnestic
drug. However, dissociating its peripheral from its CNS effects can
be challenging.
Any difference we would hope to observe between groups would
have to be mediated through central nervous effects. But
peripheral effects would not cause distinct differences in
discrimination between groups.

49. QUESTIONS?

50. THANK YOU!!