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Effects of social isolation rearing on learning in the morris water maze
BRAIN RESEARCH Brain Research7 15 (1996) I I9- I24
Research report
Effects of social isolation rearing on learning in the Morris water maze Noppamars Wongwitdecha ‘, Charles Alexander Marsden * Accepted I2 December 1995
Abstract Impaired learning has been shown as a consequence of isolation-rearing in a variety of paradigms. However, there are situations in which learning in isolation-reared rats is enhanced or unimpaired compared to socially reared rats. The present experiments investigated the effects of isolation rearing on place navigation in the Morris water maze. Two complementary paradigms were studied: isolation and socially reared rats were exposed to the water maze either without drug pretreatment or following systemic administration of scopolamine. Two conditions were examined: place learning and reversal learning. Male Lister hooded rats were either housed singly (isolation reared) or in groups of four (social reared) from weaning at 21 days of age. Six weeks later place learning and reversal learning were determined using the Morris water maze. The time taken to locate the submerged island (escape latency) was used as the measure of learning ability. The results showed that place learning. and reversal learning were enhanced in isolation reared rats compared to socially reared controls. Pretreatment with scopolamine (0.3 and 0.5 mg/kg, i.p.) produced a dose-related cognitive deficit as shown by an increase in the escape latency. Scopolamine (0.3 mg/kg) impaired both place and reversal learning but this was less pronounced in isolation compared to socially-reared rats. These results suggest that rearing in isolation may enhance spatial learning though central cholinergic mechanisms. Ke\;words:
Isolation rearing: Learning: Morris water maze; Scopolamine;Rat
1. Introduction Social isolation has been reported to produce behavioural neurochem ical and effects [8.1.5,28,30,3 1,36,41.42]. Deficit in learning has been shown as a consequence of isolation rearing in a variety of circumstances such as a spatial memory task [ 12,241, a passive avoidance task [ 161, learning a two-lever altemation schedule [31], and maze learning [ 17,191. However, in certain situations learning in isolation reared rats can be enhanced or unimpaired compared to socially reared rats. For example, in simultaneous (simple) light/dark discrimination and serial reversal learning, isolation rats were not impaired on a simultaneous discrimination in which accurate performance can be achieved simply by approaching the stimulus associated with reinforcement [21]. Housing adult rats in isolation did not impair the acquisition of schedule-induced polydipsia [23]. Isolation rearing from weaning have been shown to
. Correspondingauthor. Fax: (44) (I 15) 970-9259. ’ Present address: Department of Pharmacology. Mahidol University. Rama VI Road, Bangkok 10405, Thailand. 0006-8993/96/$15.00 6 SSDI 0006.8993(95)01578-7
1996El\evier Science B.V. All rights reserved
modify the behavioural effects of drugs in adult animals. For example, rearing rats in social isolation from weaning at 21 days of age increased the stereotyped responses produced by D-amphetamine and apomorphine [ 131, enhanced the locomotion response to D-amphetamine [20,22], increased the sensitivity to the anxiolytic-like effects of clonidine on the elevated plus-maze [43]. However, isolation reared rats are less sensitive to the anxiolytic-effects of diazepam [41] and the reinforcing properties of cocaine [3], amphetamine [44] and morphine [45] than socially reared rats. The possible neurochemical alterations that underlie the development of the isolation syndrome are still unclear. Most research on the effects of social isolation rearing have tended to concentrate on the serotonin [4,32.47] and catecholamine systems [6,15,20,34,39,46]. There has been little consideration of the possibility that isolation rearing may alter cholinergic activity or individual reactivity to amnesiac drugs. The cholinergic system plays an important role in normal learning and memory function and in certain pathological conditions in humans [ 1,2,10,37]. Scopolamine can induce amnesia in many animal models [9,11,14] and disrupt acquisition of a spatial navigation task [7.18,35]. These tasks evaluate spatial reference memory [33] which is different from the discrete or
N. Wongwitdecha, CA. Marsden/Brain
120
working memory assessed in avoidance paradigms or radial-arm maze studies. The aims of the present experiments were to investigate the effects of isolation rearing on place navigation in the Morris water maze [33], and to compare the effects of scopolamine on the spatial learning task in isolation and socially reared rats. Two complementary paradigms were studied: isolation and socially reared rats were exposed to the water maze either without drug pretreatment or following systemic administration of scopolamine. Two conditions were examined: place learning and reversal learning.
Research 715 (19961 119-124
four locations in the middle of each cardinal quadrant of the pool, submerged 1.5 cm below the water surface so that it was invisible at water level. The location of the platform was not altered throughout the training sessions. Both distal cues (i.e. standard room objects) and proximal cues (i.e. 78 X 52 cm unique black and white patterns attached to the centre of each of the four walls of the room) were used. These cues were not changed throughout the period of testing. The movement of each rat in the pool was recorded by using a video camera and computerised tracking system (Video Track CPL, Cambridge, UK). 2.3. Procedures
2. Materials
and methods
2.1. Animals
Male Lister hooded rats (Nottingham University, Medical School) were obtained at weaning (21 days postnatal). They were randomly divided into two groups and were housed either in groups of four per plastic cage (socially reared rats), or singly in plastic cage (isolation reared rats). All cages were lined with sawdust. The socially reared rats were housed in cages 52 X 32 X 20 cm high whereas the isolation reared rats were housed in cages 41 x 26 X 20 cm high. Both groups had food and water available ad libitum and were housed within the same room for 6 weeks before use. A constant dark/light cycle (on 06.00 h, off 20.00 h) was maintained in the animal house and temperature controlled at 21 f 1°C. Three experiments were performed. 1. Comparison of place and reversal learning in social and isolation reared rats (n = 12/group). The rats had been either housed socially or in isolation for 6 weeks prior to the start of the experiment. 2. Effects of scopolamine (0.5 and 0.3 mg/kg, i.p> on place learning in socially reared rats (n = 8/group). 3. Effects of scopolamine (0.3 mg/kg, i.p) on place and reversal learning in social and isolation reared rats (n = 8/group). The rats had been either housed socially or in isolation for 6 weeks prior to the start of the experiment. Each experiment was performed on separate groups of rats. 2.2. Apparatus
The Morris water maze consisted of a circular pool (200 cm in diameter, 60 cm high) with a featureless white inner surface. The pool was filled to a depth of 40 cm with 21 k 1°C water. in which powdered skimmed milk was dissolved to obscure the platform. Four points around the circumference of the pool were designated arbitrarily as points 1, 2, 3 and 4 defining the starting locations from where the rats were released. These points also divided the pool into four quadrants. The hidden escape platform was a clear Plexiglas stand (10 X 10 cm), was placed in one of
All experiments were performed between 09.00- 17.00. The procedure used was a modification of that described by Morris [33]. Two conditions were performed: one to determine place learning (acquisition) and the other reversal learning. 2.3. I. Place learning
(acquisition
phase)
On the first test day (one day before the first training trial), each rat was placed in the water maze and allowed to swim freely for 60 s without a platform. The acquisition phase began 24 h after the pre-test swim. During the acquisition phase, the hidden escape platform was located in the centre of the Northeast quadrant. Both isolation reared rats and socially reared rats were placed in the water maze in an alternative schedule (social, isolation, social, isolation, etc.). Training consisted of 20 consecutive trials (four trials each day for 5 consecutive days). The rat was placed in the water facing the pool wall at one of four randomly determined starting locations. The rat was then allowed 120 s to search for the platform. Once the rat located the platform, it was permitted to remain on it for 30 s. If the rat did not locate the platform within this time, it was placed on the platform for 30 s. Between trials rats were removed from the water and placed on a dry surface for 60 s before the start of the next trial. Escape latency was recorded in each trial and the mean time (latency) for the four trials was used as the score for a given animal, On the final day of acquisition phase, 2 min after the last learning trial, a transfer trial was given. Rats were required to swim in the pool without the escape platform for 60 s. To study the effects of scopolamine (0.5 and 0.3 mg/kg, i.p.> on place learning in social reared rats saline or scopolamine was injected i.p. 20 min before the first trial of each daily test. 2.3.2. Recersal
learning
(reversal
phase)
Both isolation and socially reared rats were first submitted to an acquisition phase, with the same training procedures outlined above. The reversal learning test was started one day after the acquisition phase. Rats were given 12 additional trials (4 trials a day for 3 consecutive days) with the hidden platform placed in the quadrant diagonally
N. Wongwitdecha, CA. Marsden/
opposite to the previous location (reversed). A final probe trial was carried out 2 min after the last reversal trial. Each rat was placed in the water maze, without the platform, for 60 s. To study the effects of scopolamine on place and reversal learning in social and isolation reared rats, the animals were treated with saline or scopolamine (0.3 mg/kg) 20 min before each daily test of the acquisition and the reversal phase.
Brain Research 715 (19961 119-124
121
120 Place Learning
H J t
100 -
$
80-
2 g
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40-
SOCIAL """"~""" IS"LAT,()N
Reversal Learning
20 -
2.4. Scopolamine studies Scopolamine HBr (Sigma) was dissolved in 0.9% saline and injected in a volume of 1 ml/kg. The doses of scopolamine (0.3 and 0.5 mg/kgl were chosen on the basis of a previous study using the Morris water maze [l 11. To study the effects of scopolamine (0.5 and 0.3 mg/kg, i.p.1 on place learning in social reared rats saline or scopolamine were injected i.p. 20 min before the first trial of each daily test. 2.5. Statistical analysis Date expressed as mean f S.E.M. Statistical analyses of the data were performed using an analysis of variance (ANOVA) procedure with repeated measures. Post hoc comparisons were assessed using Tukey’s (HSD) method. In all statistical tests a value of P < 0.05 was considered to be significant.
0-l 0
12
3
4
5
6
7
8
I 9
DAYS Fig. I. Effects of isolation rearing on place learning and reversal learning. Data represent mean ( f S.E.M.) latency to escape the platform across blocks of four trials on each day (n = 12 rats/group). * P < 0.05, significantly different from socially reared rats (ANOVA with post hoc Tukey test).
decreased performance in both groups of rats. Mean escape latencies significantly increased on the first day of reversal, as compared to the last day of the initial acquisition. Again, reversal learning was enhanced in the isolation reared rats compared to the socially reared rats. On the second day of reversal, the mean escape latencies of the isolation reared rats was significantly less than those of the socially reared rats (Fig. 15. 3.2. Effect of scopolamine on place learning in social reared rats
3. Results The results obtained from the pre-test swim showed that both socially and isolation reared rats began swimming within a few seconds of entering the water maze and were able to keep their heads above water and swim throughout the pool without apparent difficulty. In the initial acquisition phase, all rats showed learning of the Morris water maze over the 5 test days as measured by reduction in the time to find the hidden platform (escape latency). For each rat in each of the three experiments the escape latencies of the four trials on each day were grouped into one block (one block per day). 3.1. Effect of isolation learning
The escape latencies required by the scopolamine and saline control groups to locate the submerged escape platform over the 5 days of testing are shown in Fig. 2. The saline group rapidly acquired the platform location, while PLACE
LEARNING
“““‘0””
SALINE SCOPOLAMINE
0.3 m&g
-‘W-’
SCOPOLAMINE
05 mgikg
rearing on place and recersal
The escape latencies of the socially and isolation reared rats to reach the hidden platform over the 9 days of testing are shown in Fig. 1. In the initial acquisition phase (days l-3), place learning was enhanced in the isolation reared rats compared to the socially reared rats. There was significant difference in place learning between these two groups on the fourth day. Reversal of the platform position to the quadrant opposite that used for the initial acquisition phase
zo04 1
2
3
4
I 5
days Fig. 2. Effects of scopolamine (0.3 and 0.5 mg/kg i.p.1 on place learning in social reared rats. Data represent mean ( f S.E.M.) latency to escape to the platform across blocks of four trials on each day (n = 8 rats/group). * P < 0.05, * * P < 0.01, significantly different from saline-treated group (ANOVA with post hoc Tukey test).
122
N. Wongwitdecha, C.A. Marsden/Brain 60
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Place Learning (day 5)
Reversal
(day 81
50
* 40
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Learning q SALINE La scoPo-
10
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SOCIAL
ISOLATION
SOCIAL
ISOLATION
Fig. 3. Effects of scopolamine (0.3 mg/kg i.p.1 on place learning and reversal learning in socially and isolation reared rats. Data represent mean (+ S.E.M.) latency to escape the platform across blocks of four trials on each day (n = 8 rats/group). * P < 0.05, significantly different from saline-treated group (ANOVA with post hoc Tukey test).
the scopolamine treated animals were impaired with significant differences between the escape latencies of the control versus the scopolamine groups after both 0.3 mg/kg and 0.5 mg/kg (Fig. 2). 3.3. &j$ect of scopolamine on place and recersal learning in social and isolation reared rats Scopolamine (0.3 mg/kg) caused a deficit in both place and reversal learning in the socially reared rats but did not significantly impair place or reversal learning in the isolation reared rats (Fig. 3).
4. Discussion The present experiments show that after a few trials all rats typically reach the hidden platform by spatial navigation. These observations are consistent with other investigators [7,33,38]. The main findings of the present study were that the acquisition of the spatial task by adult rats was affected by rearing conditions. Our results demonstrate that young adult rats reared in isolation from weaning perform better in the water maze task than the socially reared rats. Both place learning and reversal learning were enhanced in isolation reared rats compared to socially reared rats. These results contrast with other reports showing isolation reared rats to perform less well in maze-learning situations [19,38.40]. Previous studies have shown an impairment in reversal learning of visual discriminations in the isolation reared rats compared with environmentally enriched rats, although there was no deficit in the acquisition of the original discrimination [26]. However, serial reversal learning in isolation reared animals has produced conflicting reports, for example in a light/dark discrimination task using the Krech Hypothesis Apparatus [25], isolation reared rats failed to benefit from previous experience
Research 715 (19%) 119-124
of task reversal [26]. In another report isolation reared rats did show improvement across reversals and were often only impaired on the first reversal of an obstacle manipulation task [29]. It has been suggested that reversal learning involves two stages: the inhibition of the previously rewarded strategy and the acquisition of the new strategy, these two stages are believed to involve different behavioural processes [27]. The effect of isolation rearing on reversal learning is especially interesting in view of contentions by Bitterman [5] that reversal is the best single indicator of phylogenetic differences in intelligence. Thus, the effect of isolation rearing on reversal learning may identify a true effect on intellectual ability. Our results may have implications for future studies on the influence of early environmental factors on learning. The development of a cholinergic hypothesis of age-related cognitive deficits [2] has stimulated considerable interest in the role of cholinergic systems in learning and memory. Most investigations of the role of cholinergic neurotransmission in learning and memory have focused primarily on muscarinic neurotransmission with the demonstration that muscarinic receptor antagonists impair performance in several learning paradigms [9,11,14], indicating the involvement of these receptors in cognitive processes. In the present study scopolamine significantly impaired acquisition in the Morris water maze, which was a reference memory task requiring processing of spatial information. This is similar to the impairment by scopolamine (0.5 and 1 mg/kg) in the acquisition of the helical maze, a test that also uses reference memory [14]. Comparing the effects of scopolamine on place and reversal learning in isolation and socially reared rats, we found that scopolamine (0.3 mg/kg i.p.> caused both place and reversal learning deficits in the socially reared rats but not in the isolation reared rats. This result suggests that the early social environment may influence the development of the central cholinergic system thus differentially affecting the sensitivity of the adult rats to anticholinergic drugs. In summary, isolation rearing produces long terms changes in learning reflected by changes in response to a spatial memory task. Both place and reversal learning were enhanced in isolation reared rats compared to socially reared rats, and the isolation reared rats were less sensitive to the effects of scopolamine than the socially reared rats. These changes may reflect environmentally induced alterations in central cholinergic neurotransmission. Furthermore, the isolation reared rat may be a model to investigate the possible influence of early environmental factors on learning in the human.
Acknowledgements This research was supported by an EC ‘Marie Curie’ fellowship to N.W.
N. Wongwitdecha,
CA. Marsden/Brain
Research
References [I] Bartus, R.T., Dean, R.L.. Pontecorvo, J.M. and Flicker, C., The cholinergic hypothesis: A historical overview, current perspective and future directions, Ann. NY Acad. Sci., 44 (1985) 332-358. [2] Bartus, R.T., Dean, R.L.. Beer, B. and Lippa, AS., The cholinergic hypothesis of geriatric memory dysfunction, Science, 217 (1982) 408417. [3] Berry, M.I. and Marsden, C.A., Rats reared in social isolation fail to show place preference to cocaine, J, Pswhophnrmucol., BAP Meeting Suppl. (1994) A36. [4] Bickerdike, M.J.. Wright. I.K. and Marsden, C.A., Social isolation attenuates rat forebrain 5-HT release induced by KC1 stimulation and exposure to a novel environment, Behnc. Phurmucol., 4 (1993) 231-236. 151 Bitterman. M.E.. The evolution of intelligence. Sci. Am., 212 (1965) 92- 100. [6] Blanc, G., Herve, D.. Simon, H., Lisoprawski. A.. Glowinski, J. and Tassin. J.P., Response to stress of mesocortico-frontal dopaminergic neurones in rats after long-term isolation. I%turr. 284 (1980) 265267. [7] Brandeis. R., Brandys, Y. and Yehuda. S.. The use of the Morris water maze in the study of memory and learning, ht. Neurosci.. 48 (19891 29-69. [8] Bristow. L.J.. Landon, L. and Saywell, K.L.. The glycine/NMDA receptor antagonist, L-70 1.323 reverses isolation-induced deficits in prepulae inhibition in the rat, Ps~cho~~har~~ncofog~, 1 18 (1995) 230-232. [9] Buresova. 0.. Bolhiv. J.J. and Bures, J.. Differential effects of cholinergic blockade on performance of rata in the water tank navigation task and in radial w’ater maze. Be/tat,. Neurosci., 100 (19861 476-482. [IO] Collerton, D., Cholinergic function and intellectual decline in Alzheimer’s disease, Neuroscience, 19 (1986) l-28. [ll] Cozzolino, R.. Guaraldi, D., Giuliani, A., Ghirardi, 0.. Ramacci, M.T. and Angelucci, L., Effects of concomitant nicotinic and muscarinic blockade on spatial memory disturbance in rats are purely additive: Evidence from the Morris water task. PIzvriol. Beha., 56 (19941 111-114. [12] Einon. D.. Spatial memory and response strategies in rats: age, sex and rearing differences in performance, Q. .I. Exp. Psychol., 32 (I 980) 473-489.
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Robbins. T.W., Dopaminergic and serotonergic function following isolation rearing in rats: A study of behavioural responses and Biochem. post-mortem and in viva neurochemistry, Pharmacol. Behac., 43 (19921 17-35. Jones. G.H.. Marsden, C.A. and Robbins. T.W., Behavioural rigidity and rule-learning deficits following isolation-rearing in the rat: neurochemical correlates, Behal,. Brain Res.. 43 (19911 35550. Jones, G.H., Marsden. CA. and Robbins, T.W., Increased sensitivity to amphetamine and reward-related stimuli following social isolation in rats: possible disruption of dopamine-dependent mechanisms, Psychopharmacology, 102 (19901 364-372. Jones, G.H., Robbin. T.W. and Marsden, C.A., Isolation- rearing retards the acquisition of schedule-induced polydipsia in rats. Physiol. Behac., 45 (1989) 71-77. Juraska. J.M., Henderson, C. and Muller, J., Differential rearing experience. gender and radial maze performance. Del,. Psychobiol., 17 (19841 209-215. Krech, D.. Rosenzweig, M.R. and Bennett, E.L., Dimensions of discrimination and level of cholinesterase activity in the cerebral cortex of the rat. /. Comp. Physiol. Psycho/., 49 (1956) 261-266. Krech, D.. Rosenzweig, M.R. and Bennett, E.L., Relations between brain chemistry and problem-solving among rats raised in enriched and impoverished environments, .I. Comp. Physiol. Psychol.. 55 (19621 801-807. Mackintosh, N.J. and Little. L., Selective attention and response strategies as factors in serial reversal learning, Can. J. Psychol., 23 (1969) 335-346. Meyers. R.D. and Fox, J., Differences in maze performance of group versus isolation reared rats. P~?chol. Report, 12 (19631 199-202. Morgan, M.J.. Effects of post-weaning envoironment on learning in the rat. Anim. Behal,., 21 (1973) 4299442. Morgan, M.J. and Einon, D., Incentive motivation and behavioural inhibition in socially-isolated rats, Physiol. Behal ., 15 (19751 405409.
[31] Morgan, M.J., Einon, D.F. and Nicholas, D., The effects of isolation rearing on behavioural inhibition in the rat. Q. /. Exp. Psycho/.. 27 (1975) 615-634. [32] Morinan. A. and Parker. V.. Are socially isolated rats anxious’?, Br. .I. Pharmuc,ol., 86 (19851 460. [33] Morris, R.G.M.. Developments of water maze procedure for studying spatial learning in the rat. J. Neurosci. Methods, 1 I (1984) 47-60. [34] Ojima. K., Matsumoto, K.. Tohda, M. and Watanabe, H., Hyperac-
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[I81 Hagan, J.J., Jansen, J.H.M. and Broekkamp. C.L.E.. Blockade of spatial learning by the Ml muscarmic antagonist pirenzepine. PA?-chophnrmacology, 93 (1987) 470-476. [19] Holson. R.R., Feeding neophobia: A possible explanation for the differential maze performance of rats reared in enriched or isolated environments, Physiol. Behave.. 38 (1986) 19 I-201. [20] Jones. G.H., Hernandez, T.D.. Kendall, D.A.. Marsden, C.A. and
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tivity of central noradrenergic and CRF systems is involved in social isolation-induced decrease in pentobarbital sleep, Brain Res., 684 (1995) 87-94. Pitsikas, N. and Algeri, S.. Effect of oxiracetam on scopolamine-induced amnesia in the rat in a spatial learning task, Pharmncol. Biochem. Behuc., 43 (19921 949-95 I. Reid, L.D., Gill, J.H. and Porter, P.B., Isolation rearing and HebbWilliams maze performance, Psychol. Rep., 22 (1968) 107331077. Steckler, T. and Sahgal. A.. The role of serotonergic-cholinergic interactions in the mediation of cognitive behaviour. Behuc. Brain Res., 67 (19951 165-199. Wade. S.E. and Maier, S.F.. Effects of individual housing and stressor exposure upon the acquisition of water maze escape, Learn. Motimt.. 17 (19861 287-310. Weinstock. M., Speiser. Z. and Ashkennazi, R., Changes in brain catecholamine turnover and receptor sensitivity induced by social deprivation in rats, Ps~chopharmucology, 56 (1978) 205-209. Will, B., Toniolo, G., Kelche, C.. Pallage, V.. Deluzarche. F. and Misslin. R.. The effects of postoperative physical environment on novelty seeking behaviour and maze learning in rats with hippocampal lesion. Behw. Bruin Res.. 19 (1986) 233-240. Wongwitdecha, N. and Marsden. CA., Isolation rearing reduces the anxiolytic effects of diazepam in the rats social interaction test, Behar. Pharmacol.. 6 Suppl. 1 (1995) 47-48.
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[42] Wongwitdecha, N. and Marsden, C.A.. Social isolation increases aggresstve behaviour and alters the effect of diazepam in the rats social interaction test, Behar,. Bruin Rrs., (1996) in press. [43] Wongwitdecha. N. and Marsden. C.A., Increased sensitivity to the anxiolytic effects of clonidine following social isolation in the rat. J. Psychopharmacol., 6 Suppl. (1995) A55. [44] Wongwitdecha, N. and Marsden, CA., Isolation rearing prevents the reinforcing properties of amphetamine in a conditioned place preference paradigm, Eur. J. Pharmucol., 279 (1995) 99-103. [45] Wongwitdecha. N. and Marsden, CA.. Effect of social isolation on
the reinforcing properties of morphine in a conditioned place preference test. Pharmacol. Biochrm. Behnr ., (I 996) in press. [46] Wongwitdecha. N. and Ganpinyo, P., lsolation rearing and chronic handling alter the effects of yohimbine in the forced swimming test, J. Ps,whopharmacol., 6 Suppl. (1995) A56. [47] Wongwitdecha, N.. Ganpinyo, P. and Kotchabhakdi, N., Social isolation increases the response to 8-OH-DPAT, buspirone and mCPP in the forced swimming test, J. Psychopharmacol., BAP Meeting Suppl. (1994) A8.
Research report
Effects of social isolation rearing on learning in the Morris water maze Noppamars Wongwitdecha ‘, Charles Alexander Marsden * Accepted I2 December 1995
Abstract Impaired learning has been shown as a consequence of isolation-rearing in a variety of paradigms. However, there are situations in which learning in isolation-reared rats is enhanced or unimpaired compared to socially reared rats. The present experiments investigated the effects of isolation rearing on place navigation in the Morris water maze. Two complementary paradigms were studied: isolation and socially reared rats were exposed to the water maze either without drug pretreatment or following systemic administration of scopolamine. Two conditions were examined: place learning and reversal learning. Male Lister hooded rats were either housed singly (isolation reared) or in groups of four (social reared) from weaning at 21 days of age. Six weeks later place learning and reversal learning were determined using the Morris water maze. The time taken to locate the submerged island (escape latency) was used as the measure of learning ability. The results showed that place learning. and reversal learning were enhanced in isolation reared rats compared to socially reared controls. Pretreatment with scopolamine (0.3 and 0.5 mg/kg, i.p.) produced a dose-related cognitive deficit as shown by an increase in the escape latency. Scopolamine (0.3 mg/kg) impaired both place and reversal learning but this was less pronounced in isolation compared to socially-reared rats. These results suggest that rearing in isolation may enhance spatial learning though central cholinergic mechanisms. Ke\;words:
Isolation rearing: Learning: Morris water maze; Scopolamine;Rat
1. Introduction Social isolation has been reported to produce behavioural neurochem ical and effects [8.1.5,28,30,3 1,36,41.42]. Deficit in learning has been shown as a consequence of isolation rearing in a variety of circumstances such as a spatial memory task [ 12,241, a passive avoidance task [ 161, learning a two-lever altemation schedule [31], and maze learning [ 17,191. However, in certain situations learning in isolation reared rats can be enhanced or unimpaired compared to socially reared rats. For example, in simultaneous (simple) light/dark discrimination and serial reversal learning, isolation rats were not impaired on a simultaneous discrimination in which accurate performance can be achieved simply by approaching the stimulus associated with reinforcement [21]. Housing adult rats in isolation did not impair the acquisition of schedule-induced polydipsia [23]. Isolation rearing from weaning have been shown to
. Correspondingauthor. Fax: (44) (I 15) 970-9259. ’ Present address: Department of Pharmacology. Mahidol University. Rama VI Road, Bangkok 10405, Thailand. 0006-8993/96/$15.00 6 SSDI 0006.8993(95)01578-7
1996El\evier Science B.V. All rights reserved
modify the behavioural effects of drugs in adult animals. For example, rearing rats in social isolation from weaning at 21 days of age increased the stereotyped responses produced by D-amphetamine and apomorphine [ 131, enhanced the locomotion response to D-amphetamine [20,22], increased the sensitivity to the anxiolytic-like effects of clonidine on the elevated plus-maze [43]. However, isolation reared rats are less sensitive to the anxiolytic-effects of diazepam [41] and the reinforcing properties of cocaine [3], amphetamine [44] and morphine [45] than socially reared rats. The possible neurochemical alterations that underlie the development of the isolation syndrome are still unclear. Most research on the effects of social isolation rearing have tended to concentrate on the serotonin [4,32.47] and catecholamine systems [6,15,20,34,39,46]. There has been little consideration of the possibility that isolation rearing may alter cholinergic activity or individual reactivity to amnesiac drugs. The cholinergic system plays an important role in normal learning and memory function and in certain pathological conditions in humans [ 1,2,10,37]. Scopolamine can induce amnesia in many animal models [9,11,14] and disrupt acquisition of a spatial navigation task [7.18,35]. These tasks evaluate spatial reference memory [33] which is different from the discrete or
N. Wongwitdecha, CA. Marsden/Brain
120
working memory assessed in avoidance paradigms or radial-arm maze studies. The aims of the present experiments were to investigate the effects of isolation rearing on place navigation in the Morris water maze [33], and to compare the effects of scopolamine on the spatial learning task in isolation and socially reared rats. Two complementary paradigms were studied: isolation and socially reared rats were exposed to the water maze either without drug pretreatment or following systemic administration of scopolamine. Two conditions were examined: place learning and reversal learning.
Research 715 (19961 119-124
four locations in the middle of each cardinal quadrant of the pool, submerged 1.5 cm below the water surface so that it was invisible at water level. The location of the platform was not altered throughout the training sessions. Both distal cues (i.e. standard room objects) and proximal cues (i.e. 78 X 52 cm unique black and white patterns attached to the centre of each of the four walls of the room) were used. These cues were not changed throughout the period of testing. The movement of each rat in the pool was recorded by using a video camera and computerised tracking system (Video Track CPL, Cambridge, UK). 2.3. Procedures
2. Materials
and methods
2.1. Animals
Male Lister hooded rats (Nottingham University, Medical School) were obtained at weaning (21 days postnatal). They were randomly divided into two groups and were housed either in groups of four per plastic cage (socially reared rats), or singly in plastic cage (isolation reared rats). All cages were lined with sawdust. The socially reared rats were housed in cages 52 X 32 X 20 cm high whereas the isolation reared rats were housed in cages 41 x 26 X 20 cm high. Both groups had food and water available ad libitum and were housed within the same room for 6 weeks before use. A constant dark/light cycle (on 06.00 h, off 20.00 h) was maintained in the animal house and temperature controlled at 21 f 1°C. Three experiments were performed. 1. Comparison of place and reversal learning in social and isolation reared rats (n = 12/group). The rats had been either housed socially or in isolation for 6 weeks prior to the start of the experiment. 2. Effects of scopolamine (0.5 and 0.3 mg/kg, i.p> on place learning in socially reared rats (n = 8/group). 3. Effects of scopolamine (0.3 mg/kg, i.p) on place and reversal learning in social and isolation reared rats (n = 8/group). The rats had been either housed socially or in isolation for 6 weeks prior to the start of the experiment. Each experiment was performed on separate groups of rats. 2.2. Apparatus
The Morris water maze consisted of a circular pool (200 cm in diameter, 60 cm high) with a featureless white inner surface. The pool was filled to a depth of 40 cm with 21 k 1°C water. in which powdered skimmed milk was dissolved to obscure the platform. Four points around the circumference of the pool were designated arbitrarily as points 1, 2, 3 and 4 defining the starting locations from where the rats were released. These points also divided the pool into four quadrants. The hidden escape platform was a clear Plexiglas stand (10 X 10 cm), was placed in one of
All experiments were performed between 09.00- 17.00. The procedure used was a modification of that described by Morris [33]. Two conditions were performed: one to determine place learning (acquisition) and the other reversal learning. 2.3. I. Place learning
(acquisition
phase)
On the first test day (one day before the first training trial), each rat was placed in the water maze and allowed to swim freely for 60 s without a platform. The acquisition phase began 24 h after the pre-test swim. During the acquisition phase, the hidden escape platform was located in the centre of the Northeast quadrant. Both isolation reared rats and socially reared rats were placed in the water maze in an alternative schedule (social, isolation, social, isolation, etc.). Training consisted of 20 consecutive trials (four trials each day for 5 consecutive days). The rat was placed in the water facing the pool wall at one of four randomly determined starting locations. The rat was then allowed 120 s to search for the platform. Once the rat located the platform, it was permitted to remain on it for 30 s. If the rat did not locate the platform within this time, it was placed on the platform for 30 s. Between trials rats were removed from the water and placed on a dry surface for 60 s before the start of the next trial. Escape latency was recorded in each trial and the mean time (latency) for the four trials was used as the score for a given animal, On the final day of acquisition phase, 2 min after the last learning trial, a transfer trial was given. Rats were required to swim in the pool without the escape platform for 60 s. To study the effects of scopolamine (0.5 and 0.3 mg/kg, i.p.> on place learning in social reared rats saline or scopolamine was injected i.p. 20 min before the first trial of each daily test. 2.3.2. Recersal
learning
(reversal
phase)
Both isolation and socially reared rats were first submitted to an acquisition phase, with the same training procedures outlined above. The reversal learning test was started one day after the acquisition phase. Rats were given 12 additional trials (4 trials a day for 3 consecutive days) with the hidden platform placed in the quadrant diagonally
N. Wongwitdecha, CA. Marsden/
opposite to the previous location (reversed). A final probe trial was carried out 2 min after the last reversal trial. Each rat was placed in the water maze, without the platform, for 60 s. To study the effects of scopolamine on place and reversal learning in social and isolation reared rats, the animals were treated with saline or scopolamine (0.3 mg/kg) 20 min before each daily test of the acquisition and the reversal phase.
Brain Research 715 (19961 119-124
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2.4. Scopolamine studies Scopolamine HBr (Sigma) was dissolved in 0.9% saline and injected in a volume of 1 ml/kg. The doses of scopolamine (0.3 and 0.5 mg/kgl were chosen on the basis of a previous study using the Morris water maze [l 11. To study the effects of scopolamine (0.5 and 0.3 mg/kg, i.p.1 on place learning in social reared rats saline or scopolamine were injected i.p. 20 min before the first trial of each daily test. 2.5. Statistical analysis Date expressed as mean f S.E.M. Statistical analyses of the data were performed using an analysis of variance (ANOVA) procedure with repeated measures. Post hoc comparisons were assessed using Tukey’s (HSD) method. In all statistical tests a value of P < 0.05 was considered to be significant.
0-l 0
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decreased performance in both groups of rats. Mean escape latencies significantly increased on the first day of reversal, as compared to the last day of the initial acquisition. Again, reversal learning was enhanced in the isolation reared rats compared to the socially reared rats. On the second day of reversal, the mean escape latencies of the isolation reared rats was significantly less than those of the socially reared rats (Fig. 15. 3.2. Effect of scopolamine on place learning in social reared rats
3. Results The results obtained from the pre-test swim showed that both socially and isolation reared rats began swimming within a few seconds of entering the water maze and were able to keep their heads above water and swim throughout the pool without apparent difficulty. In the initial acquisition phase, all rats showed learning of the Morris water maze over the 5 test days as measured by reduction in the time to find the hidden platform (escape latency). For each rat in each of the three experiments the escape latencies of the four trials on each day were grouped into one block (one block per day). 3.1. Effect of isolation learning
The escape latencies required by the scopolamine and saline control groups to locate the submerged escape platform over the 5 days of testing are shown in Fig. 2. The saline group rapidly acquired the platform location, while PLACE
LEARNING
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The escape latencies of the socially and isolation reared rats to reach the hidden platform over the 9 days of testing are shown in Fig. 1. In the initial acquisition phase (days l-3), place learning was enhanced in the isolation reared rats compared to the socially reared rats. There was significant difference in place learning between these two groups on the fourth day. Reversal of the platform position to the quadrant opposite that used for the initial acquisition phase
zo04 1
2
3
4
I 5
days Fig. 2. Effects of scopolamine (0.3 and 0.5 mg/kg i.p.1 on place learning in social reared rats. Data represent mean ( f S.E.M.) latency to escape to the platform across blocks of four trials on each day (n = 8 rats/group). * P < 0.05, * * P < 0.01, significantly different from saline-treated group (ANOVA with post hoc Tukey test).
122
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Fig. 3. Effects of scopolamine (0.3 mg/kg i.p.1 on place learning and reversal learning in socially and isolation reared rats. Data represent mean (+ S.E.M.) latency to escape the platform across blocks of four trials on each day (n = 8 rats/group). * P < 0.05, significantly different from saline-treated group (ANOVA with post hoc Tukey test).
the scopolamine treated animals were impaired with significant differences between the escape latencies of the control versus the scopolamine groups after both 0.3 mg/kg and 0.5 mg/kg (Fig. 2). 3.3. &j$ect of scopolamine on place and recersal learning in social and isolation reared rats Scopolamine (0.3 mg/kg) caused a deficit in both place and reversal learning in the socially reared rats but did not significantly impair place or reversal learning in the isolation reared rats (Fig. 3).
4. Discussion The present experiments show that after a few trials all rats typically reach the hidden platform by spatial navigation. These observations are consistent with other investigators [7,33,38]. The main findings of the present study were that the acquisition of the spatial task by adult rats was affected by rearing conditions. Our results demonstrate that young adult rats reared in isolation from weaning perform better in the water maze task than the socially reared rats. Both place learning and reversal learning were enhanced in isolation reared rats compared to socially reared rats. These results contrast with other reports showing isolation reared rats to perform less well in maze-learning situations [19,38.40]. Previous studies have shown an impairment in reversal learning of visual discriminations in the isolation reared rats compared with environmentally enriched rats, although there was no deficit in the acquisition of the original discrimination [26]. However, serial reversal learning in isolation reared animals has produced conflicting reports, for example in a light/dark discrimination task using the Krech Hypothesis Apparatus [25], isolation reared rats failed to benefit from previous experience
Research 715 (19%) 119-124
of task reversal [26]. In another report isolation reared rats did show improvement across reversals and were often only impaired on the first reversal of an obstacle manipulation task [29]. It has been suggested that reversal learning involves two stages: the inhibition of the previously rewarded strategy and the acquisition of the new strategy, these two stages are believed to involve different behavioural processes [27]. The effect of isolation rearing on reversal learning is especially interesting in view of contentions by Bitterman [5] that reversal is the best single indicator of phylogenetic differences in intelligence. Thus, the effect of isolation rearing on reversal learning may identify a true effect on intellectual ability. Our results may have implications for future studies on the influence of early environmental factors on learning. The development of a cholinergic hypothesis of age-related cognitive deficits [2] has stimulated considerable interest in the role of cholinergic systems in learning and memory. Most investigations of the role of cholinergic neurotransmission in learning and memory have focused primarily on muscarinic neurotransmission with the demonstration that muscarinic receptor antagonists impair performance in several learning paradigms [9,11,14], indicating the involvement of these receptors in cognitive processes. In the present study scopolamine significantly impaired acquisition in the Morris water maze, which was a reference memory task requiring processing of spatial information. This is similar to the impairment by scopolamine (0.5 and 1 mg/kg) in the acquisition of the helical maze, a test that also uses reference memory [14]. Comparing the effects of scopolamine on place and reversal learning in isolation and socially reared rats, we found that scopolamine (0.3 mg/kg i.p.> caused both place and reversal learning deficits in the socially reared rats but not in the isolation reared rats. This result suggests that the early social environment may influence the development of the central cholinergic system thus differentially affecting the sensitivity of the adult rats to anticholinergic drugs. In summary, isolation rearing produces long terms changes in learning reflected by changes in response to a spatial memory task. Both place and reversal learning were enhanced in isolation reared rats compared to socially reared rats, and the isolation reared rats were less sensitive to the effects of scopolamine than the socially reared rats. These changes may reflect environmentally induced alterations in central cholinergic neurotransmission. Furthermore, the isolation reared rat may be a model to investigate the possible influence of early environmental factors on learning in the human.
Acknowledgements This research was supported by an EC ‘Marie Curie’ fellowship to N.W.
N. Wongwitdecha,
CA. Marsden/Brain
Research
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