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Effects of Multisensory Environmental Stimulation on Contextual Conditioning in the Developing Rat
Neurobiology of Learning and Memory 74, 89–104 (2000) doi:10.1006/nlme.1999.3949, available online at http://www.idealibrary.com on
Effects of Multisensory Environmental Stimulation on Contextual Conditioning in the Developing Rat Elizabeth A. Woodcock and Rick Richardson University of New South Wales, Sydney, New South Wales 2052, Australia
The effect of increased exposure to multisensory stimulation during development on conditioned freezing to contextual cues in preweanling Sprague–Dawley rats was examined. Rats given increased environmental stimulation exhibited long-term contextual conditioning at a younger age than rats that did not receive such stimulation when there was either low or moderate levels of conditioning (Experiments 1 and 2). These differences in contextual conditioning were not a result of the stimulated rats reacting differently to shock (Experiment 4) or merely freezing more than the nonstimulated rats in all situations (Experiment 3). The role of the glucocorticoid system in the enhanced contextual learning of stimulated preweanling rats and the advantages of the contextual conditioning procedure for studying the effects of environmental stimulation are discussed. q 2000 Academic Press Key Words: multisensory stimulation; enriched environment; preweaning; contextual conditioning.
There is extensive evidence that the stimulation an animal receives from its external environment has a profound effect on its later behavior. Specifically, heightened sensory stimulation and opportunities for problem-solving enhance performance on a variety of learning and memory tasks (Renner & Rosenzweig, 1987). Most studies in this area have involved postweaning stimulation. During the postweaning stage of development, enhanced stimulation is typically provided by using an “enriched environment” procedure. In other words, the animals are placed in a large cage with various objects for the purpose of increasing the amount of sensory and social stimulation received. Rats that are raised in enriched environments show enhanced performance on problem-solving tasks such as the Hebb–Williams maze (Venable et al., 1988), the Lashley III maze (Greenough, Yuwiler, & Dollinger, 1973), and the radial arm maze (Juraska, Henderson, & Muller, 1984). Some studies in this area of research, however, have involved preweaning stimulation. Several different techniques have been used to provide enhanced stimulation during the This research was supported by a grant from the Australian Research Council to R.R. and constituted a part of the Ph.D. thesis by E.A.W., which was supported by an Australian Commonwealth Postgraduate Scholarship. Address correspondence and reprint requests to Rick Richardson, School of Psychology, University of NSW, Sydney, 2052, Australia. Fax: 61 2 9385 3641. E-mail: [email protected]. 89
1074-7427/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.
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preweaning period. Some researchers have utilized an enriched environment, like that described above, by either placing each litter into separate enriched environments (Denenberg, Woodcock, & Rosenberg, 1968; Ivinskis & Homewood, 1980) or by placing pups from different litters into a single enriched environment with one foster mother (Forgays & Read, 1962). In each case, this form of preweaning experience led to enhanced problemsolving ability on the Hebb–Williams maze during adulthood. However, since rats spend a large part of the preweaning period in close proximity to the nest, they may not take full advantage of the heightened levels of sensory stimulation provided by the enriched environment. Indeed, Denenberg et al. (1968) and Forgays and Read (1962) both found preweaning enrichment to be less effective than postweaning enrichment in terms of improving problem-solving ability. In order to provide more intensive stimulation for preweanling rats, Venable et al. (1988) individually removed the pups from the nest four times each day and exposed them to a variety of sensory experiences for 25 min. This intensive procedure provided rats with higher levels of tactile, vestibular, visual, auditory, and thermotactile stimulation and proved to be more effective in improving problemsolving ability during adulthood on the Hebb–Williams maze than postweaning stimulation. Another, more common, technique used to examine the effects of early environmental stimulation on later behavior is referred to as “handling.” All handling procedures involve some manipulation of the pups in the hands of the experimenter, but are nevertheless quite diverse. For example, handling often involves removing the animals from the nest each day and placing them into a separate holding container for durations typically ranging from 2 to 15 min (e.g., Denenberg, 1967; Escorihuela, Toben˜a, & Ferna´ndez-Terul, 1994; Meaney, Aitken, Bhatnagar, & Sapolsky, 1991). However, some researchers continue to hold the rats in their hands for this period (e.g., Levine, 1956), while others massage or “play” with the pups in their hands (Szeligo & Leblond, 1977). Handling rats during the preweaning period generally leads to reduced emotionality in mildly stressful situations, as observed by less freezing and defecation, and higher rates of exploration in adulthood (Levine, Haltmeyer, Karas, & Denenberg, 1967). However, early handling also leads to improvements during adulthood in learning and memory, as evidenced by superior performance of handled rats on tasks such as shuttlebox avoidance (Escorihuela et al., 1994) and the Morris water maze (Meaney et al., 1991). Many of the learning and memory tasks on which environmentally stimulated animals show superior performance are believed to be hippocampally mediated. Indeed, neuroanatomical analyses of the hippocampus following postweaning environmental stimulation have revealed changes in both fine and gross structure (Kempermann, Kuhn, & Gage, 1997; Olsson, Mohammed, Donaldson, Henriksson, & Seckl, 1994; Walsh, Budtz-Olsen, Penny, & Cummins, 1969). Neural modifications throughout the brain have also been reported following various forms of preweaning stimulation, including handling (Altman, Das, & Anderson, 1968), placement of the whole litter into an enriched environment (Malkasian & Diamond, 1971), and multisensory stimulation (Schapiro & Vukovich, 1970; Venable, Ferna´ndez, Dı´az, & Pinto-Hamuy, 1989). The neural changes following preweaning stimulation also include hippocampal modifications (e.g., Meaney & Aitken, 1985; Pham, Soederstroem, Henriksson, & Mohammed, 1997; Wilson, Willner, Kurz, & Nadel, 1986; Woodcock, 1994). Although the effects of preweaning environmental stimulation on neuroanatomy and
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neurochemistry have been examined both pre- and postweaning, its effects on learning and memory have only been examined after weaning. The present series of experiments therefore examined the effects of early environmental stimulation on learning and memory prior to weaning. A contextual conditioning procedure was employed for this purpose. This is particularly pertinent given the fact that contextual memory develops relatively late in the rat. That is, Rudy (1993) showed that rats 23, but not 18, days of age exhibited long-term (i.e., 24 h) memory of a context that had been paired with shock. However, these two age groups did not differ in expression of long-term memory of a discrete auditory stimulus that had been paired with shock. Further, 18-day-old rats also did not differ from 23-day-old rats in short-term contextual memory. The failure of the 18-dayold rats to exhibit long-term contextual memory was attributed to the late development of the hippocampus. Because enhanced environmental stimulation has been found to increase hippocampal development in preweanling rats (e.g., Meaney & Aitken, 1985; Pham et al., 1997; Wilson et al., 1986), we predicted that heightened environmental stimulation during the preweaning period would also accelerate the functional development of the hippocampus. In other words, the present study examined the hypothesis that preweanling rats exposed to increased environmental stimulation would exhibit long-term contextual memory at a younger age than rats that do not receive such stimulation. The contextual conditioning procedure has not previously been used with pre- or postweaning animals for the purpose of measuring the effects of environmental stimulation. In the contextual conditioning procedure rats are shocked in an environment and then reexposed to that same environment 24 h later. It is assumed that an integrated representation of the cues in the environment is constructed during the initial exposure to it. Subsequent exposure to cues belonging to this network elicits freezing responses as a result of the association between the network and shock (Kiernan, Westbrook, & Cranney, 1995; McLaren, Kaye, & Mackintosh, 1989). Animals with a better representation of the environmental cues would be predicted to show a greater degree of freezing; hence the degree of freezing can be used as a measure of the animal’s memory of the environment. GENERAL METHODS Experimental Design Each litter was assigned to one of two rearing conditions. “Stimulated” pups were exposed to an intensive sensory stimulation program for 1 h each day, from postnatal day (PND) 2 to the day prior to testing. “Nonstimulated” pups remained with their mothers and littermates during this daily 1-h period. The nonstimulated rats were not handled or removed from the nest during this period since it is believed that the stimulation received during handling—including the extra stimulation received from the mother when the pups are returned to the nest—forms a crucial component of preweaning stimulation and thus may act to reduce differences between stimulated and nonstimulated rats (see General Discussion). Nonstimulated rats tested at 26 days of age (Experiment 1) were handled the day prior to testing for 5 min in order to reduce behavioral reactivity to handling that occurs in rats of these ages (Smith, 1972). Nonstimulated rats 21 days of age and younger were not handled the day prior to testing since rats of these ages are not behaviorally reactive when handled.
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Subjects One hundred and forty-four rats from 35 litters were used in the study. Each litter was culled to 10 pups at PND 1 and housed for the duration of the experiment in a plastic maternity cage (30 3 45 3 16 cm). The cages were kept in a colony room with a 12-h light/dark cycle, and paper pellet bedding was changed once a week for all cages. Food and water were continuously available. All animals were treated in accord with the ethical guidelines of the American Psychological Association and all procedures were approved by the Animal Care and Ethics Committee at the University of New South Wales. Multisensory Stimulation Procedure In order to maximize any effects of early stimulation on the development of contextual memory, the present series of experiments used an intensive multisensory stimulation procedure based on the procedures of Venable et al. (1988, 1989) and Schapiro and Vukovich (1970). This preweaning stimulation procedure entailed removing the pups from the nest each day and exposing them to a wide range of sensory stimuli for 1 h. The stimuli were specifically chosen to stimulate auditory, visual, tactile, taste, olfactory, vestibular, and thermotactile senses. Each rat was given multisensory stimulation from PND 2 until the day prior to behavioral testing. The stimulation sessions involved placing the pups on a cold metal pan (at room temperature) and a metal plate heated to 348C and other surfaces of varying textures, such as sandpaper, brushes, and marbles, in order to stimulate their tactile sense. The olfactory sense was stimulated by exposing the pups to various odorants (e.g., lemon, peppermint) soaked on a cotton ball. The chemosensory sense was stimulated by applying various artificial tastes (e.g., lemon, vanilla) on a cotton bud to the pup’s mouth. Each day the animals were rolled slowly in a cardboard tube for 1 min for vestibular stimulation. For additional vestibular and thermotactile stimulation from PND 10 onward, the animals were placed in a bowl of warm water (348C) in which they swam for 10 s. From PND 12, a few days prior to eye-opening, visual stimulation was added to the stimulation session. This involved exposing the pups to a flashing light for 15 s. In order to ensure that the pups stayed active during this visual stimulation, they were stroked during this time. All of the sensory stimulation occurred in a 1-h period each day. At all other times, rats in the stimulation condition were kept with their mother and littermates in the standard plastic maternity cage. EXPERIMENT 1: LONG-TERM CONTEXTUAL MEMORY IN 18- AND 26-DAY-OLD RATS Experiment 1 examined the effects of preweaning environmental stimulation on contextual conditioning in 18- and 26-day-old rats. It has previously been reported that at 18 days of age rats do not show evidence of long-term contextual memory, but that this ability has emerged by 23 days of age (e.g., Rudy & Morledge, 1994). Rudy and Morledge hypothesized that the emergence of long-term contextual memory between 18 and 23 days of age results from the maturation of the hippocampus during this same period. Therefore, if environmental stimulation accelerates the maturation of the hippocampus as hypothesized, long-term contextual memory should emerge at a younger age in stimulated rats. Hence, it was predicted that 18-day-old stimulated rats in the present experiment would show long-term contextual memory.
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Method Subjects. Twenty male and female rats from each rearing condition were used in this experiment. Ten of the rats from each rearing condition were tested at 18 days of age and 10 at 26 days of age. The data of 1 18-day-old rat in the nonstimulated group that displayed levels of freezing 16 SD above the mean was omitted from the analysis, and the data of 2 26-day-old rats in each rearing group were lost due to errors in video recording. Apparatus. Testing was conducted in a rectangular chamber (9 3 9 3 13 cm) with a Perspex ceiling and back panel, a hinged Perspex door at the front, and stainless steel rods on the floor and sides. The stainless steel rods were 3 mm in diameter, placed 1 cm apart center to center. The chamber was suspended in the center of a sound-attenuating box, above a metal tray containing paper pellet bedding. Illumination was provided by a 15-W white lightbulb, and a ventilation fan provided a background noise of 60 dB. A window in the door of the sound-attenuating box enabled video recording of the subject during test. Unscrambled shock could be delivered to the grid floor of the test chamber via a custom-built constant current generator. The floor and walls of the chamber were wiped with tap water between each trial, and fresh bedding was placed in the tray. Procedure. On the day of training each rat was placed in the test chamber and after 120 s given one foot shock (1 s, 0.9 mA). Animals were removed from the chamber 30 s after the shock. Twenty-four hours later each animal was returned to the chamber and the time spent freezing over a 2-min period was recorded (from the videotape). Scoring and statistics. The percentage of time that each rat was observed to be freezing was used as the dependent variable. A 2 3 2 factorial ANOVA with two rearing conditions (stimulated and nonstimulated) and two ages (18 and 26 days) was performed to examine the effects of rearing condition and age on freezing. Each rat was treated as an independent observation in the data analysis and each of the figures presented is based on these data. Separate analyses using the litter as the unit of analysis revealed the same pattern of results for each experiment. These analyses are also presented within the Results section of each experiment. A second rater who was naive with regard to the rearing group of each subject scored a random selection of 10 rats from the video recordings. The interrater reliability between the two raters on these 10 samples was very high (r 5 .98). An initial 2 3 2 factorial ANOVA of rearing group and sex revealed that the sex of the subject had no influence on the rates of freezing (F , 1), and no interaction was found between sex and rearing condition (F , 1). Therefore, sex was not included in subsequent analyses. Results and Discussion The mean percentage of time spent freezing during the 2-min reexposure to the test chamber by the pups in each rearing condition and at each age is presented in Fig. 1. Twenty-six-day-old rats exhibited more freezing during test than did 18-day-old rats [F(1, 31) 5 38.82; p , .05; by litter F(1, 21) 5 37.31; p , .05]; however, there were no differences between stimulated and nonstimulated rats (F , 1; by litter F , 1), and no interaction between rearing condition and age [F(1, 31) 5 1.30; p . .1; by litter F(1, 21) 5 3.93; p . .05]. Given the a priori prediction concerning improved performance in the 18-day-olds given multisensory stimulation, separate ANOVAs were conducted for each age group. This indicated that 18-day-old stimulated rats exhibited more freezing
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FIG. 1. Mean (6 SEM) percentage of freezing scores for 18- and 26-day-old stimulated and nonstimulated rats.
than did 18-day-old nonstimulated rats [F(1, 17) 5 5.19; p , .05; by litter F(1, 11) 5 4.85; p , .05], but no differences were found between the two groups of 26-day-olds [F , 1; by litter F(1, 19) 5 1.65; p . .1]. These results indicate that the stimulation procedure accelerated the emergence of longterm contextual memory in preweanling rats. Given that contextual conditioning is believed to be mediated by the hippocampus (e.g., Phillips & LeDoux, 1992; Rudy, 1993), the present findings suggest that the preweaning stimulation procedure accelerated the functional development of the hippocampus. The present study is the first to test for the effects of environmental stimulation on learning and memory during the preweaning period and hence is the first to report that environmental stimulation accelerates the development of learning and memory in the preweanling rat. Other studies on the effects of environmental stimulation during the preweaning period have focused exclusively on neural effects (Schapiro & Vukovich, 1970; Venable et al., 1989) or have waited until the animals are adults to examine improvements in learning and memory (Denenberg et al., 1968; Venable et al., 1988). One potential alternative explanation for the current findings is that stimulated rats were more active in the conditioning chamber prior to shock. If this was the case they may have been exposed to more cues than nonstimulated rats, which by itself could facilitate the formation of a more complex memory representation—irrespective of any general differences in learning and memory between the two rearing groups. To test this possibility the exploratory activity of 18-day-old stimulated and nonstimulated rats was examined. The conditioning chamber was mapped by identifying 12 “focus points.” These focus points included the eight corners of the chamber, as well as the points midway between the corners along the four longest sides of the chamber. Using the video recordings of the session in which shock was delivered, the number of different focus points visited by each subject in the chamber during the 2 min prior to shock was recorded. There were no differences between stimulated and nonstimulated rats in the number of different focus points visited in this 2 min period (F , 1; by litter F , 1), nor in the total number of focus points visited (F , 1; by litter F , 1). These findings indicate that stimulated rats
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were not exposed to more contextual cues than the nonstimulated rats in the conditioning chamber prior to shock. Given these findings, the enhanced contextual conditioning of stimulated rats in the present study is more likely to be a result of enhanced learning and memory rather than heightened exploratory activity in the conditioning chamber.
EXPERIMENT 2: LONG-TERM CONTEXTUAL MEMORY FOLLOWING ONE AND THREE TRAINING SHOCKS Although the environmentally stimulated 18-day-old rats in Experiment 1 exhibited enhanced long-term contextual memory relative to 18-day-old rats reared under standard conditions, these animals still exhibited very little memory of the test context (ie., a mean freezing score of 14%). Therefore, in the present experiment we attempted to replicate this finding as well as determine whether this difference would still be observed if the level of contextual conditioning was increased by increasing the number of training shocks.
Method Subjects. Thirty 18-day-old male and female rats from each rearing condition were used. The data of 1 nonstimulated rat in the one-shock group that displayed freezing levels 6 SD deviations above the mean was omitted from the analysis, and the data of 3 additional nonstimulated rats in the one-shock group, and 2 in the three shock group were not included due to errors in video recording. This resulted in there being 11 and 13 subjects in the nonstimulated one- and three-shock groups, respectively, and 15 subjects in each of the stimulated groups. Apparatus. The apparatus used for testing was the same as that used in Experiment 1. Procedure. The procedure used was similar to that of Experiment 1 with three exceptions. First, in addition to giving one training shock to some rats, others received three training shocks. A 30-s interval separated each shock in the three-shock condition. Second, the shock intensity was reduced from 0.9 to 0.5 mA. Third, the testing period was extended from 2 to 5 min. Scoring and statistics. The percentage of time that each rat was observed to be freezing was used as the dependent variable. A 2 3 2 factorial ANOVA with 2 groups (stimulated and nonstimulated rearing) and 2 conditions (one and three shocks) was performed to examine the effects of rearing condition and number of shocks on conditioned freezing. A second rater who was naive with regard to the group or condition of each subject scored a random selection of 10 rats from the video recordings. The interrater reliability between the two raters on these 10 samples was very high (r 5 .99).
Results and Discussion The mean percentage of time spent freezing during the 5-min reexposure to the conditioning chamber by stimulated and nonstimulated rats after one and three shocks is presented
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in Fig. 2. Higher levels of freezing were observed during test when three shocks had been given than when one shock had been given [F(1, 50) 5 39.89; p , .05; by litter F(1, 23) 5 16.95; p , .05]. Also, stimulated rats displayed higher levels of freezing than nonstimulated rats {F(1, 50) 5 7.11; p , .05; by litter F(1, 23) 5 4.39; p , .05}, but there was no interaction of rearing condition and number of shocks {F , 1; by litter F , 1}. The finding that stimulated rats given one shock displayed greater levels of freezing than nonstimulated rats replicated the findings from Experiment 1 with 18-dayold rats. Furthermore, this experiment demonstrated that even when a greater amount of learning occurred (in the three-shock condition), 18-day-old stimulated rats still showed more long-term contextual memory than did nonstimulated rats. EXPERIMENT 3: EXTINCTION OF LONG-TERM CONTEXTUAL MEMORY FOLLOWING FIVE SHOCKS Pilot tests indicated that stimulated and nonstimulated rats exhibited equivalent levels of contextual memory following five training shocks. Since the observed levels of freezing following five shocks were high, they may have been equivalent only because of a ceiling effect, therefore underestimating long-term contextual memory in the stimulated rats. In the present experiment preweanling rats given five training shocks were tested over 4 consecutive days to examine extinction of conditioned freezing. Methods Subjects. Ten 21-day-old female rats from each rearing condition were used. Data of 2 rats from the stimulated group were removed from the analysis due to experimenter error. Only female rats were used in this experiment because the males from each litter had been separated to be used in a postweaning environmental enrichment experiment. However, as noted in Experiment 1, sex does not appear to affect freezing rates in the preweanling rat.
FIG. 2. Mean (6 SEM) freezing scores in 18-day-old stimulated and nonstimulated rats after one and three training shocks.
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Apparatus. The apparatus used for three rats in the nonstimulated group and two rats in the stimulated group was a chamber (20 3 12 3 13 cm) with a Perspex roof and Perspex hinged door at the front, a rear metal panel, stainless steel rod floor, and wire mesh sides. However, during testing one of the animals that was removed from the analysis escaped from this chamber so the chamber described in Experiment 1 was used for the remaining rats. Procedure. After being in the chamber for 120 s each rat received five shocks (1 s; 0.5 mA), each separated by 30 s. The rats were removed from the environment 30 s following the last shock. On each of the 4 subsequent days following training, the rats were placed back into the chamber and freezing over a 5-min period was recorded. Scoring and statistics. The percentage of time that each rat was observed to be freezing was used as the dependent variable. The data were analyzed with a mixed-design ANOVA with Group as the between-subjects variable (stimulated vs nonstimulated) and Test day as the within-subject variable. Results and Discussion The mean percentage of time freezing on each of the 4 test days by stimulated and nonstimulated rats is presented in Fig. 3. Analysis of these results showed that freezing significantly decreased over testing days [F(3, 48) 5 46.82; p , .05; by litter F(3, 18) 5 20.24; p , .05]. In other words, extinction occurred. Further, and more interestingly, stimulated rats froze significantly less overall than did nonstimulated rats [F(1, 16) 5 4.85; p , .05; by litter F(1, 6) 5 8.30; p ,. 05]. Although the interaction of rearing condition and day of testing was not significant [F(3, 48) 5 1.27; p . .1; by litter F(3, 18) 5 1.20; p . .1] posthoc comparisons revealed that the two groups did not differ on the first test day (F , 1; by litter F , 1), but they did differ on the last test day [F(1, 16) 5 9.78; p , .05; by litter F(1, 6) 5 16.36; p , .05]. That is, stimulated rats showed a faster rate of extinction than did nonstimulated rats. One possible interpretation of this outcome is that although the rate of extinction can
FIG. 3. Mean (6 SEM) freezing scores over 4 days of extinction testing in 21-day-old stimulated and nonstimulated rats following five training shocks.
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be assumed to be a result of the strength of the original memory trace, it also involves the learning of a new memory (i.e., context and “no shock”). Hence, the observed rate of extinction may represent an interaction between the strength of the original memory trace and the ability of the animal to learn during extinction trials that the context no longer signals shock. This conceptualization of extinction is similar to that offered by Bouton (1993), who described extinction as a process involving retroactive interference, whereby the learning that occurs during extinction interferes with the memory that was formed during training. From this perspective, the greater extinction observed in the stimulated rats may be a result of their enhanced ability to learn that the context no longer signals shock. In other words, the present findings may be another instance of stimulated rats demonstrating superior performance on a task involving retroactive interference, like discrimination reversal learning (e.g., Krech, Rosenzweig, & Bennett, 1962). The results of the present experiment may also be another illustration of the idea that the superior learning ability of stimulated animals is more apparent on difficult tasks (assuming that learning that a previously dangerous place is now safe is more difficult than learning that an unfamiliar place is dangerous). In any case, the results of this experiment are consistent with the finding that adult rats reared in an enriched environment show faster extinction on an active avoidance task (Klicperova & Novakova, 1976). Finally, it should be noted that the results of the present experiment have an important practical implication. Specifically, the results of this experiment rule out any interpretations of the results of Experiments 1 and 2 that are based on the idea that stimulated rats simply freeze more than nonstimulated rats. EXPERIMENT 4: EFFECTS OF PREWEANING STIMULATION ON SHOCK SENSITIVITY In Experiments 1 and 2 preweanling rats given multisensory stimulation exhibited enhanced long-term contextual memory compared to nonstimulated rats. This effect was attributed to general improvements in learning and memory processes. However, this effect might have been the result of differences between stimulated and nonstimulated rats in shock sensitivity. More specifically, rats placed into a novel environment are often reported to exhibit an analgesic response, whereby pain thresholds are elevated (Bardo & Hughes, 1979). Given that the stimulated rats were repeatedly exposed to novel objects and environments, it could be argued that the test chamber was less novel to them, and, therefore, it elicited less novelty-induced analgesia. This difference in novelty-induced analgesia at the time of training would result in the shocks being perceived as more intense by the stimulated rats. This may then lead to enhanced long-term contextual memory (for an example of the effects of shock intensity on contextual conditioning see Bevins, McPhee, Rauhut, & Ayres, 1997). To test this possibility, a shock sensitivity experiment was conducted. Method Subjects. Twelve 18- to 19-day-old female rats from each rearing condition were used. Apparatus. The apparatus used for testing was the same as that used in Experiment 1.
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Procedure. Each rat was placed into the conditioning chamber and after 20 s exposed to a sequence of 32 shocks (each 1 s in duration) increasing in intensity from 0.15 to 0.3 mA. Each shock was separated by 3 s and the intensity was increased by 0.01 mA every second shock. The thresholds for the following three behavioral responses to shock were determined by an observer who was nai¨ve of the rearing condition of each rat: flinching, vocalization, and shuffling. Flinching was defined as a sudden, brief, muscle contraction, not involving any locomotion, while shuffling was a more pronounced movement involving locomotion. Vocalization was defined as any audible vocal sound. Jumping responses were also recorded, but these data were not included because only two rats exhibited this response. Thresholds were defined as the lowest shock intensity at which the behavior was exhibited. One rat that did not vocalize or shuffle at any point during the session was assigned the maximum value (0.3 mA) for those behaviors. Results and Discussion The thresholds for the three behavioral responses to shock in stimulated and nonstimulated rats are shown in Table 1. No significant differences between stimulated and nonstimulated rats were found for vocalization (F , 1; by litter F , 1), shuffling (F , 1; by litter F , 1) or flinching (F , 1; by litter F , 1). A repeated measures analysis revealed that the threshold for shuffling was significantly higher than that for flinching [F(1, 22) 5 45.72, p , .05; by litter F(2, 12) 5 14.98; p , .05]. Other investigators have also reported the highest thresholds for shuffling and lowest for flinching (e.g., Fessler & Beatty, 1976; Marks & Hobbs, 1972). These findings indicate that stimulated and nonstimulated preweanling rats experience the shock as equally aversive, thereby excluding differences in novelty-induced analgesia as an explanation for the observed differences in conditioned freezing between stimulated and nonstimulated rats. GENERAL DISCUSSION The primary result of this study was that preweaning rats given daily multisensory stimulation exhibited long-term contextual memory at a younger age than nonstimulated rats (Experiments 1 and 2). No effect of rearing condition was observed in slightly older rats (i.e., 26 days of age compared to 18; Experiment 1). One interpretation of this latter result is that exposure to multisensory stimulation during the preweaning period merely accelerates the functional maturation of the hippocampus; once that structure becomes fully functional in the nonstimulated rats (i.e., by 26 days of age), then there are no detectable differences between rats from the two rearing conditions in terms of contextual
TABLE 1 Mean (6SEM) Thresholds (in mA) for Shock-Elicited Vocalization, Shuffling, and Flinching in 18- to 19-Day-Old Stimulated and Nonstimulated Rats Group Nonstimulated Stimulated
Flinching
Vocalization
Shuffling
0.17 (0.0096) 0.17 (0.0076)
0.20 (0.0146) 0.20 (0.0077)
0.23 (0.0153) 0.23 (0.0131)
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conditioning. This interpretation is unlikely, however, given the plethora of studies showing beneficial effects of environmental enrichment on memory and problem-solving ability in adult rats. A more probable interpretation of this age-related effect is that the contextual conditioning procedure used in the present study is relatively simple for the 26-day-olds, and is, therefore, rather insensitive in detecting the altered learning and memory abilities of the stimulated rats. This notion is supported by reports that the learning abilities of adult enriched- and standard-reared rats are more easily discriminated when learning is assessed with complex, rather than simple, tasks (e.g., Krech, Rosenzweig, & Bennett, 1962; Ough, Beatty, & Khalili, 1972; Renner & Rosenzweig, 1987). From this perspective, a more difficult contextual conditioning procedure would be more sensitive in detecting the enhanced learning and memory abilities of stimulated rats. Support for this prediction is provided by a study by Woodcock and Richardson (2000) in which the time between placement in the training context and shock administration was varied. When this interval was long (e.g., 120 s), there were no differences between adult rats reared in enriched or standard environments in subsequent context freezing. However, when this interval was short (e.g., 16 s.; see Fanselow, 1986), standard-reared rats did not acquire a context–shock association but enriched-reared rats did. Reducing the time between placement in the context and shock administration reduced the time available to encode a representation of the environmental cues to associate with the shock. This reduction in the time available to encode a representation of the context attenuated learning in the standard-reared rats but not the enriched-reared rats. In other words, the enriched-reared rats appeared to be able to form a representation of the training context more rapidly than the standard-reared rats. Although the mechanisms mediating the enhanced learning following environmental stimulation are still uncertain, they most likely involve cellular changes induced by the increased neuronal activity resulting from the stimulation procedure. Of these cellular changes, one that is particularly relevant to the present study is an alteration in the hippocampal glucocorticoid receptor system, which has been implicated in contextual conditioning (Pugh, Fleshner, & Rudy, 1997a; Pugh, Tremblay, Fleshner, & Rudy, 1997b). For example, Pugh et al. (1997a) found that a pretraining injection of a glucocorticoid receptor antagonist disrupted contextual conditioning. From this, one might suggest that increasing the sensitivity of glucocorticoid receptors would enhance contextual conditioning, especially in animals that have an “impaired” system (e.g., preweanling rats that do not yet have a functionally mature system). Since rearing animals within an enriched environment has been shown to increase hippocampal glucocorticoid receptor expression (Mohammed et al., 1993), which in turn increases sensitivity to glucocorticoids, this appears to be a likely mechanism for the observed stimulation effects on long-term contextual memory. A final point to consider involves whether the effects of the present stimulation procedure on learning and memory can be attributed solely to the effects of handling. As outlined in the Introduction, there is a substantial literature on the effects of handling during the preweaning period (e.g., Denenberg, 1967; Meaney et al., 1991). One of the primary physiological effects of handling is a modification of the hypothalamic–pituitary–adrenal system. That is, handled rats are reported to have increased hippocampal glucocorticoid receptor numbers (Meaney & Aitken, 1985) and secrete less glucocorticoid in response
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to stress (Levine, 1957). These physiological effects of handling, and the behavioral effects described in the Introduction, are usually attributed to “stress” associated with being handled as well as changes in mother–infant interactions (Levine, 1975). This is in marked contrast to the interpretations of other early stimulation procedures where the effects (on learning, memory, and the brain) are usually attributed to increased opportunities to learn about and interact with stimuli in the environment. However, handling and other forms of environmental stimulation are remarkably similar in terms of the nature of the stimulation that each provides and their effects on the brain and behavior of the animal. For example, handling, like other early stimulation procedures, increases stimulation of the tactile, vestibular, kinesthetic, visual, and olfactory senses. In addition, the mothers of handled pups spend more time licking and grooming their pups which further increases the sensory stimulation received by the pup (Denenberg, 1999; Liu et al., 1997). Given the fact that handling and other forms of environmental stimulation increase stimulation of multiple sensory systems, it is not surprising that they all have similar effects on behavior and the brain. That is, handling and other stimulation procedures enhance learning and memory— particularly spatial memory (e.g., Meaney et al., 1991; Venable et al., 1988)—and lead to similar neural changes (Altman et al., 1968; Meaney, Aitken, Bhatnagar, Van Berkel, & Sapolsky, 1988; Olsson et al., 1994; Szeligo & Leblond, 1977). These findings suggest that handling is a particular form of environmental enrichment or stimulation. However, although handling stimulates many of the same sensory modalities as other stimulation procedures do, the multisensory stimulation procedure used in the present series of experiments provides a greater range and intensity of sensory experiences than handling alone and is therefore likely to enhance learning and memory ability and increase neural growth to a greater degree. This notion is supported by findings that both preweaning enrichedrearing and postweaning environmental enrichment combined with handling are more effective in improving learning and memory than handling alone (Greenough, Madden, & Fleischman, 1972; Ivinskis & Homewood, 1980). These findings—together with the known effects of isolated-rearing, superenriched environments, and seminatural environments—indicate that environmental stimulation represents a continuum from severe sensory and social isolation to intense levels of stimulation and learning opportunities. Comparisons of any two rearing procedures on the continuum generally show that the greatest neural changes and the most beneficial changes in learning and memory result from the procedure that provides the greater amount of environmental stimulation (Krech et al., 1960). On this continuum, handling appears to lie in between standard-rearing and other more intensive forms of environmental stimulation (e.g., multisensory stimulation or enriched-rearing). In addition, the finding that preweaning enrichment without handling (the entire litter is reared within an enriched environment) enhances problem-solving ability relative to standard-rearing (Denenberg et al., 1968; Forgays & Read, 1962) suggests that the present results are not solely due to the effects of handling. Therefore, in answer to our query of whether the present results are due to multisensory stimulation or to handling, it appears that the answer is both. That is, the effects observed are probably due to increased sensory stimulation received through handling and mother–infant interactions, which is enhanced by additional stimulation provided by other components of the enrichment procedure.
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Levine, S. (1957). Infantile experience and resistence to physiological stress. Science, 126, 405–406. Levine, S. (1975). Psychosocial factors in growth and development. In L. Lennart (Ed.), Society, stress and disease (Vol. 2): Childhood and adolescence (pp. 43–50). London: Oxford Univ. Press. Levine, S., Haltmeyer, G. C., Karas, G. G., & Denenberg, V. H. (1967). Physiological and behavioral effects of infantile stimulation. Physiology & Behavior, 16, 965–968. Liu, D., Diorio, J., Tannenbaum, B., Caldji, C., Francis, D., Freedman, A., Sharma, S., Pearson, D., Plotsky, P. M., & Meaney, M. J. (1997). Maternal care, hippocampal glucocorticoid receptors, and hypothalamicpituitary-adrenal responses to stress. Science, 277, 1659–1662. Malkasian, D. R., & Diamond, M. C. (1971). The effects of environmental manipulation on the morphology of the neonate rat brain. International Journal of Neuroscience, 2, 161–170. Marks, H. E., & Hobbs, S. H. (1972). Changes in stimulus reactivity following gonadectomy in male and female rats of different ages. Physiology & Behavior, 8, 1113–1119. McLaren, I. P. L., Kaye, H., & Mackintosh, N. J. (1989). An associative theory of the representation of stimuli: Applications to perceptual learning and latent inhibition. In R. G. M. Morris (Ed.), Parallel distributed processing—Implications for psychology and neurobiology (pp. 102–130). Oxford: Oxford Univ. Press. Meaney, M. J., & Aitken, D. H. (1985). The effects of early postnatal handling on hippocampal glucocorticoid receptor concentrations: Temporal parameters. Developmental Brain Research, 22, 301–304. Meaney, M. J., Aitken, D. H., Bhatnagar, S., Van Berkel, C., & Sapolsky, R. M. (1988). Postnatal handling attenuates neuroendocrine, anatomical, and cognitive impairments related to the aged hippocampus. Science, 238, 766–768. Meaney, M. J., Aitken, D. H., Bhatnagar, S., & Sapolsky, R. M. (1991). Postnatal handling attenuates certain neuroendocrine, anatomical, and cognitive dysfunctions associated with aging in female rats. Neurobiology of Aging, 12, 31–38. Mohammed, A. K., Henriksson, B. G., So¨derstro¨m, S., Ebendal, T., Olsson, T., & Seckl, J. R. (1993). Environmental influences on the central nervous system and their implications for the aging rat. Behavioural Brain Research, 57, 183–191. Olsson, T., Mohammed, A. H., Donaldson, L. F., Henriksson, B. G., & Seckl, J. R. (1994). Glucocorticoid receptor and NGFI-A gene expression are induced in the hippocampus after environmental enrichment in adult rats. Molecular Brain Research, 23, 349–353. Ough, B. R., Beatty, W. W., & Khalili, J. (1972). Effects of isolation and enriched rearing on response inhibition. Psychonomic Science, 27, 293–294. Pham, T. M., Soederstroem, S., Henriksson, B. G., & Mohammed, A. H. (1997). Effects of neonatal stimulation on later cognitive function and hippocampal nerve growth factor. Behavioural Brain Research, 86, 113–120. Phillips, R. G., & LeDoux, J. E. (1992). Differential contribution of amygdala and hippocampus to cued and contextual fear condtioning. Behavioral Neuroscience, 106, 274–285. Pugh, C. R., Fleshner, M., & Rudy, J. W. (1997a). Type II glucocorticoid receptor antagonists impair contextual but not auditory-cue fear conditioning in juvenile rats. Neurobiology of Learning and Memory, 67, 75–79. Pugh, C. R., Tremblay, D., Fleshner, M., & Rudy, J. W. (1997b). A selective role for corticosterone in contextualfear conditioning. Behavioral Neuroscience, 111, 503–511. Renner, M. J., & Rosenzweig, M. R. (1987). Enriched and impoverished environments: Effects on brain and behavior. New York: Springer-Verlag. Rudy, J. W. (1993). Contextual conditioning and auditory cue conditioning dissociate during development. Behavioral Neuroscience, 107, 887–891. Rudy, J. W., & Morledge, P. (1994). Ontogeny of contextual fear conditioning in rats: Implications for consolidation, infantile amnesia, and hippocampal system function. Behavioral Neuroscience, 108, 227–234. Schapiro, S., & Vukovich, K. R. (1970). Early experience effects upon cortical dendrites: A proposed model for development. Science, 167, 292–294. Smith, H. V. (1972). Effects of environmental enrichment on open-field activity and Hebb-Williams problem solving in rats. Journal of Comparative and Physiological Psychology, 80, 163–168.
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Szeligo, F., & Leblond, C. P. (1977). Response of the three main types of glial cells of cortex and corpus callosum in rats handled during suckling or exposed to enriched, control, and impoverished environments during weaning. Journal of Comparative Neurology, 172, 247–264. Venable, N., Ferna´ndez, V., Dı´az, E., & Pinto-Hamuy, T. (1989). Effects of preweaning environmental enrichment on basilar dendrites of pyramidal neurons in occipital cortex: A golgi study. Developmental Brain Research, 49, 140–144. Venable, N., Pinto-Hamuy, T., Arraztoa, J. A., Contador, M. T., Chellew, A., Pera´n, C., & Valenzuela, X. (1988). Greater efficacy of preweaning than postweaning environmental enrichment on maze learning in adult rats. Behavioural Brain Research, 31, 89–92. Walsh, R. N., Budtz-Olsen, O. E., Penny, J. E., & Cummins, R. A. (1969). The effects of environmental complexity on the histology of the rat hippocampus. Journal of Comparative Neurology, 137, 361–366. Wilson, D. A., Willner, J., Kurz, E. M., & Nadel, L. (1986). Early handling increases hippocampal long-term potentiation in young rats. Behavioural Brain Research, 21, 223–227. Woodcock, E. A. (1994). The enriched environment as a tool for investigating environmentally-mediated behavioural and neural plasticity. Unpublished honours’ thesis, Univ. of New South Wales, Sydney. Woodcock, E. A., & Richardson, R. (2000). Effects of environmental enrichment on rate of contextual processing and discriminative ability in adult rats. Neurobiology of Learning and Memory, 73, 1–10.
Effects of Multisensory Environmental Stimulation on Contextual Conditioning in the Developing Rat Elizabeth A. Woodcock and Rick Richardson University of New South Wales, Sydney, New South Wales 2052, Australia
The effect of increased exposure to multisensory stimulation during development on conditioned freezing to contextual cues in preweanling Sprague–Dawley rats was examined. Rats given increased environmental stimulation exhibited long-term contextual conditioning at a younger age than rats that did not receive such stimulation when there was either low or moderate levels of conditioning (Experiments 1 and 2). These differences in contextual conditioning were not a result of the stimulated rats reacting differently to shock (Experiment 4) or merely freezing more than the nonstimulated rats in all situations (Experiment 3). The role of the glucocorticoid system in the enhanced contextual learning of stimulated preweanling rats and the advantages of the contextual conditioning procedure for studying the effects of environmental stimulation are discussed. q 2000 Academic Press Key Words: multisensory stimulation; enriched environment; preweaning; contextual conditioning.
There is extensive evidence that the stimulation an animal receives from its external environment has a profound effect on its later behavior. Specifically, heightened sensory stimulation and opportunities for problem-solving enhance performance on a variety of learning and memory tasks (Renner & Rosenzweig, 1987). Most studies in this area have involved postweaning stimulation. During the postweaning stage of development, enhanced stimulation is typically provided by using an “enriched environment” procedure. In other words, the animals are placed in a large cage with various objects for the purpose of increasing the amount of sensory and social stimulation received. Rats that are raised in enriched environments show enhanced performance on problem-solving tasks such as the Hebb–Williams maze (Venable et al., 1988), the Lashley III maze (Greenough, Yuwiler, & Dollinger, 1973), and the radial arm maze (Juraska, Henderson, & Muller, 1984). Some studies in this area of research, however, have involved preweaning stimulation. Several different techniques have been used to provide enhanced stimulation during the This research was supported by a grant from the Australian Research Council to R.R. and constituted a part of the Ph.D. thesis by E.A.W., which was supported by an Australian Commonwealth Postgraduate Scholarship. Address correspondence and reprint requests to Rick Richardson, School of Psychology, University of NSW, Sydney, 2052, Australia. Fax: 61 2 9385 3641. E-mail: [email protected]. 89
1074-7427/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.
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preweaning period. Some researchers have utilized an enriched environment, like that described above, by either placing each litter into separate enriched environments (Denenberg, Woodcock, & Rosenberg, 1968; Ivinskis & Homewood, 1980) or by placing pups from different litters into a single enriched environment with one foster mother (Forgays & Read, 1962). In each case, this form of preweaning experience led to enhanced problemsolving ability on the Hebb–Williams maze during adulthood. However, since rats spend a large part of the preweaning period in close proximity to the nest, they may not take full advantage of the heightened levels of sensory stimulation provided by the enriched environment. Indeed, Denenberg et al. (1968) and Forgays and Read (1962) both found preweaning enrichment to be less effective than postweaning enrichment in terms of improving problem-solving ability. In order to provide more intensive stimulation for preweanling rats, Venable et al. (1988) individually removed the pups from the nest four times each day and exposed them to a variety of sensory experiences for 25 min. This intensive procedure provided rats with higher levels of tactile, vestibular, visual, auditory, and thermotactile stimulation and proved to be more effective in improving problemsolving ability during adulthood on the Hebb–Williams maze than postweaning stimulation. Another, more common, technique used to examine the effects of early environmental stimulation on later behavior is referred to as “handling.” All handling procedures involve some manipulation of the pups in the hands of the experimenter, but are nevertheless quite diverse. For example, handling often involves removing the animals from the nest each day and placing them into a separate holding container for durations typically ranging from 2 to 15 min (e.g., Denenberg, 1967; Escorihuela, Toben˜a, & Ferna´ndez-Terul, 1994; Meaney, Aitken, Bhatnagar, & Sapolsky, 1991). However, some researchers continue to hold the rats in their hands for this period (e.g., Levine, 1956), while others massage or “play” with the pups in their hands (Szeligo & Leblond, 1977). Handling rats during the preweaning period generally leads to reduced emotionality in mildly stressful situations, as observed by less freezing and defecation, and higher rates of exploration in adulthood (Levine, Haltmeyer, Karas, & Denenberg, 1967). However, early handling also leads to improvements during adulthood in learning and memory, as evidenced by superior performance of handled rats on tasks such as shuttlebox avoidance (Escorihuela et al., 1994) and the Morris water maze (Meaney et al., 1991). Many of the learning and memory tasks on which environmentally stimulated animals show superior performance are believed to be hippocampally mediated. Indeed, neuroanatomical analyses of the hippocampus following postweaning environmental stimulation have revealed changes in both fine and gross structure (Kempermann, Kuhn, & Gage, 1997; Olsson, Mohammed, Donaldson, Henriksson, & Seckl, 1994; Walsh, Budtz-Olsen, Penny, & Cummins, 1969). Neural modifications throughout the brain have also been reported following various forms of preweaning stimulation, including handling (Altman, Das, & Anderson, 1968), placement of the whole litter into an enriched environment (Malkasian & Diamond, 1971), and multisensory stimulation (Schapiro & Vukovich, 1970; Venable, Ferna´ndez, Dı´az, & Pinto-Hamuy, 1989). The neural changes following preweaning stimulation also include hippocampal modifications (e.g., Meaney & Aitken, 1985; Pham, Soederstroem, Henriksson, & Mohammed, 1997; Wilson, Willner, Kurz, & Nadel, 1986; Woodcock, 1994). Although the effects of preweaning environmental stimulation on neuroanatomy and
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neurochemistry have been examined both pre- and postweaning, its effects on learning and memory have only been examined after weaning. The present series of experiments therefore examined the effects of early environmental stimulation on learning and memory prior to weaning. A contextual conditioning procedure was employed for this purpose. This is particularly pertinent given the fact that contextual memory develops relatively late in the rat. That is, Rudy (1993) showed that rats 23, but not 18, days of age exhibited long-term (i.e., 24 h) memory of a context that had been paired with shock. However, these two age groups did not differ in expression of long-term memory of a discrete auditory stimulus that had been paired with shock. Further, 18-day-old rats also did not differ from 23-day-old rats in short-term contextual memory. The failure of the 18-dayold rats to exhibit long-term contextual memory was attributed to the late development of the hippocampus. Because enhanced environmental stimulation has been found to increase hippocampal development in preweanling rats (e.g., Meaney & Aitken, 1985; Pham et al., 1997; Wilson et al., 1986), we predicted that heightened environmental stimulation during the preweaning period would also accelerate the functional development of the hippocampus. In other words, the present study examined the hypothesis that preweanling rats exposed to increased environmental stimulation would exhibit long-term contextual memory at a younger age than rats that do not receive such stimulation. The contextual conditioning procedure has not previously been used with pre- or postweaning animals for the purpose of measuring the effects of environmental stimulation. In the contextual conditioning procedure rats are shocked in an environment and then reexposed to that same environment 24 h later. It is assumed that an integrated representation of the cues in the environment is constructed during the initial exposure to it. Subsequent exposure to cues belonging to this network elicits freezing responses as a result of the association between the network and shock (Kiernan, Westbrook, & Cranney, 1995; McLaren, Kaye, & Mackintosh, 1989). Animals with a better representation of the environmental cues would be predicted to show a greater degree of freezing; hence the degree of freezing can be used as a measure of the animal’s memory of the environment. GENERAL METHODS Experimental Design Each litter was assigned to one of two rearing conditions. “Stimulated” pups were exposed to an intensive sensory stimulation program for 1 h each day, from postnatal day (PND) 2 to the day prior to testing. “Nonstimulated” pups remained with their mothers and littermates during this daily 1-h period. The nonstimulated rats were not handled or removed from the nest during this period since it is believed that the stimulation received during handling—including the extra stimulation received from the mother when the pups are returned to the nest—forms a crucial component of preweaning stimulation and thus may act to reduce differences between stimulated and nonstimulated rats (see General Discussion). Nonstimulated rats tested at 26 days of age (Experiment 1) were handled the day prior to testing for 5 min in order to reduce behavioral reactivity to handling that occurs in rats of these ages (Smith, 1972). Nonstimulated rats 21 days of age and younger were not handled the day prior to testing since rats of these ages are not behaviorally reactive when handled.
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Subjects One hundred and forty-four rats from 35 litters were used in the study. Each litter was culled to 10 pups at PND 1 and housed for the duration of the experiment in a plastic maternity cage (30 3 45 3 16 cm). The cages were kept in a colony room with a 12-h light/dark cycle, and paper pellet bedding was changed once a week for all cages. Food and water were continuously available. All animals were treated in accord with the ethical guidelines of the American Psychological Association and all procedures were approved by the Animal Care and Ethics Committee at the University of New South Wales. Multisensory Stimulation Procedure In order to maximize any effects of early stimulation on the development of contextual memory, the present series of experiments used an intensive multisensory stimulation procedure based on the procedures of Venable et al. (1988, 1989) and Schapiro and Vukovich (1970). This preweaning stimulation procedure entailed removing the pups from the nest each day and exposing them to a wide range of sensory stimuli for 1 h. The stimuli were specifically chosen to stimulate auditory, visual, tactile, taste, olfactory, vestibular, and thermotactile senses. Each rat was given multisensory stimulation from PND 2 until the day prior to behavioral testing. The stimulation sessions involved placing the pups on a cold metal pan (at room temperature) and a metal plate heated to 348C and other surfaces of varying textures, such as sandpaper, brushes, and marbles, in order to stimulate their tactile sense. The olfactory sense was stimulated by exposing the pups to various odorants (e.g., lemon, peppermint) soaked on a cotton ball. The chemosensory sense was stimulated by applying various artificial tastes (e.g., lemon, vanilla) on a cotton bud to the pup’s mouth. Each day the animals were rolled slowly in a cardboard tube for 1 min for vestibular stimulation. For additional vestibular and thermotactile stimulation from PND 10 onward, the animals were placed in a bowl of warm water (348C) in which they swam for 10 s. From PND 12, a few days prior to eye-opening, visual stimulation was added to the stimulation session. This involved exposing the pups to a flashing light for 15 s. In order to ensure that the pups stayed active during this visual stimulation, they were stroked during this time. All of the sensory stimulation occurred in a 1-h period each day. At all other times, rats in the stimulation condition were kept with their mother and littermates in the standard plastic maternity cage. EXPERIMENT 1: LONG-TERM CONTEXTUAL MEMORY IN 18- AND 26-DAY-OLD RATS Experiment 1 examined the effects of preweaning environmental stimulation on contextual conditioning in 18- and 26-day-old rats. It has previously been reported that at 18 days of age rats do not show evidence of long-term contextual memory, but that this ability has emerged by 23 days of age (e.g., Rudy & Morledge, 1994). Rudy and Morledge hypothesized that the emergence of long-term contextual memory between 18 and 23 days of age results from the maturation of the hippocampus during this same period. Therefore, if environmental stimulation accelerates the maturation of the hippocampus as hypothesized, long-term contextual memory should emerge at a younger age in stimulated rats. Hence, it was predicted that 18-day-old stimulated rats in the present experiment would show long-term contextual memory.
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Method Subjects. Twenty male and female rats from each rearing condition were used in this experiment. Ten of the rats from each rearing condition were tested at 18 days of age and 10 at 26 days of age. The data of 1 18-day-old rat in the nonstimulated group that displayed levels of freezing 16 SD above the mean was omitted from the analysis, and the data of 2 26-day-old rats in each rearing group were lost due to errors in video recording. Apparatus. Testing was conducted in a rectangular chamber (9 3 9 3 13 cm) with a Perspex ceiling and back panel, a hinged Perspex door at the front, and stainless steel rods on the floor and sides. The stainless steel rods were 3 mm in diameter, placed 1 cm apart center to center. The chamber was suspended in the center of a sound-attenuating box, above a metal tray containing paper pellet bedding. Illumination was provided by a 15-W white lightbulb, and a ventilation fan provided a background noise of 60 dB. A window in the door of the sound-attenuating box enabled video recording of the subject during test. Unscrambled shock could be delivered to the grid floor of the test chamber via a custom-built constant current generator. The floor and walls of the chamber were wiped with tap water between each trial, and fresh bedding was placed in the tray. Procedure. On the day of training each rat was placed in the test chamber and after 120 s given one foot shock (1 s, 0.9 mA). Animals were removed from the chamber 30 s after the shock. Twenty-four hours later each animal was returned to the chamber and the time spent freezing over a 2-min period was recorded (from the videotape). Scoring and statistics. The percentage of time that each rat was observed to be freezing was used as the dependent variable. A 2 3 2 factorial ANOVA with two rearing conditions (stimulated and nonstimulated) and two ages (18 and 26 days) was performed to examine the effects of rearing condition and age on freezing. Each rat was treated as an independent observation in the data analysis and each of the figures presented is based on these data. Separate analyses using the litter as the unit of analysis revealed the same pattern of results for each experiment. These analyses are also presented within the Results section of each experiment. A second rater who was naive with regard to the rearing group of each subject scored a random selection of 10 rats from the video recordings. The interrater reliability between the two raters on these 10 samples was very high (r 5 .98). An initial 2 3 2 factorial ANOVA of rearing group and sex revealed that the sex of the subject had no influence on the rates of freezing (F , 1), and no interaction was found between sex and rearing condition (F , 1). Therefore, sex was not included in subsequent analyses. Results and Discussion The mean percentage of time spent freezing during the 2-min reexposure to the test chamber by the pups in each rearing condition and at each age is presented in Fig. 1. Twenty-six-day-old rats exhibited more freezing during test than did 18-day-old rats [F(1, 31) 5 38.82; p , .05; by litter F(1, 21) 5 37.31; p , .05]; however, there were no differences between stimulated and nonstimulated rats (F , 1; by litter F , 1), and no interaction between rearing condition and age [F(1, 31) 5 1.30; p . .1; by litter F(1, 21) 5 3.93; p . .05]. Given the a priori prediction concerning improved performance in the 18-day-olds given multisensory stimulation, separate ANOVAs were conducted for each age group. This indicated that 18-day-old stimulated rats exhibited more freezing
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FIG. 1. Mean (6 SEM) percentage of freezing scores for 18- and 26-day-old stimulated and nonstimulated rats.
than did 18-day-old nonstimulated rats [F(1, 17) 5 5.19; p , .05; by litter F(1, 11) 5 4.85; p , .05], but no differences were found between the two groups of 26-day-olds [F , 1; by litter F(1, 19) 5 1.65; p . .1]. These results indicate that the stimulation procedure accelerated the emergence of longterm contextual memory in preweanling rats. Given that contextual conditioning is believed to be mediated by the hippocampus (e.g., Phillips & LeDoux, 1992; Rudy, 1993), the present findings suggest that the preweaning stimulation procedure accelerated the functional development of the hippocampus. The present study is the first to test for the effects of environmental stimulation on learning and memory during the preweaning period and hence is the first to report that environmental stimulation accelerates the development of learning and memory in the preweanling rat. Other studies on the effects of environmental stimulation during the preweaning period have focused exclusively on neural effects (Schapiro & Vukovich, 1970; Venable et al., 1989) or have waited until the animals are adults to examine improvements in learning and memory (Denenberg et al., 1968; Venable et al., 1988). One potential alternative explanation for the current findings is that stimulated rats were more active in the conditioning chamber prior to shock. If this was the case they may have been exposed to more cues than nonstimulated rats, which by itself could facilitate the formation of a more complex memory representation—irrespective of any general differences in learning and memory between the two rearing groups. To test this possibility the exploratory activity of 18-day-old stimulated and nonstimulated rats was examined. The conditioning chamber was mapped by identifying 12 “focus points.” These focus points included the eight corners of the chamber, as well as the points midway between the corners along the four longest sides of the chamber. Using the video recordings of the session in which shock was delivered, the number of different focus points visited by each subject in the chamber during the 2 min prior to shock was recorded. There were no differences between stimulated and nonstimulated rats in the number of different focus points visited in this 2 min period (F , 1; by litter F , 1), nor in the total number of focus points visited (F , 1; by litter F , 1). These findings indicate that stimulated rats
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were not exposed to more contextual cues than the nonstimulated rats in the conditioning chamber prior to shock. Given these findings, the enhanced contextual conditioning of stimulated rats in the present study is more likely to be a result of enhanced learning and memory rather than heightened exploratory activity in the conditioning chamber.
EXPERIMENT 2: LONG-TERM CONTEXTUAL MEMORY FOLLOWING ONE AND THREE TRAINING SHOCKS Although the environmentally stimulated 18-day-old rats in Experiment 1 exhibited enhanced long-term contextual memory relative to 18-day-old rats reared under standard conditions, these animals still exhibited very little memory of the test context (ie., a mean freezing score of 14%). Therefore, in the present experiment we attempted to replicate this finding as well as determine whether this difference would still be observed if the level of contextual conditioning was increased by increasing the number of training shocks.
Method Subjects. Thirty 18-day-old male and female rats from each rearing condition were used. The data of 1 nonstimulated rat in the one-shock group that displayed freezing levels 6 SD deviations above the mean was omitted from the analysis, and the data of 3 additional nonstimulated rats in the one-shock group, and 2 in the three shock group were not included due to errors in video recording. This resulted in there being 11 and 13 subjects in the nonstimulated one- and three-shock groups, respectively, and 15 subjects in each of the stimulated groups. Apparatus. The apparatus used for testing was the same as that used in Experiment 1. Procedure. The procedure used was similar to that of Experiment 1 with three exceptions. First, in addition to giving one training shock to some rats, others received three training shocks. A 30-s interval separated each shock in the three-shock condition. Second, the shock intensity was reduced from 0.9 to 0.5 mA. Third, the testing period was extended from 2 to 5 min. Scoring and statistics. The percentage of time that each rat was observed to be freezing was used as the dependent variable. A 2 3 2 factorial ANOVA with 2 groups (stimulated and nonstimulated rearing) and 2 conditions (one and three shocks) was performed to examine the effects of rearing condition and number of shocks on conditioned freezing. A second rater who was naive with regard to the group or condition of each subject scored a random selection of 10 rats from the video recordings. The interrater reliability between the two raters on these 10 samples was very high (r 5 .99).
Results and Discussion The mean percentage of time spent freezing during the 5-min reexposure to the conditioning chamber by stimulated and nonstimulated rats after one and three shocks is presented
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in Fig. 2. Higher levels of freezing were observed during test when three shocks had been given than when one shock had been given [F(1, 50) 5 39.89; p , .05; by litter F(1, 23) 5 16.95; p , .05]. Also, stimulated rats displayed higher levels of freezing than nonstimulated rats {F(1, 50) 5 7.11; p , .05; by litter F(1, 23) 5 4.39; p , .05}, but there was no interaction of rearing condition and number of shocks {F , 1; by litter F , 1}. The finding that stimulated rats given one shock displayed greater levels of freezing than nonstimulated rats replicated the findings from Experiment 1 with 18-dayold rats. Furthermore, this experiment demonstrated that even when a greater amount of learning occurred (in the three-shock condition), 18-day-old stimulated rats still showed more long-term contextual memory than did nonstimulated rats. EXPERIMENT 3: EXTINCTION OF LONG-TERM CONTEXTUAL MEMORY FOLLOWING FIVE SHOCKS Pilot tests indicated that stimulated and nonstimulated rats exhibited equivalent levels of contextual memory following five training shocks. Since the observed levels of freezing following five shocks were high, they may have been equivalent only because of a ceiling effect, therefore underestimating long-term contextual memory in the stimulated rats. In the present experiment preweanling rats given five training shocks were tested over 4 consecutive days to examine extinction of conditioned freezing. Methods Subjects. Ten 21-day-old female rats from each rearing condition were used. Data of 2 rats from the stimulated group were removed from the analysis due to experimenter error. Only female rats were used in this experiment because the males from each litter had been separated to be used in a postweaning environmental enrichment experiment. However, as noted in Experiment 1, sex does not appear to affect freezing rates in the preweanling rat.
FIG. 2. Mean (6 SEM) freezing scores in 18-day-old stimulated and nonstimulated rats after one and three training shocks.
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Apparatus. The apparatus used for three rats in the nonstimulated group and two rats in the stimulated group was a chamber (20 3 12 3 13 cm) with a Perspex roof and Perspex hinged door at the front, a rear metal panel, stainless steel rod floor, and wire mesh sides. However, during testing one of the animals that was removed from the analysis escaped from this chamber so the chamber described in Experiment 1 was used for the remaining rats. Procedure. After being in the chamber for 120 s each rat received five shocks (1 s; 0.5 mA), each separated by 30 s. The rats were removed from the environment 30 s following the last shock. On each of the 4 subsequent days following training, the rats were placed back into the chamber and freezing over a 5-min period was recorded. Scoring and statistics. The percentage of time that each rat was observed to be freezing was used as the dependent variable. The data were analyzed with a mixed-design ANOVA with Group as the between-subjects variable (stimulated vs nonstimulated) and Test day as the within-subject variable. Results and Discussion The mean percentage of time freezing on each of the 4 test days by stimulated and nonstimulated rats is presented in Fig. 3. Analysis of these results showed that freezing significantly decreased over testing days [F(3, 48) 5 46.82; p , .05; by litter F(3, 18) 5 20.24; p , .05]. In other words, extinction occurred. Further, and more interestingly, stimulated rats froze significantly less overall than did nonstimulated rats [F(1, 16) 5 4.85; p , .05; by litter F(1, 6) 5 8.30; p ,. 05]. Although the interaction of rearing condition and day of testing was not significant [F(3, 48) 5 1.27; p . .1; by litter F(3, 18) 5 1.20; p . .1] posthoc comparisons revealed that the two groups did not differ on the first test day (F , 1; by litter F , 1), but they did differ on the last test day [F(1, 16) 5 9.78; p , .05; by litter F(1, 6) 5 16.36; p , .05]. That is, stimulated rats showed a faster rate of extinction than did nonstimulated rats. One possible interpretation of this outcome is that although the rate of extinction can
FIG. 3. Mean (6 SEM) freezing scores over 4 days of extinction testing in 21-day-old stimulated and nonstimulated rats following five training shocks.
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be assumed to be a result of the strength of the original memory trace, it also involves the learning of a new memory (i.e., context and “no shock”). Hence, the observed rate of extinction may represent an interaction between the strength of the original memory trace and the ability of the animal to learn during extinction trials that the context no longer signals shock. This conceptualization of extinction is similar to that offered by Bouton (1993), who described extinction as a process involving retroactive interference, whereby the learning that occurs during extinction interferes with the memory that was formed during training. From this perspective, the greater extinction observed in the stimulated rats may be a result of their enhanced ability to learn that the context no longer signals shock. In other words, the present findings may be another instance of stimulated rats demonstrating superior performance on a task involving retroactive interference, like discrimination reversal learning (e.g., Krech, Rosenzweig, & Bennett, 1962). The results of the present experiment may also be another illustration of the idea that the superior learning ability of stimulated animals is more apparent on difficult tasks (assuming that learning that a previously dangerous place is now safe is more difficult than learning that an unfamiliar place is dangerous). In any case, the results of this experiment are consistent with the finding that adult rats reared in an enriched environment show faster extinction on an active avoidance task (Klicperova & Novakova, 1976). Finally, it should be noted that the results of the present experiment have an important practical implication. Specifically, the results of this experiment rule out any interpretations of the results of Experiments 1 and 2 that are based on the idea that stimulated rats simply freeze more than nonstimulated rats. EXPERIMENT 4: EFFECTS OF PREWEANING STIMULATION ON SHOCK SENSITIVITY In Experiments 1 and 2 preweanling rats given multisensory stimulation exhibited enhanced long-term contextual memory compared to nonstimulated rats. This effect was attributed to general improvements in learning and memory processes. However, this effect might have been the result of differences between stimulated and nonstimulated rats in shock sensitivity. More specifically, rats placed into a novel environment are often reported to exhibit an analgesic response, whereby pain thresholds are elevated (Bardo & Hughes, 1979). Given that the stimulated rats were repeatedly exposed to novel objects and environments, it could be argued that the test chamber was less novel to them, and, therefore, it elicited less novelty-induced analgesia. This difference in novelty-induced analgesia at the time of training would result in the shocks being perceived as more intense by the stimulated rats. This may then lead to enhanced long-term contextual memory (for an example of the effects of shock intensity on contextual conditioning see Bevins, McPhee, Rauhut, & Ayres, 1997). To test this possibility, a shock sensitivity experiment was conducted. Method Subjects. Twelve 18- to 19-day-old female rats from each rearing condition were used. Apparatus. The apparatus used for testing was the same as that used in Experiment 1.
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Procedure. Each rat was placed into the conditioning chamber and after 20 s exposed to a sequence of 32 shocks (each 1 s in duration) increasing in intensity from 0.15 to 0.3 mA. Each shock was separated by 3 s and the intensity was increased by 0.01 mA every second shock. The thresholds for the following three behavioral responses to shock were determined by an observer who was nai¨ve of the rearing condition of each rat: flinching, vocalization, and shuffling. Flinching was defined as a sudden, brief, muscle contraction, not involving any locomotion, while shuffling was a more pronounced movement involving locomotion. Vocalization was defined as any audible vocal sound. Jumping responses were also recorded, but these data were not included because only two rats exhibited this response. Thresholds were defined as the lowest shock intensity at which the behavior was exhibited. One rat that did not vocalize or shuffle at any point during the session was assigned the maximum value (0.3 mA) for those behaviors. Results and Discussion The thresholds for the three behavioral responses to shock in stimulated and nonstimulated rats are shown in Table 1. No significant differences between stimulated and nonstimulated rats were found for vocalization (F , 1; by litter F , 1), shuffling (F , 1; by litter F , 1) or flinching (F , 1; by litter F , 1). A repeated measures analysis revealed that the threshold for shuffling was significantly higher than that for flinching [F(1, 22) 5 45.72, p , .05; by litter F(2, 12) 5 14.98; p , .05]. Other investigators have also reported the highest thresholds for shuffling and lowest for flinching (e.g., Fessler & Beatty, 1976; Marks & Hobbs, 1972). These findings indicate that stimulated and nonstimulated preweanling rats experience the shock as equally aversive, thereby excluding differences in novelty-induced analgesia as an explanation for the observed differences in conditioned freezing between stimulated and nonstimulated rats. GENERAL DISCUSSION The primary result of this study was that preweaning rats given daily multisensory stimulation exhibited long-term contextual memory at a younger age than nonstimulated rats (Experiments 1 and 2). No effect of rearing condition was observed in slightly older rats (i.e., 26 days of age compared to 18; Experiment 1). One interpretation of this latter result is that exposure to multisensory stimulation during the preweaning period merely accelerates the functional maturation of the hippocampus; once that structure becomes fully functional in the nonstimulated rats (i.e., by 26 days of age), then there are no detectable differences between rats from the two rearing conditions in terms of contextual
TABLE 1 Mean (6SEM) Thresholds (in mA) for Shock-Elicited Vocalization, Shuffling, and Flinching in 18- to 19-Day-Old Stimulated and Nonstimulated Rats Group Nonstimulated Stimulated
Flinching
Vocalization
Shuffling
0.17 (0.0096) 0.17 (0.0076)
0.20 (0.0146) 0.20 (0.0077)
0.23 (0.0153) 0.23 (0.0131)
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conditioning. This interpretation is unlikely, however, given the plethora of studies showing beneficial effects of environmental enrichment on memory and problem-solving ability in adult rats. A more probable interpretation of this age-related effect is that the contextual conditioning procedure used in the present study is relatively simple for the 26-day-olds, and is, therefore, rather insensitive in detecting the altered learning and memory abilities of the stimulated rats. This notion is supported by reports that the learning abilities of adult enriched- and standard-reared rats are more easily discriminated when learning is assessed with complex, rather than simple, tasks (e.g., Krech, Rosenzweig, & Bennett, 1962; Ough, Beatty, & Khalili, 1972; Renner & Rosenzweig, 1987). From this perspective, a more difficult contextual conditioning procedure would be more sensitive in detecting the enhanced learning and memory abilities of stimulated rats. Support for this prediction is provided by a study by Woodcock and Richardson (2000) in which the time between placement in the training context and shock administration was varied. When this interval was long (e.g., 120 s), there were no differences between adult rats reared in enriched or standard environments in subsequent context freezing. However, when this interval was short (e.g., 16 s.; see Fanselow, 1986), standard-reared rats did not acquire a context–shock association but enriched-reared rats did. Reducing the time between placement in the context and shock administration reduced the time available to encode a representation of the environmental cues to associate with the shock. This reduction in the time available to encode a representation of the context attenuated learning in the standard-reared rats but not the enriched-reared rats. In other words, the enriched-reared rats appeared to be able to form a representation of the training context more rapidly than the standard-reared rats. Although the mechanisms mediating the enhanced learning following environmental stimulation are still uncertain, they most likely involve cellular changes induced by the increased neuronal activity resulting from the stimulation procedure. Of these cellular changes, one that is particularly relevant to the present study is an alteration in the hippocampal glucocorticoid receptor system, which has been implicated in contextual conditioning (Pugh, Fleshner, & Rudy, 1997a; Pugh, Tremblay, Fleshner, & Rudy, 1997b). For example, Pugh et al. (1997a) found that a pretraining injection of a glucocorticoid receptor antagonist disrupted contextual conditioning. From this, one might suggest that increasing the sensitivity of glucocorticoid receptors would enhance contextual conditioning, especially in animals that have an “impaired” system (e.g., preweanling rats that do not yet have a functionally mature system). Since rearing animals within an enriched environment has been shown to increase hippocampal glucocorticoid receptor expression (Mohammed et al., 1993), which in turn increases sensitivity to glucocorticoids, this appears to be a likely mechanism for the observed stimulation effects on long-term contextual memory. A final point to consider involves whether the effects of the present stimulation procedure on learning and memory can be attributed solely to the effects of handling. As outlined in the Introduction, there is a substantial literature on the effects of handling during the preweaning period (e.g., Denenberg, 1967; Meaney et al., 1991). One of the primary physiological effects of handling is a modification of the hypothalamic–pituitary–adrenal system. That is, handled rats are reported to have increased hippocampal glucocorticoid receptor numbers (Meaney & Aitken, 1985) and secrete less glucocorticoid in response
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to stress (Levine, 1957). These physiological effects of handling, and the behavioral effects described in the Introduction, are usually attributed to “stress” associated with being handled as well as changes in mother–infant interactions (Levine, 1975). This is in marked contrast to the interpretations of other early stimulation procedures where the effects (on learning, memory, and the brain) are usually attributed to increased opportunities to learn about and interact with stimuli in the environment. However, handling and other forms of environmental stimulation are remarkably similar in terms of the nature of the stimulation that each provides and their effects on the brain and behavior of the animal. For example, handling, like other early stimulation procedures, increases stimulation of the tactile, vestibular, kinesthetic, visual, and olfactory senses. In addition, the mothers of handled pups spend more time licking and grooming their pups which further increases the sensory stimulation received by the pup (Denenberg, 1999; Liu et al., 1997). Given the fact that handling and other forms of environmental stimulation increase stimulation of multiple sensory systems, it is not surprising that they all have similar effects on behavior and the brain. That is, handling and other stimulation procedures enhance learning and memory— particularly spatial memory (e.g., Meaney et al., 1991; Venable et al., 1988)—and lead to similar neural changes (Altman et al., 1968; Meaney, Aitken, Bhatnagar, Van Berkel, & Sapolsky, 1988; Olsson et al., 1994; Szeligo & Leblond, 1977). These findings suggest that handling is a particular form of environmental enrichment or stimulation. However, although handling stimulates many of the same sensory modalities as other stimulation procedures do, the multisensory stimulation procedure used in the present series of experiments provides a greater range and intensity of sensory experiences than handling alone and is therefore likely to enhance learning and memory ability and increase neural growth to a greater degree. This notion is supported by findings that both preweaning enrichedrearing and postweaning environmental enrichment combined with handling are more effective in improving learning and memory than handling alone (Greenough, Madden, & Fleischman, 1972; Ivinskis & Homewood, 1980). These findings—together with the known effects of isolated-rearing, superenriched environments, and seminatural environments—indicate that environmental stimulation represents a continuum from severe sensory and social isolation to intense levels of stimulation and learning opportunities. Comparisons of any two rearing procedures on the continuum generally show that the greatest neural changes and the most beneficial changes in learning and memory result from the procedure that provides the greater amount of environmental stimulation (Krech et al., 1960). On this continuum, handling appears to lie in between standard-rearing and other more intensive forms of environmental stimulation (e.g., multisensory stimulation or enriched-rearing). In addition, the finding that preweaning enrichment without handling (the entire litter is reared within an enriched environment) enhances problem-solving ability relative to standard-rearing (Denenberg et al., 1968; Forgays & Read, 1962) suggests that the present results are not solely due to the effects of handling. Therefore, in answer to our query of whether the present results are due to multisensory stimulation or to handling, it appears that the answer is both. That is, the effects observed are probably due to increased sensory stimulation received through handling and mother–infant interactions, which is enhanced by additional stimulation provided by other components of the enrichment procedure.
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