Effects of Ovarian Hormones and Environment on Radial Maze and Water Maze Performance of Female Rats

Effects of Ovarian Hormones and Environment on Radial Maze and Water Maze Performance of Female Rats

Physiology & Behavior, Vol. 66, No. 1, pp. 11–20, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/99/$–see front m...

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Physiology & Behavior, Vol. 66, No. 1, pp. 11–20, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/99/$–see front matter

PII S0031-9384(98)00272-8

Effects of Ovarian Hormones and Environment on Radial Maze and Water Maze Performance of Female Rats JILL M. DANIEL,* SHARON L. ROBERTS* AND GARY P. DOHANICH†1 *Department of Psychology, Tulane University, †Department of Psychology and Neuroscience Program, Tulane University, 2007 Percival Stern Hall, New Orleans, LA 70118 Received 8 June 1998; Accepted 31 August 1998 DANIEL, J. M., S. L. ROBERTS AND G. P. DOHANICH. Effects of ovarian hormones and environment on radial maze and water maze performance of female rats. PHYSIOL BEHAV 66(1) 11–20, 1999.—The effects of gonadal hormones and environment on performance in an eight-arm radial maze and in the Morris water maze were determined in female rats. Long–Evans female rats were ovariectomized or sham ovariectomized at 35 days of age, and housed in complex environments or in isolation for the duration of the study. One month following surgeries, spatial working memory performance in the radial maze was assessed. Exposure to complex environmental conditions independently enhanced performance, as indicated by increased arm choice accuracy during 20 days of maze training. Additionally, gonadally intact females significantly outperformed ovariectomized females before cyclicity was disrupted by food deprivation. Following radial maze training, spatial reference memory performance was assessed in the same females utilizing the Morris water maze. Gonadally intact females housed in isolation performed significantly more poorly during 16 days of place training trials and displayed significantly shorter times in the platform quadrants and fewer target crossings during probe trials than gonadally intact and ovariectomized females housed in complex environments and ovariectomized rats housed in isolation. Consequently, acquisition and retention of the water maze was impaired by the presence of ovaries, and this impairment was counteracted by exposure to complex environments. Performance did not differ between groups on cued trials, indicating that sensorimotor and motivational functions did not differ between groups. Results of these experiments indicate that endogenous gonadal hormones can differentially affect performance on tasks of spatial working and spatial reference memory, and that environmental conditions can interact with gonadal hormones to affect behavior. © 1999 Elsevier Science Inc. Working memory Reference memory Estradiol Estrogen Progesterone

Learning Radial maze Complex environment

Ovarian hormones

ioral effects of exposure to complex environments. Four studies to date have included females. In the first report, performance during acquisition of an eight-arm radial maze with all arms baited was enhanced in both gonadally intact male and female rats following exposure to complex environmental conditions (12). In a later study, it was found that both gonadally intact male and female rats housed in complex conditions made fewer errors than their isolated littermates during the acquisition of a 17-arm radial maze with all arms baited (36). In a study that examined the effects of exposure to complex environmental conditions on working and reference memory performance utilizing a 17-arm radial maze with 11 arms baited, gonadally intact rats of both sexes reared in complex environments made fewer working and reference memory er-

ENVIRONMENTAL and hormonal conditions can influence the expression of a variety of mammalian behaviors, including learning and memory. Environmental conditions can be manipulated by comparing subjects reared in a complex environment, in which rodents are housed in social groups with a changing set of stimuli, to subjects reared in an isolated environment, in which rodents are housed individually in standard stainless steel laboratory cages (55). A large body of evidence indicates that rodents housed in complex environmental conditions display enhanced performance compared to rodents housed in isolated environmental conditions on tasks that measure learning and memory, including a variety of dry-land mazes (6,17,29,31,32,61) and the Morris water maze (48). Male rodents primarily have been used to examine the behav1To

Water maze

whom requests for reprints should be addressed. E-mail: [email protected]

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12 rors than did their littermates reared in isolation during 24 days of maze training (56). Additionally, female mice reared in complex environments displayed significantly shorter swim paths than did females reared in isolation on a place training task in the Morris water maze (38). Recently, it has been demonstrated that the gonadal hormone, estrogen, can also exert effects on learning and memory performance. In contrast to the consistently positive effects of exposure to complex environmental conditions, the effects of estrogen on learning and memory in mammals vary with task and gender, as well as levels and schedules of estrogen exposure. Available evidence indicates that estrogen enhances performance on tasks that are dependent on spatial working memory, usually measured in T mazes and radialarm mazes. For example, in an eight-arm radial maze, estradiol delivered via Silastic capsules improved working memory performance in male rats when delays were instituted between the fourth and fifth arm choices on posttraining tests (45). Young gonadectomized females with Silastic implants that produced proestrous levels of estradiol (90 pg/mL) failed to display similar improvements, although capsules that generated diestrous levels of estradiol (15 pg/mL) enhanced performance (43). In another series of experiments, both low (40 pg/mL) and high levels of estradiol (200 pg/mL) delivered via Silastic implants to young and aged ovariectomized rats resulted in an improvement of working memory on a 12-arm radial maze task with eight arms baited and four arms unbaited (71). In our laboratory, estradiol delivered by Silastic capsules at diestrous levels for 4 weeks prior to eight-arm radial maze acquisition, during acquisition, or both, also improved choice accuracy in ovariectomized rats (10), an enhancement limited to working memory (15). Additionally, systemic administration of a physiological dose of estrogen significantly improved choice accuracy during acquisition of reinforced T-maze alternation in ovariectomized rats (14). Despite the dendritic proliferation in hippocampal morphology reported at proestrus (73,74), no differences in the acquisition or performance of an eight-arm radial maze were found across the estrous cycle in female rats, although sensorimotor or motivational factors were impaired at proestrus (62). No study to date has compared the performance of gonadally intact female rats to ovariectomized rats on a task dependent upon spatial working memory. The effects of ovarian hormones on performance in the Morris water maze are dependent on both treatment regimen and behavioral measure. Although posttraining administration of a water-soluble form of estradiol within 1 h after a place training session in the Morris water maze improved performance on a retention test 24 h later in both gonadally intact male and gonadectomized female rats (52–54), most studies indicate that ovarian hormones fail to enhance acquisition or retention in the Morris water maze, when employed conventionally as a measure of spatial reference memory (4,59). In some studies, the presence of endogenous or exogenous ovarian hormones in female rodents actually impaired escape latency during place training in the water maze (18,19,40,69). Interestingly, when the water maze was adapted to assess working memory utilizing a water-escape, matching-to-sample task, estradiol administered to ovariectomized rats enhanced performance (51). The objectives of the current experiments were to compare the independent effects and to examine the interactive effects of two factors—ovarian hormones and environment—each of which has been demonstrated to influence learning and memory performance. No previous study has manipulated envi-

DANIEL, ROBERTS AND DOHANICH ronmental and gonadal hormone conditions concurrently. In the first experiment, spatial working memory performance in an eight-arm radial maze was assessed in ovariectomized and gonadally intact female rats housed in either complex or isolated environmental conditions. In the second experiment, spatial reference memory performance in the Morris water maze was assessed in the same animals. METHODS

Subjects Forty Long–Evans hooded females rats, approximately 25 days of age, were purchased from Harlan–Sprague–Dawley, Inc. (Indianapolis, IN). Animal care was in accordance with guidelines set by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were housed in a temperature-controlled vivarium under a 12-h light–dark cycle (lights on at 0700 h). At 35 days of age, 20 of the animals were ovariectomized and 20 underwent sham surgeries, while under anesthesia induced by injection of ketamine (100 mg/kg i.m.; Bristol Laboratories, Syracuse, NY) and xylazine (7.0 mg/kg i.m., Miles Laboratories, Shawnee, KS). Six Long– Evans male rats were housed in pairs in the same vivarium with the females throughout the course of the experiments to promote cyclicity. Environments One week after surgeries, females under each hormone condition were assigned to either an isolated environment or a complex environment. Those assigned to the isolated environment were housed individually in stainless steel cages (35 3 18 3 20 cm) and received minimal handling. Females assigned to the complex environment were housed in groups of 10 (five rats of each hormone condition) in 75-gallon aquaria (122 3 46 3 50 cm) containing various stimuli that were changed daily. Stimuli included wheels, blocks, ladders, and tubes. Assigned housing conditions were maintained throughout the course of the experiments. All rats were allowed free access to water and Purina Laboratory Rat Chow (Purina Mills, St. Louis, MO) prior to initiation of food deprivation (see below). Experiment 1 Beginning 1 month after exposure to these environmental conditions, all rats were food deprived to 90% of their freefeeding weight. A 5-g per week weight gain was allowed. The effect of food deprivation on the estrous cycles of the gonadally intact females was monitored by daily examination of vaginal cytology from smears collected between 1300 and 1400 h, beginning 10 days prior to initiation of food deprivation and continuing throughout the experiment. After 4 days of food deprivation, radial maze training began. Females were trained to obtain food rewards (Kellogg’s Froot Loops) from the arms of an elevated eight-arm radial maze obtained commercially from Lafayette Instruments (Lafayette, IN). The maze consisted of black metal floors and clear Plexiglas walls. The eight arms (10 cm wide 3 70 cm long 3 20 cm high) were separated from an octagonal center compartment (33 cm across) by guillotine doors that could be opened or closed silently by mechanical relay. The maze was located in the same room in which the animals were housed. The room contained many fixed extramaze cues. To begin each trial, the rat was placed in the center compartment of the maze with the doors of the arms closed. All doors were then

OVARIAN HORMONES AND ENVIRONMENT opened, and the rat was allowed to enter any of the arms. An arm choice was scored if the rat traveled one-half of the length of the arm. The animal was allowed to choose arms in any order until all of the arms were visited or until 5 min elapsed. An error was scored if a rat reentered an arm previously visited. Accuracy was scored by two measures—number of correct choices in the first eight visits and number of correct choices until the first error. Each rat was tested once daily between 0900 and 1200 h for 20 consecutive days. Experiment 2 Immediately after completion of radial maze training, all females were again allowed free access to food. The estrous cycles of the gonadally intact females were monitored throughout Experiment 2. Beginning 1 month after completion of radial maze training, all females were trained in a water maze that consisted of a black plastic circular pool, 160 cm in diameter and 47 cm in height, filled to a level of 28 cm with an opaque solution of white tempura paint at a temperature of 258C (8,47). The water maze was located in a different room from which the animals were housed. The room contained many fixed extramaze cues. During place training sessions (acquisition) conducted over 16 days, the time required to find a platform, 12 cm in diameter and submerged 2 cm below the solution surface, was recorded. On each daily session, each animal was placed into the tank from two random points and allowed a maximum of 2 min to escape to the platform. If the platform was not found and mounted during a trial, the animal was manually guided through the water to it. Females received two place training trials daily, with a 2-min intertrial interval. Following the second place training trial on every other day, the platform was removed for a 1-min probe trial (retention), during which time spent in each of the four quadrants of the tank and the number of target crossings over the previous location of the platform was recorded by a video camera with a timer located above the tank. On Days 17–20, each female received two cued trials per day, during which latency to escape to a platform marked by a small flag was recorded to assess possible sensorimotor or motivational impairments. RESULTS

Experiment 1 Figure 1 illustrates that females housed in complex environmental conditions outperformed females housed in isolated environmental conditions during acquisition of a working memory task in an eight-arm radial maze. Choice accuracy data collected over 20 days were analyzed by a three-way ANOVA (hormone condition, environment, 4-day block) with repeated measures on block. Both gonadally intact and ovariectomized females housed in complex environmental conditions achieved a significantly higher mean number of correct arm choices in the first eight choices, F(1, 136) 5 33.57, p , 0.0001 (see Fig. 1A), and achieved a significantly higher mean number of correct choices before making an error, F(1, 136) 5 47.18, p , 0.0001 (see Table 1), than gonadally intact and ovariectomized females housed in isolated environments. There was a significant block effect, F(4, 544) 5 15.90, p , 0.0001 for mean number correct of first eight choices, and F(5, 680) 5 16.92, p , 0.0001 for mean number correct before the first error, indicating that the performance of all groups improved over time (see Fig. 1B). There was a significant environment 3 block interaction, F(4, 544) 5 4.45,

13 p , 0.002, indicating that the effect of environment differed across blocks. As illustrated in Fig. 1C, the effect of environment was not expressed until the second 4-day block. Results from daily examination of vaginal cytology from smears collected from the gonadally intact females indicated that while all displayed normal 4–5-day estrous cycles prior to initiation of food deprivation, major disruptions in estrous cycles were evident following implementation of food deprivation. Of the 20 intact females, 13 displayed complete cessation of cycles and seven displayed lengthening of cycles of up to 7 days. These disruptions took place by the eighth day of maze training. Although no significant main effect of hormone or hormone 3 block interaction were revealed, analysis of the effect of hormone on performance during each block was conducted to investigate the effect of the disruption of estrous cycles on performance. On the first 4-day block, before cyclicity was disrupted, gonadally intact females achieved a significantly higher mean number of correct arm choices in the first eight choices than did ovariectomized females, F(1, 136) 5 7.22, p , 0.05 (see Fig. 1D). There was no significant environment 3 hormone interaction. Experiment 2 Figure 2 illustrates that gonadally intact and ovariectomized females housed in complex environments and ovariectomized rats housed in isolation outperformed gonadally intact females housed in isolation during place training trials in a water maze. Escape latency data collected over 16 days with two trials per day were analyzed by a three-way ANOVA (hormone condition, environment, 4-day block) with repeated measures on block. A significant main effect of hormone condition, F(1, 148) 5 14.23, p , 0.0002, indicated that ovariectomized rats outperformed gonadally intact females in place training trials. A significant main effect of environment, F(1, 148) 5 30.84, p , 0.0001, indicated that females housed in complex environments outperformed females housed in isolation. Additionally, a significant interaction of hormone condition and environment, F(1, 148) 5 9.13, p , 0.003, followed by group mean comparisons (see Fig. 2A), indicated that gonadally intact females housed in isolation displayed the longest escape latencies compared to ovariectomized rats housed in isolation or females of either hormone condition housed in complex environments. There was a significant block effect F(3, 444) 5 61.89, p , 0.0001, indicating that the performance of all groups improved over time (see Fig. 2B). No significant interactions of block with hormone condition or environment were revealed. No significant treatment effects were revealed on the first trial (data not shown). TABLE 1 EFFECTS OF OVARIAN HORMONES AND ENVIRONMENT ON RADIAL ARM MAZE PERFORMANCE, MEAN NUMBER OF CORRECT CHOICES UNTIL FIRST ERROR AVERAGED OVER 20 DAYS OF TRAINING Group

Intact/complex environment Ovariectomized/complex environment Intact/isolated environment Ovariectomized/isolated environment

Overall Mean 6 SEM

6.01 6 0.116 5.89 6 0.135 4.97 6 0.129 4.83 6 0.113

Ovariectomized and gonadally intact females were housed in either complex environmental conditions or isolated conditions for 30 days prior to and throughout maze training. A main effect of environment was revealed (p , 0.0001).

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FIG. 1. Effects of endogenous ovarian hormones and environment on radial maze performance of female rats. Rats were ovariectomized (OVX) or sham ovariectomized (INT), and housed in complex environments (EC) or isolated environments (IC). (A) Mean number of correct arm choices in the first eight visits (6SEM) averaged over 20 days of training (*p , 0.01 versus IC/INT and IC/OVX). (B) Effect of hormone and environment on acquisition over 20 days, presented in blocks of 4 days. (C) Effect of environment on acquisition over 20 days, presented in blocks of 4 days (*p , 0.001, complex environment versus isolated environment). (D) Effect of hormone on acquisition over 20 days, presented in blocks of 4 days (*p , 0.05, intact versus ovariectomized).

Figure 3 illustrates that gonadally intact females housed in isolation spent significantly shorter times in the platform quadrants and had significantly fewer target crossings than did gonadally intact and ovariectomized females housed in complex environments and ovariectomized rats housed in isolation. Data collected over eight probe trials were analyzed by three-way ANOVA (hormone condition, environment, trial) with repeated measures on trial. Significant main effects of environment indicated that females housed in complex conditions spent significantly more time in the platform quadrants, F(1, 34) 5 5.98, p , 0.02, and had significantly more target

crossings, F(1, 34) 5 15.50, p , 0.0004, compared to females housed in isolation. No significant main effects of hormone condition were revealed. Significant interactions for hormone condition and environment, F(1, 34) 5 4.61, p , 0.04 for quadrant times, and F(1 ,34) 5 4.51, p , 0.05, for target crossings, followed by group mean comparisons revealed that gonadally intact females housed in isolation spent significantly less time in the platform quadrants (see Fig. 3A) and had significantly fewer target crossings (see Fig. 3C) than any of the other three groups. Also, ovariectomized females housed in isolation had significantly fewer target crossings than intact

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FIG. 2. Effects of endogenous ovarian hormones and environment on performance during place training trials in the Morris water maze. Rats were ovariectomized (OVX) or sham ovariectomized (INT), and housed in complex environments (EC) or isolated environments (IC). (A) Mean latency to escape platform (6SEM) averaged over 16 days, with two trials per day (*p , 0.01 versus EC/OVX, EC/INT, and IC/OVX). (B) Effect of hormone and environment on acquisition over 16 days, presented in blocks of 4 days.

females housed in complex environments. Significant main effects of trial, F(7, 238) 5 5.06, p , .0001, for quadrant times (see Fig. 3B), and F(7, 238) 5 4.16, p , 0.0002 for target crossings (see Fig. 3D), indicated the performance of all the groups changed across trials. No significant interactions of trial with hormone condition or environment were revealed. Figure 4 illustrates that there were no significant differences between the performances of any of the four groups during cued sessions conducted to assess possible sensorimotor or motivational deficits. Escape latency data collected over 4 days of cued trials with two trials per day were analyzed by three-way ANOVA (hormone condition, environment, day) with repeated measures on day. A main effect of day, F(3, 102) 5 7.49, p , 0.0001, indicated that the performance of all groups improved across days. No significant interactions or additional main effects were revealed. Results from daily examination of vaginal cytology from smears collected from gonadally intact females indicated that all displayed normal 4–5 day estrous cycles throughout Experiment 2. DISCUSSION

The results of these experiments indicate that ovarian hormones and environmental conditions can independently and interactively exert influence on learning and memory performance. In Experiment 1, which assessed spatial working memory utilizing an eight-arm radial maze, exposure to complex environmental conditions independently enhanced performance. Additionally, gonadally intact females significantly outperformed ovariectomized females during the early phase of radial maze acquisition before food deprivation disrupted cyclicity. In Experiment 2, which assessed spatial reference memory utilizing the Morris water maze, gonadally intact females housed in isolation performed significantly more poorly on place training trials and on probe trials compared to intact

females housed in a complex environment or ovariectomized females housed in either environment, indicating that acquisition and retention of the water maze was impaired by the presence of ovaries, and that this impairment was counteracted by exposure to complex environmental conditions. The results of this study suggest that the ovarian hormones present in gonadally intact female rats can enhance performance during acquisition of a radial maze, a task dependent upon working memory. Previously, we have demonstrated that a constant delivery of exogenous estrogen to ovariectomized rats enhanced working memory performance throughout radial maze acquisition (10). In the present experiment, enhancement in performance was present during the early phase of radial maze acquisition, but did not persist across 20 days of acquisition. Food deprivation can disrupt the estrous cycles of female rodents (5), although not everyone has reported major disruptions (62). In spite of attempts of promote cyclicity by housing males in the room with females and by keeping food restriction to a minimum, all intact females displayed major disruptions in their estrous cycles. These disruptions took place by the eighth day of acquisition. Interestingly, this corresponds to the point at which intact females no longer displayed improved arm choice accuracy compared to ovariectomized females suggesting that circulating ovarian hormone in normally cycling female rats can exert positive influences on radial maze performance. Clearly, further research into the effects of endogenous ovarian hormones on working memory performance is indicated. It is not surprising that the presence of ovaries affected performance on tasks of learning and memory. Ovarian hormones have been demonstrated to modulate structural, biochemical, and electrical activity in the hippocampus, an area of the brain implicated in learning and memory. Progestin receptor mRNA was detected in neurons in CA1 and CA3 of the hippocampus (30) and estrogen receptors are located in CA1, CA3, and the dentate gyrus (41,46). In the hippocam-

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FIG. 3. Effects of endogenous ovarian hormones and environment on performance during probe trials in the Morris water maze. Rats were ovariectomized (OVX) or sham ovariectomized (INT) and housed in complex environments (EC) or isolated environments (IC). (A) Mean time spent in platform quadrants (6SEM) averaged over eight trials (*p , 0.05 versus EC/INT, EC/OVX, and IC/OVX). (B) Mean time spent in platform quadrants across eight trials. (C) Mean number of target crossings (6SEM) averaged over eight trials (*p , 0.05 versus EC/INT, EC/ OVX, and IC/OVX; **p , 0.05 versus EC/INT). (D) Mean number of target crossings across eight trials.

pus, estrogen receptor immunoreactive cells are located in the greatest concentrations in the hilus of the dentate gyrus and the stratum radiatum of CA1 (70). Results of a recent in situ hybridization study revealed that although both the classical estrogen receptor-alpha mRNA and the newly cloned estrogen receptor-beta mRNA are expressed in the hippocampus, estrogen receptor-beta mRNA was more abundant (58). Dissociated hippocampal neurons grown in culture doubled their dendritic spine density following exposure to estradiol (49). The density of apical dendritic spines on pyramidal neurons in the CA1 region (73) and the density of synapses in the stratum radiatum of CA1 (74) positively correlated with fluctua-

tions of estrogen and progesterone levels during the estrous cycles of female rats. Similar results were reported following administration of exogenous hormones to ovariectomized rats (28). Recent reports indicate a positive relationship between NMDA receptor binding and increased synapse and spine density in CA1 pyramidal cells (75). Estradiol treatment increased the sensitivity of CA1 pyramidal cells to NMDA receptor-mediated synaptic input, and this sensitivity positively correlated with dendritic spine density. Furthermore, exogenous estrogen administered to ovariectomized rats (9) and elevated levels of endogenous estrogen present during proestrus (68) were associated with enhanced induction of long-term

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FIG. 4. Effects of endogenous ovarian hormones and environment on performance during cued trials in the Morris water maze. Rats were ovariectomized (OVX) or sham ovariectomized (INT), and housed in complex environments (EC) or isolated environments (IC). Results were averaged over 4 days, with two trials per day. No significant differences between the performances of the groups were revealed.

potentiation in CA1 in response to Schaffer collateral stimulation, while hippocampal multiple unit activity and CA1 population-evoked response were increased during proestrus (37,72). Numerous studies have implicated acetylcholine as a major neurotransmitter in the regulation of learning and memory (13). In doubling labeling experiments, 50–80% of the cholinergic neurons in the medial septum, horizontal limb of the diagonal band, and nucleus basalis magnocellularis expressed estrogen receptor immunoreactivity (16,21,63). Recent evidence suggests that ovarian hormones can modulate cholinergic neurochemistry in these basal forebrain areas and in associated cortical and hippocampal projection sites. For example, estrogen replacement increased potassium-stimulated acetylcholine release in the hippocampus and overlying cortex (25). Estrogen also increased the number of basal forebrain neurons expressing immunoreactivity for the synthetic enzyme for acetylcholine, choline acetyltransferase, or choline acetyltransferase mRNA in ovariectomized rats (20,22,24) and increased the activity of choline acetyltransferase in the horizontal limb and projection sites in the hippocampus and cortex (42,44,59). Additionally, high affinity transport of the precursor, choline, was reduced in the frontal cortex and hippocampus following ovariectomy, an effect reversed by estrogen replacement (59). The modulation of cholinergic neurochemistry by ovarian hormones may be related to the ability of estrogen and progesterone to influence expression of specific neurotrophins and their receptors (20,24,26). For example, levels of trkA mRNA in the medial septum and brainderived neurotrophic factor mRNA in the hippocampus are affected by fluctuating levels of endogenous ovarian hormones and are elevated by estrogen and progesterone replacement (23). Results of the current study indicate that, unlike the initial enhancement in performance in the radial maze, a task dependent upon spatial working memory (Experiment 1), the presence of ovaries failed to enhance performance during ac-

17 quisition and retention in the water maze, tasks dependent upon spatial reference memory (Experiment 2). Studies to date report either no effect of ovarian hormones on performance during water maze acquisition (4,59) or an impairment in performance (18,19,40,69). In our laboratory, estrogen selectively improved working memory, but not reference memory, during acquisition of a radial maze task with four baited and four unbaited arms (15). Recent reports employing the highly specific cholinergic immunotoxin, 192 IgG-saporin, indicate cholinergic projections from the basal forebrain to the hippocampus regulate spatial working memory (57,67), but not spatial reference memory (1–3,64). Thus, if ovarian hormones modulate learning and memory performance via interactions with basal forebrain and hippocampal cholinergic neurons, selective improvement of working but not reference memory would be expected. In our experiments, the effects of ovarian hormones on nonmnemonic processes must also be considered. In a recent report, estrogen-treated ovariectomized gerbils had increased swim times, but similar swim distances compared to ovariectomized controls during acquisition of the water maze (39). It was concluded that the prolonged swim times in the estrogentreated females were most likely due to impairments in motor function and not deficits in spatial discrimination. However, other studies have reported increased swim distances in estrogen-treated rodents in the water maze (18,69). In the present study, no significant differences were found between gonadally intact groups and controls during the first place training trial or during cued trials, indicating no sensorimotor or motivational impairments. Additionally, the probe trials, on which the gonadally intact females performed more poorly than the ovariectomized females, provided a measure of retention that was independent of escape latency or swim speed. Furthermore, it is possible that during probe trials the hormonal status of the animals influenced resistance to extinction. Our results confirm previous reports (12,36,56) that exposure to complex environmental conditions can positively influence performance of female rats on tasks of learning and memory. This effect was demonstrated during acquisition of a radial maze task and during acquisition and retention of a water maze task. This enhanced performance may be related to reported structural changes in cortical and hippocampal neurons. Exposure to complex environmental conditions for varied durations has measurable effects on brain structure. Rodents housed in complex conditions relative to those housed in isolated conditions usually exhibit increased neuronal size, dendritic branching, dendritic spines, synaptic contacts, glial cells, and vascularization in various brain areas (11,27,60,65). Interestingly, female rodents are particularly susceptible to changes in the hippocampus as a result of environmental manipulations. Female rats housed in complex environments showed significant increases in the dendritic branching in the hippocampal granule cells of the dentate gyrus relative to females housed in isolated environments (34). In the CA3 hippocampal region, females exposed to a complex environment had more dendritic material in the apical tree proximal to the cell body compared to controls housed in isolated conditions (35). Finally, female mice reared in complex environments displayed significantly more new neurons in the dentate gyrus compared to littermates reared in standard laboratory cages (38). The significant interaction between hormone condition and environment revealed in Experiment 2 for place training trials and probe trials indicate that the impairment in water maze performance induced by ovarian hormones can be pre-

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vented by exposure to complex environmental conditions. Although the neural mechanisms that mediate this response are unknown, there are interesting possibilities. It is currently unclear how circulating ovarian hormones affect many cortical areas. The impairment in water maze performance reported here and in other studies (18,19,40,69) may be related to changes induced by ovarian hormones in cortical areas other than the hippocampal region. The positive neural effects resulting from exposure to complex conditions may be able to compensate for such changes. Additionally, stress response can impair learning and memory performance (50). Levels of endogenous and exogenous ovarian hormones positively correlate with levels of corticosterone and adrenocorticotrophins following restraint stress (7,66). It may be that in the place training and probe trials in the aversively motivated water maze, a stress response is elicited in the gonadally intact females resulting in impaired performance. Interestingly, the hippocampus, an area of the brain that undergoes structural changes in females rats following exposure to complex environments (34,35), regulates adrenocortical activity (33). Thus, the positive changes in the hippocampus resulting from expo-

sure to a complex environment may have compensated for stress-induced impairment in performance in gonadally intact females. In conclusion, the present data support the hypothesis that ovarian hormones can differentially affect performance on tasks of working and reference memory. The ability of exposure to a complex environment to prevent the impairment on a reference memory task induced by endogenous ovarian hormones demonstrates that environmental conditions can interact with hormonal status to affect behavior. Our results also indicate that environmental factors are a more powerful determinant of cognitive performance than hormonal conditions. ACKNOWLEDGEMENTS

This research was supported by National Science Foundation award IBN-9514816 to Gary P. Dohanich and a Ruiz Award for Research in Psychology to Jill M. Daniel. The authors especially thank Lydia Weathers (Facility Manager) and Diana Plaisance (Animal Technician) for excellent animal husbandry, and Amy Wecker, Samuel Wolff, Peter Gallogly, and Lucianne Marin for assistance in data collection.

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