Spatial and Nonspatial Learning Across the Rat Estrous Cycle

Copyright 1997 by the American Psychological Association, Inc. 0735-7044/97/$3.00

Behavioral Neuroscience 1997, Vol. I l l , No. 2, 259-266

Spatial and Nonspatial Learning Across the Rat Estrous Cycle Stacey G. Warren and Janice M. Juraska

This document is copyrighted by the American Psychological Association or one of its allied publishers. This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.

University of Illinois at Urbana-Champaign Recent evidence has demonstrated that there are fluctuations in both the anatomy and physiology of the hippocampus across the estrous cycle of the female rat. In the present study we examined the behavioral implications of these changes by testing females on either a hippocampal or nonhippocampal version of the Morris water maze during the various phases of the estrous cycle. Males were also tested on these tasks. Although there was little variance on the nonhippocampal cue task, females in proestrus performed significantly better than those in estrus. Optimal female performance on the spatial version of the task occurred during the phase of estrus, whereas the least efficient performance occurred during proestrus. These results do not support the traditional view that hippocampal long-term potentiation is positively correlated with spatial learning.

Lewis, & Rawitch, 1980) and EPSP amplitude (Wong & Moss, 1992) as well. Collectively, these data indicate that the physiological activity of this region is sensitive to gonadal hormone fluctuation. With such notable structural and physiological modifications one must wonder whether there are corresponding behavioral transformations. A variety of behaviors fluctuate across the estrous cycle in the rat, for example, running wheel activity (Finger, 1969), open field locomotion (Birke & Archer, 1975), active avoidance (Diaz-Veliz, Soto, Dussaubat, & Mora, 1989; Sfikakis, Spyraki, Sitaras, & Varonos, 1978), and object exploration (Birke, 1979); however, the hippocampus is not necessarily involved with these behaviors. The question of interest here is whether a form of learning which requires the hippocampus will vary across the estrous cycle in a manner consistent with the anatomical and electrophysiological fluctuations. Reports of a positive correlation between LTP and spatial learning (Barnes, 1979), as well as between CA1 hippocampal spine density and spatial learning (Moser, Trommald, & Andersen, 1994), suggest that spatial learning should vary significantly across the estrous cycle. Two previous reports of changes in spatial learning across the estrous cycle have been unable to answer the question of whether the anatomical and electrophysiological fluctuations are reflected in spatial behavior changes (Frye, 1995; Warren, 1993). Neither study confined testing to one hormonal condition within each animal, which made it impossible to make comparisons between groups. In addition, all phases of the estrous cycle were not tested and the classification of the estrous cycle phases was not consistent with the classifications used in the anatomical or electrophysiological studies (Frye, 1995), rendering it difficult to make comparisons between studies. These studies do suggest that spatial learning varies across the estrous cycle, however. There are numerous reports of sex differences in spatial learning in rats, with males often performing better than females (for review, see Williams & Meek, 1991). There are also reports in which no sex difference has been found on tasks that sometimes do result in sex differences (Bucci,

The hippocampal formation is known to play an essential role in memory (O'Keefe & Nadel, 1978; Squire, 1992), but only an ancillary role in reproductive behavior. Curiously, this structure exhibits dramatic morphological and electrophysiological changes across the estrous cycle in the female rat. The density of dendritic spines and of axo-spinous synapses in hippocampal CA1 stratum radiatum and s. lacunosum moleculare varies with plasma estrogen levels in female rats (Gould, Woolley, Frankfurt, & McEwen, 1990; Woolley, Gould, Frankfurt, & McEwen, 1990; Woolley & McEwen, 1992, 1993). When estrogen is high, during proestrus, synapse number is at its highest; and when estrogen is low, during estrus, synapse number is at its lowest. The decline in spine and synapse number is, on average, greater than 30%. The electrophysiological properties of the hippocampus also vary with the estrous cycle. Long-term potentiation (LTP) mirrors the fluctuation in synaptic density, with greatest increases in posttetanic excitatory postsynaptic potential (EPSP) slope evident during the afternoon of proestrus and minimal increases during estrus (Warren, Humphreys, Juraska, & Greenough, 1995). The increase in synapse number does not translate directly into a larger baseline EPSP slope, but rather it is the response to the tetanizing stimulus that is enhanced. High estrogen levels in females have also been associated with lowered seizure threshold (Buterbaugh & Hudson, 1991; Teresawa & Timiras, 1968; Woolley & Tirniras, 1962) and greater EPSP duration (Wong & Moss, 1992). Topical administration of estrogen increases the population spike (Teyler, Vardaris, Stacey G. Warren and Janice M. Juraska, Department of Psychology, University of Illinois. This research was supported by National Science Foundation Grants IBN9310945 and HD07333. Correspondence concerning this article should be addressed to Janice M. Juraska, Department of Psychology, 603 E. Daniel Street, University of Illinois at Urbana-Champaign, Champaign, Illinois 61820. Electronic mail may be sent via Internet to Stacey G. Warren at s warren ©s.psych.uiuc.edu or to Janice M. Juraska at [email protected].

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Chiba, & Gallagher, 1995; Juraska, Hendersen, & Miiller, 1984). Given the morphological and electrophysiological changes that occur in the hippocampus across the estrous cycle, it is likely that the cycle influences how females perform spatial tasks. It may be that there are points during the cycle when females perform quite well and other points when they perform more poorly. Depending on the task, the estrous cycle could be a highly relevant factor contributing to whether or not a sex difference is found. In the present article we examined the behavioral implications of the morphological and electrophysiological changes that occur across the estrous cycle of the female rat. In order to make meaningful comparisons with the anatomy and physiology, the classification of the cycle and time of day during which testing was conducted were consistent with previous studies (e.g., Gould et al., 1990; Warren et al., 1995). We tested females on either a hippocampal dependent, spatial version or a nonhippocampal, nonspatial version of the Morris water maze. Females were tested during only one phase of their cycle, and males were also tested. All subjects received pretraining in an attempt to reduce the contribution of stress to performance. We hypothesized that females in proestrus (i.e., animals that exhibited the greatest LTP and that had the highest number of synapses) would perform the best on the spatial version of the water maze and that there should be no differences on the cue version (nonhippocampal dependent) of the task. We further predicted, on the basis of a report by Frye (1995), that differences would be evident only on the initial trials and that groups would perform similarly in the later trials. Method

Subjects Adult male and female Long-Evans hooded rats at 120-150 days of age, which were first generation descendants from Simonsen Laboratory (Gilroy, CA) stock, were used. Subjects were housed in same-sex pairs in standard clear Plexiglas laboratory cages. Food and water were available ad libitum, and the colony was maintained on a 12-hr light-dark cycle with the lights on at 0700. Females were tested in one of three phases of the estrous cycle. On the place version of the task 11 females in the phase of proestrus, 12 in estrus, 12 in diestrus, and 12 males were tested. On the cue version of the task a separate group of animals was tested: 5 females in proestrus, 6 in estrus, 5 in diestrus, and 6 males.

Handling and Estrous Cycle Determination All animals received daily handling, in the afternoon, for at least 2 weeks prior to pretraining. The estrous cycle of the females was monitored daily, at the same time as the handling, and only those exhibiting two or more consecutive 4- or 5-day cycles were included in the study. To determine the phase of the estrous cycle a sample of vaginal cells was obtained using the lavage technique, and the cell cytology was examined under low power with a light microscope. All vaginal cell samples were obtained and classified by the same investigator as in Warren et al. (1995) and categorized as described by Feder (1981). Specifically, in proestrus the majority of cells were large, round, and nucleated; in estrus the majority of cells were cornified; and in diestrus there were a variety of cell

types along with leukocytes. The phase of the cycle was not held constant or counterbalanced during pretraining, so the phase of the cycle during which pretraining took place was not necessarily the same as that during which testing took place.

Apparatus and Procedure: Pretraining Maze and room. A small pink wading pool (122 cm in diameter, 36 cm deep) with a metal liner insert was used for pretraining. There was a cylindrical shaped escape platform (19 cm tall and 9 cm diameter) placed in the center of the pool. The pool was filled with water to a depth of 1 cm below the surface of the platform, rendering the platform visible to the swimming rat. The pool was located in a room in which numerous extramaze visual cues were available. The experimenter remained at the start location, approximately 0.5 m away from the outside edge of the tank, on each trial. Procedure. All subjects received pretraining in the small pink maze 3-7 days prior to testing in the larger Morris water maze. Subjects were first placed on the visible platform for 20 s. They then received three trials in which they were placed into the water, with the nose facing the edge of the pool, and given 60 s in which to locate and climb onto the visible platform. If an animal did not locate the platform within this time it was guided there by the investigator. Each animal remained on the platform for 15 s, was dried off, and then placed back into the water for the next trial. After completing all three trials the subject was returned to its home cage.

Apparatus and Procedure: Testing Maze and room. The testing water tank, 175 cm in diameter and 74 cm deep, was painted white and filled with water at 26 °C (± 1). White nontoxic water color paint was added to the water to make it opaque. A submerged platform (58 cm tall and 10 cm square) was located in one of four possible quadrant locations of the tank. The tank was located in a different room than that used in pretraining. Numerous extramaze visual cues were visible from within the maze. The experimenter remained at the start location, approximately 0.5 m away from the outside edge of the tank, on each trial . A video camera was mounted above the center of the tank and each trial was recorded. A tracking system (Chromotrak, Version 3.0) was used to analyze the movement of each rat. Place task. All testing began at 1500 hr. Each subject received 16 trials, followed by a probe trial on the same day. The submerged platform was located in one of four possible quadrants of the tank and remained there throughout training. On each trial the subject was placed in the water, facing the edge of the tank, in one of four start locations. The order of the start locations was varied in a quasi-random fashion such that in each block of four trials the subject started from each location one time and never started from the same place on any two consecutive trials. If the subject did not locate the platform within 60 s it was guided there by the experimenter and allowed to remain on the platform for 15 s. The intertrial interval was 3-5 min, during which the subject remained in its home cage in a different room. After the eighth trial there was a 1-hr break followed by the final eight trials and a probe trial. During the probe trial the platform was removed from the tank, and the subject was required to swim for 60 s. The purpose of the probe trial was to provide an additional method of evaluating the subject's knowledge of where the platform was located by quantifying the amount of time spent in the quadrant where the platform was previously situated. In addition, the probe trial provides the

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ESTROUS CYCLE AND SPATIAL LEARNING opportunity to examine the subject's response to the absence of the platform. Cue task. The testing room, the tank, and the procedure for the cue task were identical to that used for the place task with two exceptions. A black ball was hung above the location of the submerged platform on each trial and the location of the platform and ball moved, together, on each trial. This task requires the rat to ignore the extramaze information and to use the ball as a cue or beacon to indicate the location of the submerged escape platform. On the probe trial the cue was hung but the platform was removed. Lesions to the fornix do not impair performance on this cue task, however spatial performance (the place task) is impaired (for review, see Brandeis, Brandys, & Yehuda, 1989; Warren, 1993). As we ran the cue task, we found little variance between subjects and therefore did not need a large number of subjects in this task.

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Statistical Analysis

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The latency, path length, and swim speed were analyzed with repeated measures analysis of variance (ANOVA). The data were grouped into blocks consisting of four trials per block. Planned comparisons were made between proestrus and estrus. Previous work indicated that the difference might be apparent only initially (Frye, 1995), so the first set of eight trials and second set of eight trials were analyzed separately. We predicted, a priori, that females in the proestrous phase of the cycle would perform better than females in the estrous phase only on the initial eight trials. With additional training we predicted that the difference would diminish. The time and distance spent in the target quadrant on the probe trial was analyzed with an ANOVA to determine whether all groups would exhibit the same spatial bias for this quadrant. Additionally, we examined whether the groups responded differently to the discovery that the platform was no longer present by comparing the amount of time and the distance spent in the outer annulus of the tank (outer third of the tank).

Results

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5 Block 1 Block2 Blocks Block4 Trial Block Figure 1. Latency on the place version of the water maze with all trials, except the probe, and groups represented. Trial block is the mean of four consecutive trials. Error bars are SEMs.

proestrus group, which indicated that those in the phase of estrus performed better than those in proestrus. Post hoc analysis with ANOVA indicated that on measures of both latency, F(l, 21) = 6.3, p < .05, and path length, F(l, 21) = 6.47, p < .05, the mean for the estrus group was significantly lower than the mean for the proestrus group on Block 3, but there was no significant difference between the groups on Block 4. On the probe trial there were no differences between groups in the amount of time or distance swum in the target quadrant (the fourth of the tank in which the platform had been during training) or in the outer perimeter of the tank (outer 30% of the tank). There were also no significant differences in the total number of platform crossings. At the suggestion of a reviewer, we conducted post hoc analyses on Trials 9 to 16 to establish whether the difference between estrus and proestrus groups following the 1-hr break quickly disappeared or was consistent across several trials. One-tailed unpaired t tests on both the latency and the path length measures were used. We are aware that this practice violates statistical convention and capitalizes on chance; however, it is for exploratory purposes only, and the p values should be interpreted cautiously. As shown in Figure 3, the difference between these two groups is large on several of the trials following the 1-hr break. The difference is significant (p < .05) or there is a trend (p = .06) on Trials 9, 10, 12, and 13. There were no differences on Trials 11 and 14-16.

There was no main effect of hormone condition (estrus, proestrus, diestrus, male) and no hormone by block interaction on any measure (Figure 1). There was a main effect of block (p < .0001) for all measures: latency, F(3, 129) = 39.13; path length, F(3, 129) = 42.69; swim speed, F(3, 129) = 6.88. For both the latency and path length measures this indicated that all subjects improved in their ability to reach the platform as the trials progressed. All subjects also reduced their swimming speed with advancing trials. Planned comparisons revealed that on the measure of swim speed there was no main effect of hormone or hormone by block interaction on the first two blocks or the second two blocks. For the measures of latency and path length, there was no main effect of group and no group by block interaction on the first two blocks. On the last two blocks, however, there was a significant main effect of hormone group for the measure of path length, F(l, 21) = 4.321, p = Cue Task .05, and a hormone group by block interaction for both latency, F(l, 21) = 5.07, p < .05 (Figure 2A), and path There was no main effect of hormone condition (estrus, length, F(l, 21) = 5.33, p < .05 (Figure 2B). On each of proestrus, diestrus, male) and no hormone by block interacthese measures the estrus group had lower means than the tion on any of the measures (latency, path length, or swim

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trials progressed. All subjects also reduced their swimming speed with successive trials. As with the planned comparisons of the place task we compared all measures of the estrus and proestrus groups in a separate repeated measures ANOVA. On the measure of swim speed there was no main effect of hormone or hormone by block interaction. For the measures of latency and path

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speed; Figure 4). There was a main effect of block (p < .0005) for all measures: latency, F(3, 54) = 36.72; path length, F(3,54) = 40.04; swim speed, F(3,54) = 3.02. Both the latency and path length measures indicated that all subjects improved in their ability to reach the platform as the

Figure 3. Latency (A), measured in seconds, and path length (B), measured in centimeters, on the place version of the water maze. Trials 9 through 16 are presented individually for both the estrus and proestrus groups for better clarification of the group differ-

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length, there was no main effect of group and no group by block interaction on the first two blocks. On the last two blocks there were no main effects of hormone group; however, there was a hormone group by block interaction for both latency, F(l, 9) = 6.27, p < .05 (Figure 5A), and path length, F(l, 9) = 8.08, p < .05 (Figure 5B). On each of these measures the proestrus group had lower means than the estrus group. Note that this is the opposite of what was found with the place task. On the probe trial there were no significant differences between groups on any measure.


Discussion These results demonstrate that performance on the place version of the Morris water maze varies significantly across the estrous cycle of the rat, with females in the estrus phase of the cycle outperforming those in the proestrus phase. Although the difference is relatively small, it is contrary to our prediction that females in proestrus would perform better than those in estrus, on the basis of previous anatomical (Gould et al., 1990; Woolley et al., 1990; Woolley & McEwen, 1992, 1993) and electrophysiological studies (Warren et al., 1995). Noncognitive factors such as motor abilities were ruled out by the lack of group differences in swim speed and by the superior performance of the females in proestrus on the nonspatial cue task. The cue task has the same motivational and motor components as the place version but differs by requiring the subject to attend to different information in order to find the hidden platform. The superior performance of the females in proestrus on the cue task demonstrates that the poorer performance of the proestrus females on the "hippocampal" version of the task cannot be attributed to the fact that these females are more

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