BEHAVIORAL AND NEURAL BIOLOGY
33, 45-58 (1981)
Ovarian Hormones and Retention of Learned Fear in Rats 1 DEBBIE L. EBNER, RICK RICHARDSON, AND DAVID C . RICCIO Department of Psychology, Kent State University, Kent, Ohio 44242 The present series of investigations was designed to evaluate whether naturally occurring or exogenously induced hormonal changes might influence memory in a state-dependent paradigm. Rats in four experiments received Pavlovian fear conditioning to static environmental cues, followed by passive avoidance testing. In Experiment 1 animals were trained and tested in either the same stages of the estrous cycle (E-E, P-P) or in different stages (E-D, D-E, P-D, D-P). In Experiment 2 rats were trained during one stage of pregnancy, and tested for retention when hormonal conditions were presumably similar (same state) or altered (mismatched). No convincing evidence was found which would indicate that naturally occurring changes in hormonal levels associated with either stages of estrous (Exp 1) or stages of pregnancy (Exp 2) modulate retention of fear. Exogenous administrations of progesterone were employed in Experiments 3 and 4 in order to permit more direct experimental control over internal states and to enhance the salience of changes in internal environment. With a 7-day retention interval progesterone manipulations failed to influence retention performance in a state-dependent manner (Experiment 3), but with a 1-day interval asymmetrical state-dependent effects were obtained. Implications of these findings with respect to boundary conditions on the role of internal contextual cues in memory retrieval were discussed.
That retention is influenced importantly by the similarity between contextual or background stimuli present at acquisition and at later testing has long been recognized (McGeoch, 1942; Spear, 1978). While a number of studies have examined the role ,of similarity of external contextual cues, in recent years there has been increasing interest in the possibility that internal physiological states also may modulate retention. Druginduced state-dependent retention provides a salient illustration of the influence of one type of internal stimulus. For example, using pentobarbital Overton (1964) demonstrated that rats trained and tested while Supported in part by a Sigma Xi Grant-In-Aid to the first author and NIMH Grant MH 30223 to the third author. Several of the experiments constituted the basis for a thesis submitted by Debbie L. Ebner for the M.A. degree at Kent State University. A portion of these data were presented at the Eastern Psychological Association meeting, April 1980. The authors gratefully acknowledge the generous contribution of Nan Page and Robert Estill in collection of the data, and the assistance of Theresa Rittinger on one of the experiments. Reprints may be requested from any of the authors. 45 0163-1047/81/090045-14502.00/0 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
EBNER, RICHARDSON, AND RICCIO
in the same drug state were superior in retention to those tested in a drug condition different from training. This finding has since been extended to include a variety of exogenously administered chemical agents. Characteristic similarities among these state-dependent agents include their ability to pass the blood-brain barrier, CNS effects such as anesthesia or activation, discriminability, and association with mood changes (cf. Spear, 1978). Does the influence of internal cues on memory extend to situations where the "state" is produced by naturally occurring changes? In contrast with the burgeoning literature on state-dependent drug effects, it appears that relatively few studies have examined whether endogenous changes in internal state might also function as contextual cues affecting memory. Among the few studies in this area is one by Holloway and Wansley (1973) showing that retention waxes and wanes in correspondence with internal periodicities. Their finding appears to corroborate an earlier report by Stroebel (1967) which suggested that circadian rhythms are capable of modulating memory. These types of evidence suggest that other endogenous stimuli associated with periodic and discriminable internal states might modulate retention. One pervasive source of fluctuations in endogenous stimuli are the hormonally related changes associated with various aspects of the female reproductive cycle. Although these hormonal changes can affect such nonreproductive behaviors as emotionality (Anderson, 1940; Birke & Archer, 1975; Burke & Broadhurst, 1966), mood (Paige, 1971), and, like state-dependent agents, the hormones can cross the blood-brain barrier to influence the CNS, little is known about these hormonal states v i s a vis their role as contextual cues for memory retrieval. Whether the biologically important states associated with ovarian hormones can become linked with nonreproductive behavioral processes, such as retention of learned fear, is addressed in Experiments 1 and 2. EXPERIMENT 1
The marked changes in levels of estrogen and progesterone which occur during the rat's estrous cycle (Butcher, Collins, & Fugo, 1974) would seem to provide a convenient preparation with which to evaluate a natural form of state dependency. Despite the fact that the estrous cycle is often mentioned in connection with memory loss from an altered internal state (Spear, 1978), the only directly pertinent research appears to be a study by Gray that may have been relatively insensitive to detecting effects of stimulus change. In this regard, Stroebel (1967) reported that Pavlovian fear conditioning (CER) was sensitive to circadian changes, but active avoidance was not. Moreover, Holloway and Wansley (1973) have successfully used passive avoidance to demonstrate changes in retention associated with circadian rhythms.
OVARIAN HORMONES AND RETENTION OF FEAR
Another consideration is based upon the particular stage of estrous cycle compared. In Gray's study estrous and diestrous states were examined. However, a greater magnitude of change appears to exist in ovarian hormones between proestrous and diestrous; and thus a potentially more salient stimulus change might result if these phases were used as the endogenous states. In view of these considerations, further examination of possible effects of estrous cycle changes upon retention of learned behavior seemed warranted. In the following experiment Pavlovian fear conditioning to static environmental cues provided the learning task. After a delay interval, subjects were administered a test in which passive avoidance of the fear stimuli provided the index of retention.
Method Subjects. The subjects were 38 adult female albino rats purchased from the Holtzman Company. They were housed in groups of six and maintained on a 16-hr-on/8-hr-off, light/dark cycle. Subjects were given ad lib food and water throughout the study. Apparatus. The training and testing apparatus consisted of a Plexiglas chamber (47.5 x 19 x 22 cm) divided into two equal compartments. A guillotine door could be lowered or raised in front of an opening (6.5 x 8 cm) which permitted passage between the two areas. One compartment had black walls, an opaque lid, and in addition contained a small black Plexiglas restraining box (20.8 x 17.5 x 10.5 cm) which fitted over the subjects during shocks. The other compartment had white walls and a clear Plexiglas lid. Identical flooring in both compartments consisted of 0.6-cm grids spaced approximately 1 cm apart. Only the grids on the black side of the box were connected to the shock source. A Scientific Prototype scrambled AC shocker was programmed to deliver a 100-V shock (nominal measurement at output). Pilot investigations determined that with this type of shocker, flinch thresholds were in the range of 50-70 V. The experimental room was illuminated by a 15-W light bulb suspended 20 cm above the white half of the chamber. Stopwatches were used to record latency and total time on white measures. Procedure. All animals were earpunched for identification purposes, gang-caged, and randomly assigned to one of six groups. Two same state groups received both training and testing in either estrous or proestrous [E-E (n = 6), P-P (n = 9)] while four mismatched groups received training and testing in different states of estrous, proestrous, or diestrous [E-D (n = 7), D-E (n = 6), P-D (n = 3), D-P (n = 7)]. (A larger sample size for the P-D condition was intended, but subjects cycling patterns failed to accommodate us.) The letters designating groups refer to phases at training and testing, respectively, with E representing estrous, P, proestrous, and D, diestrous.
EBNER, RICHARDSON, AND RICCIO
Stages of estrous were determined by daily vaginal smears. All smears were taken at 8:00 AMdaily throughout the experiment and the procedure was begun 2 weeks prior to training in order to allow cyclic adjustment. Smears were stained with methylene blue and stage of estrous was determined by relative proportion of cornified cells, nucleated epithelial, and epithelial cells. The criterion for "estrous" was that more than threefourths of the cells in the smear had to be cornified. The criterion for proestrous was that more than three-fourths of the cells in the smear had to be nucleated epithelial. Diestrous was determined when threefourths of the cells were epithelial. Stages of estrous were recorded daily until an animal's smear met criterion for her group. All animals reaching criterion on any 1 day received training by the early afternoon of that same day. Training consisted of placing the subject in the black compartment away from the door separating the two chambers and lowering a black Plexiglas restraining container over the animal. Three shocks were then administered during 60 sec of confinement to the black area. These shocks occurred 10, 30, and 40 sec after the exposure began. Following the l-min exposure to black, animals were placed in the white (safe) compartment for 2 min. This entire procedure was repeated once, so that by the end of the training session, subjects had received Pavlovian differential conditioning consisting of six shocks during 2-min exposure to black and 4 min without shock in the white compartment. Subjects were then returned to their home cages. Vaginal smears were continued daily during one to two complete cycles until the subject's smear met the established criterion for testing. For all animals reaching criterion on any 1 day, testing was completed by the early afternoon of that same day. During testing, subjects were placed in the white compartment facing away from the door separating the two chambers. Ten seconds later the door was removed and the time taken for the rat to cross into the black (fear) side (all four feet) was recorded as initial latency. The total time the animal spent on the white side was also recorded for the 15-min test duration. Those animals not crossing within 15 min were assigned a test latency and total time on white score of 900 sec. Due to time and apparatus limitations, the subjects were run in several replications. Results and Discussion Neither test measure provided evidence of differential forgetting among the groups, although as expected the scores did indicate acquisition of learned fear. The median cross-through latencies for the same-state groups were 900 (E-E) and 464 (P-P) sec. For three mismatched states the comparable scores were 455 (E-D), 234 (D-E), and 662 (D-P) sec. Because only three subjects fell into the P-D condition, little credence
OVARIAN HORMONES AND RETENTION OF FEAR
can be put in the median of 22 sec for this group. The pattern of results for the groups was highly similar for the total time on white (safe) side scores. As there were a large number of ceiling scores at testing, nonparametric analyses were performed. Kruskal-Wallis one-way analysis of variance failed to reveal a significant effect of treatment with either dependent variable Co > .2). In an attempt to gain greater statistical sensitivity by increasing overall sample size, and given the specific predictions made, latency data from the same-state conditions were pooled to form one group and data from the several mismatched conditions were combined to form another group. Even with this approach no evidence of differences in retention was obtained (Mann-Whitney U test, onetailed, z = 1.29, p > .09). It appears that under the conditions employed in this experiment, stages of estrous do not function as contextual retrieval cues for a fear response in a state-dependent manner. Given Gray's (1977) similar findings for active avoidance, these negative results with respect to endogenously controlled state-dependent memory extend the sample of task variables to include passive avoidance following Pavlovian fear conditioning. EXPERIMENT 2 While neither Experiment 1 nor Gray's (1977) study provides support for the notion that stages of estrous modify retention in a state-dependent fashion, it seemed possible that other ovarian-related conditions might produce such an effect. Because gestation results in marked alterations in hormonal levels and other internal conditions, in Experiment 2 we attempted to use stage of pregnancy in rats as a source of endogenous contextual cues. Although a study by Kristal, Axelrod, and Noonan (1978) reported no evidence that reproductive conditions affected acquisition or retention of learned behavior, their experimental design was not intended to assess possible state-dependent effects associated with pregnancy. A pilot study in our lab indicated that state of pregnancy might modulate retention of fear in a state-dependent fashion but the possibility that performance artifacts differentially influenced test scores could not be ruled out. The general strategy of this experiment involved training rats at one stage of pregnancy and testing for retention when hormonal conditions were presumably similar (same state) or when the hormonal levels were altered (mismatched). In order to determine whether the retention differences found in the pilot study reflected a state-dependentlike effect or performance artifacts, the experimental and control groups were chosen so that: (1) the day of training would be held constant for one same-state and one mismatched group; (2) retention interval would b e held constant for one same-state and one mismatched-state group.
EBNER, RICHARDSON, AND RICCIO
Method Subjects. A total of 50 adult female Holtzman albino rats were earpunched, randomly assigned to groups, and housed with one male in single cages. Vaginal smears were taken at 8:00 AM every morning thereafter until a sperm positive or a vaginal plug was found. Lighting and feeding were as in the other experiments. Apparatus and procedure. The apparatus used was the same as described in Experiment 1. Training and testing procedures were also identical to those in the preceding experiment with the exception that they took place between 2:00 and 5:00 PM. Group Same (n = 11) was trained on Day 15 and tested on Day 19 of pregnancy, while group Mismatch1 (n = 10) was trained on Day 15 and tested on Day 21. Any retention differences between these two could not be based on different conditions at acquisition. In order to equate retention interval another group in the mismatch designation (Mismatch-2, n = 9) was trained on Day 17 and tested on Day 21. In order to evaluate effects of changes in activity level on test scores we attempted to measure cross-through latencies for untrained animals on Day 19 (Activity-l, n = 5). A second set of rats (Activity-2, n = 5) trained and tested on Day 21 was employed to determine whether expected long latencies would be disrupted by maternal behaviors around time of parturition. Finally, a shorter retention interval was used for a sixth group (n = 10) to represent what might be termed " I n t e r m e d i a t e Mismatch" in hormonal levels. For this condition training was administered on Day 13 and testing on Day 16 of pregnancy. If there is a continuum of contextual similarity governing m e m o r y retrieval, and the manipulation results in a moderate but perceptible change in internal stimuli, then retention in this group might be intermediate between that of the same and mismatched states despite the shortened retention interval.
Results and Discussion Median total spatial avoidance scores on the retention test were 722 sec for the same-state condition, 773 and 589 sec for the two mismatched groups, and 248 sec for the " i n t e r m e d i a t e " mismatch. The sample size in Activity-1 proved too small for meaningful data analysis because of inaccurate determination of pregnancy stage and experimental error. For the other performance evaluation group (Activity-2) the median test score was 549 sec. A Kruskal-Wallis one-way analysis of variance applied to the four retention groups failed to reveal any significant treatment effect (H(3) = 7.39, p > .05). While the distribution of scores in the intermediate condition appears quite different from those in the same-state group, neither the logic of the design nor the overall statistical analysis would seem to justify singling out this comparison. Thus, under more
OVARIAN HORMONES AND RETENTION OF FEAR
well controlled conditions the findings of our pilot experiment were not replicated. EXPERIMENT 3
Experiments 1 and 2 implicitly attest to the difficulties inherent in studies in which the major independent variable involves naturally occurring internal states. (See Mineka , for example, on the issue of establishing conditioned hunger.) In the present case, the relatively gradual onset and offset of internal change conceivably increased the difficulty of discriminating such change. A further source of variability was introduced by the use of indirect measures (e.g., vaginal smears) as indices of hormonal levels. Also, the nature of endogenous cycles often precludes holding constant the retention intervals. For instance, the retention interval is necessarily confounded for same- and mismatched-state groups if both are trained at the same point in the cycle but the simple remedy for this problem introduces potential confoundings at either the time of training or testing. Thus, the use of exogenous administration of chemicals normally produced in the body has obvious appeal in terms of experimental control, even though it begs the original question about endogenous hormonal conditions. In addition, evidence from state-dependent paradigms that administration of corticosteroid hormone or ACTH can modify retention of fear conditioning (Pappas & Gray, 1971; Gray, 1975) indicates the potential usefulness of such a paradigm. Thus, the general strategy of Experiment 3 was to manipulate ovarian hormonal state by administering progesterone at training, testing, or both. In order to decrease the possible masking, or at least complicating, influence of ongoing variation in endogenous levels of progesterone, subjects were ovariectomized prior to the experiment. While Stewart, Krebs, and Kaczender (1967) have reported progesterone dosages appropriate for drug discrimination learning in a T maze employing escape from shock, we found that the sedative nature of those hormone levels precluded their use in the conditioning paradigm where testing occurred without shock. Accordingly, on the basis of pilot work, we chose a lower dose (25 mg/kg) which produced observable behavioral changes in most subjects but did not completely sedate the animals. Determination of a dose-response relationship was not undertaken since 25 mg/kg represented the highest dose (and presumably maximal change) which did not introduce behavioral complications. The mismatch condition of major interest (hormone to saline: P-S) and the two types of same state condition (P-P; S-S) constituted the groups employed in this initial experiment. Given a limited sample of subjects, we reasoned that of the two possible mismatch conditions (P-S; S-P) the P-S condition could provide the more compelling evidence for state-dependent retention (i.e., performance unclouded by artifacts such as drowsiness during testing).
EBNER, RICHARDSON, AND RICCIO
Method Subjects. Twenty-three female adult Holtzman rats were ovariectomized and randomly assigned to three treatment groups (P-P, n = 7; S-S, n = 8; P-S, n = 8). The subjects were housed in groups of six and maintained on a 16-hr-on/8-hr-off, light-dark cycle. Approximately 2 weeks after ovariectomy, subjects received handling for 3 consecutive days preceding the experiment. Ad lib food and water were available throughout the study. Procedure and surgery. Subjects were anesthesized with chlorapent. In order to remove each ovary, a dorsal incision was made posterior to the rib cage and approximately 5-10 mm lateral to the midline. When the incision extended through the muscular layers of the body wall, scissors were inserted to spread the opening approximately 10 mm. This directly exposed the ovaries which were then extracted, ligated, and cut. The uterus was then replaced in the body cavity and the incision was sutured with wound clips. Approximately 2 weeks following surgery, all subjects received ip injections of either aqueous progesterone or saline 5 min prior to both training and testing. Subjects were run between approximately 10:00 AM and 3:00 PM. The apparatus, as well as training and testing procedures, was the same as in the previous two experiments. A 7-day retention interval was used. Results and Discussion The median total time on white scores were 880, 745, and 869 sec for the S-S, P-P, and P-S groups, respectively. As might be expected, Kruskal-WaUis one-way analysis of variance revealed no significant effect of state on retention (H(3) = .28, p > . 10). (Although time on safe side is presented here, the pattern of results and statistical outcome is virtually the same with latency measures.) A number of factors might explain the fact that the putative stimulus changes failed to alter memory. For instance, in Experiments 1 and 2, attention to endogenous ovarian changes may have been overshadowed by external black/white apparatus cues as predictors of shock, thus reducing or preventing contextual stimulus control by ovarian cues. In addition, the present findings may indicate that rats have difficulty associating internal contextual changes with external shocks. It is known, for example, that for rats in a Pavlovian conditioning paradigm, internal gustatory stimuli paired with external shock do not readily come to serve as conditioned stimuli, nor do exteroceptive cues easily become associated with internal malaise (Garcia & KoeUing, 1966). These findings on differential associabiiity raise the question of whether the concept of preparedness or belongingness is applicable also with respect to contextual or background cues.
OVARIAN HORMONES AND RETENTION OF FEAR
While any or all of the preceding considerations may be important, failure of subjects to attend to their internal contextual state would also provide a parsimonious and testable explanation for the lack of memory effects. As a number of studies of stimulus generalization have shown that prior discrimination training along an irrelevant (extradimensional) dimension increases the steepness of generalization gradients along a second continuum (cf. Honig, 1969; Thomas, 1970), attention to ovarian contextual stimuli might be enhanced by introducing an initial phase of orthogonal discrimination training to the hormonal states. A pilot study was conducted to explore this possibility. We assumed that rats receiving prior discrimination training in an operant chamber (with the presence or absence of progesterone being the S + or S- for bar pressing) might subsequently be more sensitive to the internal state present at the time of Pavlovian fear conditioning. Even though after 35 days of training the discrimination based on the hormonal state (exogenous administrations of progesterone in ovariectomized animals) was still not established, the Pavlovian conditioning phase of the experiment was instituted. With testing given after a 1-day retention interval suggestive evidence for statedependent retention was obtained. Whether animals in the mismatch groups had had prior experience with progesterone or not, they tended to perform more poorly than animals in the same-state group. These data suggest that the removal of a hormonal state that had been present at training may impair retention. The use of a 1-day rather than 1-week retention interval may have accounted for the difference between the outcome of this pilot and that of Experiment 3. There is evidence which suggests that the influence of contextual change upon retention is somewhat dependent on the length of the retention interval. McAllister and McAllister (1963) and Pesselman and Riccio (1980) are among those who have reported that apparatus changes produce performance decrements at short retention intervals, whereas the same changes have very little effect after retention intervals of 1 week. Experiment 4 examined more fully the effects on memory of changes in exogenous progesterone after a 24-hr retention interval.
Method Subjects and apparatus. Thirty-nine female adult Holtzman rats were ovariectomized, earpunched, and randomly assigned to four treatment groups. Group P-P had 9 subjects; all other groups had 10. The subjects were housed in single cages and received the same lighting, feeding, and handling experiences as in Experiment 1. The Pavlovian fear conditioning apparatus used in all previous experiments was also used in Experiment 4.
EBNER, RICHARDSON, A N D RICCIO
Procedure. Subjects received ip injections of either aqueous progesterone or saline 35-55 min prior to both training and testing. Fear conditioning and testing procedures were identical to those used in Experiment 3 except that a 24-hr retention interval separated training and testing. Results and Discussion
Figure 1 presents the median total time on white measures for all groups. Kruskal-Wallis one-way analysis of variance indicated a significant treatment effect whether latencies (H(3) = 8.45, p < .05) or total time on white (H(3) = 7.8, p = .05) served as the measure. Subsequent comparisons of latencies between the groups provided some support for an asymmetrical state-dependent effect. As the figure suggests, both same-state groups showed latencies significantly longer than those of the group trained in progesterone but tested without (P-P vs P-S, U = 19, p < .05; S-S vs P-S, U = 20, p < .05). Also, the two mismatched groups differed, with the saline-to-progesterone condition superior (S-P vs P-S, U = 17, p < .02), but the two same-state groups were comparable (S-S vs P-P, U = 38, p > .10). This pattern of results is consistent with the interpretation that presence of progesterone at training provides an internal contextual cue the removal of which is detrimental to retention. An alternative but less interesting explanation is that progesterone impairs initial learning; the poor performance of the P-S condition would reflect lack of learning, not memory loss. While the strong retention in the P-P group appears to refute such an explanation, it was possible that scores in this condition were spuriously elevated due to a direct effect of the hormone on performance. Accordingly, we subsequently administered the same dose 300
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FIG. 1. Median passive avoidance scores for groups trained and/or tested with exogenous progesterone (PRO) or saline (SAL) in a state-dependent retention design. The effect of progesterone on response latencies is assessed by the untrained group shown at right.
OVARIAN HORMONES AND RETENTION OF FEAR
level of progesterone to another group (n = 5) of ovariectomized rats which had received no previous training and measured their crossthrough latencies in the test apparatus. As Fig. 1 also shows, progesterone per se did not affect the tendency (or ability) of rats to enter the black compartment shortly after placement in the white side. The data from Experiment 4 suggest that exogenous progesterone can exert an asymmetrical state-dependent effect on retrieval at short retention intervals. At longer retention intervals (Experiment 3), however, the mismatch in drug state fails to produce an observable decrement in performance. These findings appear to extend the generality of other studies which have shown that changes in external background stimuli disrupt retention performance after short intervals, but have little effect following more prolonged training to testing delays. GENERAL DISCUSSION In general, these findings provide little support for our hypothesis that ovarian hormonal states might modulate retention of learned responses which are unrelated to reproductive behaviors. Experiments 1 and 2 obtained no reliable evidence that endogenous changes associated with stages of estrous or pregnancy at training interacted with the phase at testing to determine memory retrieval. Experiments 3 and 4 provided some evidence that exogenous manipulation of progesterone level could influence memory, but the effects were obtained only under a relatively restricted set of conditions. A variety of explanations for the singular lack of effectiveness of endogenous states upon memory can be considered. For example, it might be proposed that these negative results would be expected in terms of evolutionary considerations. While we would agree that it seems unadaptive for female rats to undergo changes in memory of learned episodes as their hormonal levels fluctuate, such biological constraints cannot necessarily be assumed a priori. Neither mood states during learning nor the environmental milieu in which information is acquired would seem relevant to later retention of that information, yet "mismatches" in these conditions can markedly degrade memory retrieval in humans (Bower, Monteiro, & Gilligan, 1978; Smith, Glenburg, & Bjork, 1978; Smith, 1979) as can mismatches in circadian cycles in animals (Holloway & Wansley, 1973). Thus, it seems prudent to regard the issue of whether endogenous stimuli can function as contextual cues as an empirical question. Although negative results may reflect the particular choice of parameter values, or lack of task sensitivity, other considerations suggest that the conditions used were appropriate. By testing under extinction conditions we sought to rule out any masking effects of physically aversive stimulation. The mismatch in endogenous conditions was as disparate as
EBNER, RICHARDSON, AND RICCIO
possible, and even included the additional contribution (Experiment 2) of the gross morphological changes following parturition. Furthermore, Pavlovian fear conditioning is reported to be more sensitive than several other aversive tasks in detecting circadian rhythm effects on memory (Stroebel, 1967). More directly, exogenous progesterone effects were detected in Experiment 4 and the same test has been used in our laboratory to demonstrate ontogenetic differences in retention (Schulenburg, Riccio, & Stikes, 1971). Also, Holloway and Wansley (1973), using a similar test procedure, have demonstrated fluctuations in memory depending on internal rhythms. Perhaps endogenous changes function as contextual cues for memory in tasks more closely relevant to biological and ecological demands, such as conditioned taste aversion. While we have begun to examine this possibility, the present outcome has value in delineating boundary conditions on the stimuli that act as contextual cues.
It should be noted that other learning processes could work against endogenous hormonal states functioning as contextual cues. Since prior experience with to-be-conditioned stimuli retards later learning to those stimuli, i.e., "latent inhibition," previous estrous cycles may have impaired the establishment of ovarian levels as contextual stimuli. The relatively slow rate of change of endogenous internal stimuli may also diminish their salience as cues. However, these same characteristics would be expected t o exist for circadian rhythms, yet, as mentioned above, these internal characteristics have been found to impair retention. From a broader perspective, the present study raises several basic questions about the nature of contextual cues in memory retrieval. The endogenous bodily conditions were present at training but irrelevant to the reinforcement contingencies; however, unlike other contextual stimuli, they exerted little influence on retention. Thus, the findings help to define boundary conditions on the role of contextual cues; naturally occurring ovarian states were in a nominal but not functional relationship to memory retrieval. At the same time, the outcome poses a question previously recognized by Spear (1978): "Must contextual cues be attended to, or is their contiguity with training conditions sufficient?" This issue is a hoary one in the history of research on other types of stimulus control of responding. One way of increasing the salience of, or attention to, stimuli has been through discriminative training. Our difficulty in establishing exogenous progesterone as a discriminative cue leaves the issue unresolved. However, we think this strategy may prove useful in evaluating the parallelisms between contextual stimuli and traditional discriminative stimuli in search of the interesting possibility that they represent a common process in different guises. Finally, evidence that mismatches in exogenous progesterone produce a decrement in test performance after brief but not long retention intervals
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is consistent with research showing that exteroceptive stimulus generalization gradients become flatter as a function of time. Both these classes of finding can be interpreted as indicating that changes in stimulus conditions (e.g., internal milieu) between training and testing are more readily detected after short delays, but they also pose a paradox for models of forgetting which propose that slightly altered stimulus situations have impaired retrieval: If explicit changes in stimuli become less perceptible over time, how can subtle and implicit shifts in contextual conditions become the source of retrieval failure (cf. Riccio & Ebner, 1981)?
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