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Social modulation of behavioral reproductive senescence in female rats
Physiology&Behavior,Vol. 52, pp. 603-608, 1992
0031-9384/92 $5.00 + .00 Copyright © 1992PergamonPressLtd.
Printed in the USA.
Social Modulation of Behavioral Reproductive Senescence in Female Rats JUDITH
A. L E F E V R E ~ A N D M A R T H A
K. M c C L I N T O C K 2
Department of Psychology, The University of Chicago, Chicago, I L 60637 R e c e i v e d l l F e b r u a r y 1991 LEFEVRE, J. A. AND M. K. McCLINTOCK. Social modulation of behavioral reproductivesenescence infemale rats. PHYSIOL BEHAV 52(3) 603-608, 1992.--During aging, the lordosis reflex of female rats changes from a state of cyclic lordosis, when the lordosis reflex can be elicited by manual palpation only on proestrus, to a state of constant lordosis (CL), when it can be elicited daily. Social signals from other females altered this pattern of behavioral senescence. Group living decreased the lordosis reflex intensity in both old and young rats and delayed the onset of CL. Long-term group living delayed CL until late in the life span, while temporary group living delayed it only until rats returned to isolation. Long-term group living also changed the temporal relationship between CL and the acyclic, anovulatory state of constant estrus. Finally, the lordosis reflex intensity, measured by manual palpation, predicted the old rats' receptivity to a male, indicating that constant lordosis is a biomarker for other aspects of behavioral reproductive senescence. Aging
Sexual behavior
Rats
Social environment
R E P R O D U C T I V E senescence has typically been described in terms of changes in vaginal cytology as well as hypothalamic, pituitary, and ovarian function. These neuroendocrine changes are associated with a sequence of reproductive states in the female rat: regular estrous cycles, irregular cycles, constant estrus (the acyclic state which only half of females enter), persistent diestrus, and finally anestrus (2,9,13,20). Less frequently, reproductive senescence has been described in terms of female sexual behavior. During aging, the temporal coordination between sexual receptivity and the ovarian cycle changes. Receptivity is characterized by a lordosis reflex in response to male mounts, a behavioral posture which facilitates intromission (24). Young rats are receptive only on proestrus, just prior to ovulation, maximizing the likelihood of conception (15,22). Middle-aged rats that are in constant estrus, as defined by vaginal cytology, are often receptive repeatedly on consecutive days (5,12). Finally, old rats that are in persistent diestrus (e.g., repetitive pseudopregnancies) are rarely receptive, if at all (5). A similar pattern, of increase and then decrease, emerges when the lordosis reflex is elicited by manual palpation and measured longitudinally throughout reproductive senescence (13). The frequency of the lordosis reflex increases until the reflex can be elicited on every day of the cycle, the state of constant lordosis. Then the frequency of the lordosis reflex decreases, until the reflex can no longer be elicited. In isolated rats, this process of behavioral reproductive senescence precedes and predicts changes in the other components
Social isolation
Lordosis reflex
of the estrous cycle. As the lordosis reflex increases in frequency, it decouples from the other estrous-cycle components that are still cycling. For example, when the lordosis reflex becomes acyclic, rats still have regular or irregular cycles in vaginal cytology (13). In fact, the onset of constant lordosis predicts when rats will stop cycling and enter constant estrus, a n acyclic, anovulatory state (13,14). In young rats, social signals from other females enhance the temporal coordination of the lordosis reflex with other estrous cycle components. They modulate the frequency and intensity of the lordosis reflex during the estrous cycle, as well as modulating cycle length, regularity, and the frequency of ovulation ( 1,6,17,18). However, their effect on behavioral reproductive senescence is unknown. There is some evidence that the effect of social signals on behavior declines during aging. For example, social signals from males have less effect on the approach behavior of old than young females (4). It is u n k n o w n , however, whether social signals from other females continue to modulate the lordosis reflex of old rats, just as they modulate that of young rats (14,17). Thus, one purpose of this research was to determine whether living in groups with other females affects the lordosis reflex of old rats to the same extent that it affects that of young rats. If social signals continue to modulate the lordosis reflex during aging, they may delay the onset of constant lordosis, the acyclic behavioral state. However, there may be a limit to how long social signals can delay constant lordosis, just as there is a limit
1Present address: Andrus Gerontology Center, Leonard Davis School of Gerontology, University of Southern California, University Park, MC0191, Los Angeles, CA 90089-0191. 2 Requests for reprints should be addressed to Dr. Martha K. McClintock, Department of Psychology, The University of Chicago, 5730 South Woodlawn Ave., Chicago, IL 60637.
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to their effect on another biomarker of aging, the acyclic state of constant estrus (14). To first determine whether social signals can delay the onset of constant lordosis, we examined the effect of group living during the period when isolated animals begin to enter the state. Next, we determined how long social signals can delay constant lordosis, by maintaining group living throughout the lifespan. Then, we determined whether differences in the effect of group living on constant lordosis and constant estrus alters the temporal relationship or prevalence of the two biomarkers of reproductive aging during the lifespan. Finally, we investigated whether the lordosis reflex measured by manual palpation is a biomarker for other aspects of behavioral reproductive senescence. Because our longitudinal studies necessitated daily assessment of the lordosis reflex, it was measured by manual palpation. However, it is unknown whether this measure is associated with receptivity to a male in old rats, as it is in young rats (17,24). Thus, a final purpose was to determine whether the relationship between the lordosis reflex measured by manual palpation and receptivity is different in old and young rats. GENERAL METHOD
Animals Female Sprague-Dawley rats (n = 172) were received from Charles River Breeding Laboratories at 1-1/2to 2 months of age (n = 34) or bred from Charles River-derived females (n = 138). Rats were maintained in the laboratory throughout the life span [note that these cohorts of rats are different from those used in previous studies (13,14,17)]. Females were group housed five per cage in stainless steel cages (18 X 24 X 14") or isolated in plastic cages with wire bottoms ( 10 × 9 × 8-1/2"),as required, for each study. Light intensity and the amount of space per rat were the same in the isolation and group cages. None of the rats were ever mated, except as specified in Study 3. Colony rooms were lit with a reversed light cycle (14L: 10D; middark at 1330 h with dim red illumination during the dark phase) and maintained at 22 _+ 2°C, with relative humidity 3555%. Food (Teklad rat chow, 4% fat) and tap water were supplied ad lib. Animal health was monitored during daily data collection and biweekly health checks. Individuals were sacrificed at the first sign of respiratory or other disease.
Daily Measures and Coding The lordosis reflex and vaginal smear were measured once daily from 2 or 4 months of age throughout the life span. The intensity of the lordosis reflex was measured at middark by manual palpation. Intensity was scored on a ten-point scale based on two measures: strength and habituation. Each level of strength (0-4, ranging from a hunched posture to intense dorsiflexion of the back) was combined with one of two levels of habituation (0-1, reflex habituates vs. does not habituate with repeated stimulation) (13,19). Interrater reliability was 91%. These daily measures were then analyzed to determine the lordosis state of the animal (13). The daily scores were averaged to obtain a mean for each vaginal-smear cycle. From the record of mean cycle scores, the state of constant lordosis (CL) was identified when the mean was >5 for at least three vaginal-smear cycles spanning at least 16 days. In our laboratory, this criterion corresponds to a condition in which the lordosis reflex can be elicited on every day. A mean of <5 indicated the state of cyclic lordosis. The vaginal smears were made from vaginal lavages taken daily at middark. They were examined immediately and classified
according to the stage of the estrous cycle (2,13,15). Interrater reliability was 87%. The daily vaginal smear records were then used to code vaginal smear cycles and reproductive states as previously described: regular cycles, irregular cycles, constant estrus, and persistent diestrus (13).
Analysis Frequencies were analyzed with the Pearson chi-square test and Fisher's Exact test (11,26). The onset, duration, and termination of constant lordosis and constant estrus were analyzed with the Log-Rank test in the SAS Life Test procedure for survival analysis (25). Changes in the lordosis reflex intensity were analyzed with a three-way analysis of variance (2 × 2 X 2 factorial model: age and social environment, with time period as a repeated measure) and univariate analyses for each time period, using the SAS General Linear Models Procedure (25). Associations between the lordosis reflex elicited by manual palpation and that elicited by male mounts were analyzed with the SAS linear regression procedure (25). Summary statistics are presented as the means and SEM. STUDY 1: SOCIAL MODULATIONOF THE LORDOSIS REFLEX INTENSITY IN OLD RATS Although social signals modulate the lordosis reflex during the estrous cycle of young rats, it is unknown whether they continue to do so during aging. This study determined whether group living affects the lordosis reflex intensity of old rats to the same extent that it affects that of young females.
Method All females were housed alone at 4 months and were maintained in isolation until the start of the study. Their vaginalsmear cycles were continuously monitored beginning at 3-1h months of age. The old rats (15-L/2 months of age; n = 40) all had irregular vaginal-smear cycles (of varying lengths), although many had passed through the acyclic state of constant estrus (63%). The young rats (4-Vz months of age; n = 53) had regular cycles, 4 days in length (42% of rats) or irregular cycles of varying lengths (58% of rats). Rats remained alone during a baseline period (28 days). Females were then divided into experimental and control groups. In the young rats, the two groups were matched by cycle length during the baseline period in order to standardize the time during which the average lordosis reflex intensity was calculated. However, we did not include a detailed analysis of vaginal-smear cycle length in this behavioral study because a preliminary analysis of these data confirmed our previous reports (17,19) that the effect of isolation on the lordosis reflex is independent of any change in vaginal-smear cycle length. During the experimental period (28 days), the experimental females (young rats, n = 25; old rats, n = 22) were moved to group housing. The control rats (young rats, n = 28; old rats, n = 18) remained isolated to control for the effects of aging during the course of the experiment. Each rat's average lordosis reflex intensity was calculated separately for the baseline and experimental periods, using data from complete vaginal-smear cycles
(20). Results As expected, there were age differences in the lordosis reflex during the baseline isolation period. Almost half of the old rats (48%) were in CL, but none of the young rats were (Pearson chisquare test, p _< 0.001). In general, the old rats had an elevated
SOCIAL M O D U L A T I O N OF B E H A V I O R A L A G I N G average lordosis reflex intensity which was higher than that in the young rats [old rats = 5.1 _+ 0.3; young rats = 3.0 + 0.2; main effect of age during the baseline period, F(1, 89) = 36.86, p ~< 0.0001]. Despite these initial age differences, group living had the same effect on the behavior of old and young females, decreasing their lordosis reflex intensity to a similar extent from baseline intensity (see Fig. 1). In contrast, the lordosis reflex intensity of the control rats maintained in isolation did not change. STUDY 2: SOCIAL MODULATION OF THE BIOMARKER CONSTANT LORDOSIS
In Study 1, group living decreased the lordosis reflex intensity in old rats to the same extent that it decreased it in young rats. This finding suggested that living in groups for a long time would delay the onset of the biomarker constant lordosis (CL) during aging, particularly because it is a biomarker for constant estrus and other aspects of the reproductive aging process (13,14). To test this hypothesis, we first grouped rats at the age when isolated females begin to enter CL. Then we determined how long social signals could delay the onset of CL, by grouping animals throughout their whole life span. Finally, we assessed the effects of long-term group living on the temporal relationship of CL and constant estrus (CE), to determine whether social signals alter the sequence or prevalence of these two biomarkers o f reproductive aging.
Method In part 1 of this study, the young rats that were grouped in Study 1 continued living in groups until the age when isolated rats typically enter CL (temporary group living; 5.5-7 months of age; n = 25). The young females that were controls in Study 1 continued in isolation (n = 28). In the part 2 of the study, females from a second cohort were either grouped throughout the whole life span (long-term group living; n = 25) or isOlated from 2 months of age on (n = 24), when data collection began.
Resu#s Part 1. Temporary group living. Although temporary group living had no effect on the incidence of CL (percent of rats entering CL: grouped rats = 56%, isolated rats - 64%, Pearson chi-square test, NS), it did delay the onset of CL among rats that entered the state (age of CL onset: grouped rats = 9.6 + 0.5 months; isolated rats = 8.0 + 0.4 months; L o g - R a n k test, p _< 0.04; see Fig. 2A). The onset of CL was delayed until rats returned to isolation. Then rats entered CL at a rate comparable to that seen in females that were always isolated (as indicated by the parallel slopes of the two curves in Fig. 2A; the rate of entry into CL is proportional to the slope of the log survivor curve). Part 2. Long-term group living. In contrast to temporary group living, group living throughout the whole life span decreased the incidence of CL (incidence of CL: grouped rats, 24%; isolated rats, 79%; Pearson chi-square test, p < 0.001). A m o n g rats that entered CL, long-term group living dramatically delayed its onset (onset of CL: grouped rats - 23 _+ 0.8 months; isolated rats = 8.6 + 0.9 months; L o g - R a n k test, p _< 0.0002; see Fig. 2B). This delay was much greater than the delay in temporary group living (Log-Rank test, p _< 0.0001; there was no difference in CL onset in isolation in the two studies, L o g - R a n k test, NS). Long-term group living also delayed the termination of CL (termination of CL: grouped rats = 27.0 _+ 1.0 months; isolated rats = 15.8 _+ 1.1 months; L o g - R a n k test, p < 0.0002). However, it did not
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FIG. 1. The effect of the social environment on the lordosis reflex intensity. (A) Effect on young rats. (B) Effect on old rats. In both graphs, the lordosis reflex intensity represents the mean intensity during complete vaginal-smear cycles. Main effect of social environment, F(1, 89) = 15.77, p -< 0.0001; main effect of age, F( 1, 89) = 35.15, p _<0.0001; interaction of social environment and age, F(1, 89) = 0.23, NS; period (baseline vs. experimental), F(1, 89) - 32.88, p _< 0.0001; interaction of period and social environment, F(I, 89) = 70.01, p _< 0.0001; interaction of period and age, F(1, 89) = 5.19, p _< 0.0252; interaction of period and social environment and age, F(1, 89) = 1.21, NS. alter the duration of the state (duration of CL: grouped rats = 3.9 _+ 1.6 months; isolated rats = 5.8 + 0.9 months; L o g - R a n k test, NS). Group living throughout the whole life span also changed the temporal relationship between CL and CE. Although group living delayed CE (onset of CE: grouped rats = 12.9 _+ 1.3 months; isolated rats = 8.8 + 0.9 months; L o g - R a n k test, p _< 0.05), the delay was less than for CL. Consequently, the onset of CL actually followed that of CE in rats that entered both states, reversing the temporal relationship that occurred in isolated rats (CL follows CE: grouped rats = 100%; isolated rats = 31%; Fisher's Exact test, p -< 0.05). G r o u p living also shortened the duration of CE, in contrast to its effects on CL (duration of CE: grouped rats = 2.4 _+ 0.4 months; isolated rats = 5.0 + 1.2 months; L o g - R a n k test, p -< 0.008), perhaps as a consequence of the delayed onset reported above (20,21). Consequently, group living altered the prevalence of both states during the life span (see Fig. 3). STUDY 3: LORDOSIS REFLEX AND RECEPTIVITY TO A MALE IN OLD RATS
The previous longitudinal studies focused on the lordosis reflex measured noninvasively by manual palpation, independent
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mission and subsequent pregnancy or pseudopregnancy. Tests were carried out during the second half of the dark period when male mounting is most frequent (10). Tests took place in mating arenas (30 × 30 × 9") filled with clean bedding. The arenas were left open so that the female could escape the male by climbing its sides, and each contained an open plastic shelter (10 × 9 × 8-1/2"). Prior to testing, sexually experienced males were tested for vigor with young females and then adapted to the mating arenas for an hour. Then each female was tested for 5 minutes with each of two males, and behaviors were scored by a trained observer. Interrater reliability was 92%. A daily receptivity score was calculated for each female by determining the lordosis quotient, the percentage of mounts she responded to with a lordosis reflex (24). For analysis, data from one complete vaginal-smear cycle were used.
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The temporal coordination between the lordosis response to manual palpation and to a male was altered in old rats. Although approximately half of the old rats (52%) maintained a cyclic
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(A) The effectof temporary group living. The arrow indicates resumption of isolation. (B) The effect of long-term group living. In both graphs, the data points represent the percentage of rats that remained in cycliclordosis at a given age (i.e., had not yet entered CL). (Note that there is no significant difference between the two cohorts of isolated rats, Log-Rank test NS.)
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The association between lordosis reflexes elicited by manual palpation and by a male rat's mount was compared in old and young rats. Old females (n = 21; 17 months of age) had passed through CE (63% of old rats), and all old females had irregular vaginal-smear cycles with extended days of cornifled epithelial cells and/or leukocytes in the vaginal cytology. The young rats (n = 9; 5 months of age) had regular cycles. The lordosis reflex in response to manual palpation was measured daily during the life span as described in the General Method section. The lordosis reflex in response to a male was measured during 11 consecutive days of mating tests. Because of repeated testing, vaginal masks were used to prevent intro-
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of stimuli from a male. To investigate whether this measure is a biomarker for other aspects of behavioral reproductive senescence, this study assessed the relationship between the lordosis reflex elicited by manual palpation and that elicited by a male rat.
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Age (Months) FIG. 3. The prevalence of constant lordosis (CL) and constant estrus (CE) in rats during isolation and long-term group living. The vertical dotted lines indicate the beginning of data collection.
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SOCIAL MODULATION OF BEHAVIORAL AGING lordosis reflex pattern similar to that manifested by all young rats, these old rats were less likely to have both their maximum lordosis reflex intensity to manual palpation and their maximum receptivity to a male occur on the same day (temporal coordination of the two responses: old rats with a cyclic reflex = 33%; young rats = 100%; Pearson chi-square test, p -< 0.01). Furthermore, the rest of the old rats did not have a cyclic lordosis reflex to manual palpation and instead maintained the same intensity across the cycle, which was either tonically low (maximum intensity _< 1.5; range _<2; 38% of all old rats) or tonically high (maximum intensity ~ 7, range _< 2; 10% of all old rats). Thus, these old rats did not have an identifiable day of a maximum lordosis reflex intensity. Consequently, their maximum lordosis reflex elicited by a male and manual palpation were also no longer temporally coordinated. Despite this loss of temporal coordination in the old rats, the average intensity of the lordosis reflex to manual palpation predicted the magnitude of their receptivity to a male. The mean lordosis reflex intensity to manual palpation, averaged over the vaginal-smear cycles of each old rat, predicted both the percentage of cycle days that the rats were receptive (r 2 - 0.45, linear regression, n - 21, p _< 0.0008; see Fig. 4) and the mean lordosis quotient, averaged over cycle days (r 2 = 0.35, linear regression, n = 21, p _< 0.0047). Comparable correlations could not be calculated for young rats because they did not have the variance that old females had in these measures of mating with the male: all young females were receptive to the male only on proestrus and had a high lordosis quotient. Thus, the two behaviors remained associated in old rats, although they were no longer temporally coordinated within a vaginal-smear cycle, as they were in young rats. GENERAL DISCUSSION
Social signals from other females dramatically alter behavioral reproductive senescence in female rats. Group living throughout the whole life span decreased the incidence of the acyclic behavioral state, constant lordosis (CL), by almost 70% compared to isolation. It also greatly delayed its onset and termination in rats that entered the state. Consequently, the state began at the end of the life span, rather than during the first half. Thus, social signals continued to modulate the lordosis reflex intensity of rats as they aged. In fact, group living had as strong an effect on the behavior of old rats as on that of young rats. These findings in old rats extend previous reports on young rats that social signals from females modulate the lordosis reflex and enhance its coordination with other components of the estrous cycle (14,17). They also replicate our earlier findings in different cohorts of rats that the lordosis reflex intensity increases during aging, particularly in isolation (13,14). Social signals delayed behavioral acyclicity for almost as long as rats lived in groups, even when females stopped having estrous cycles and entered constant estrus (CE). Conversely, isolating rats after temporary group living initiated the onset of CL even when rats were still cycling. Thus, the social environment had a greater effect on behavior than on vaginal cyclicity, although it affected both components of the estrous cycle. Since group living delayed behavioral acyclicity more than vaginal acyclicity, it consequently reversed the temporal relationship between CL and CE. Group living also altered their prevalence during the lifespan. These findings demonstrate that both of these biomarkers of aging are highly responsive to the social environment, rather than developmentally fixed. The two components of the estrous cycle may be affected by different social signals. Olfactory signals alter vaginal cyclicity,
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FIG. 4. The relationship between the lordosis reflex elicited by manual palpation (mean intensity during the vaginal-smear cycle of each old rat) and by a male, as measured by either (A) the percentage of cycle days rats displayed lordosisreflexes in responseto a male or (B) the mean lordosis quotient during the vaginal-smearcycle. but do not affect the lordosis component of the estrous cycle (17). Instead, preliminary results suggest that body contact modulates the lordosis reflex (17). The exact nature of the signals provided by body contact, as well as their transduction pathways, need to be further investigated. However, it is clear that the lordosis reflex and cyclicity are affected by different social signals when animals are young, and presumably this difference continues during aging. If body contact is the social signal that affects the lordosis reflex, rather than pheromonal or other signals, it would explain why old and young rats are equally affected by group living. The potency of body contact is not likely to be reduced by the aging of the source rats, as is the potency of pheromones (23). Likewise, our data suggest that response to body contact does not depend on age or the endocrine state of the recipient animal. Thus, social modulation of the lordosis reflex was age independent, rather than age dependent. This distinction between age-independent and age-dependent effects is important for understanding when during the life span the social environment modulates reproductive function. The effect of social signals on the lordosis reflex may be mediated by ovarian hormones. It is not possible to measure levels of ovarian hormone daily over a long period of time, as would be required to demonstrate endocrine mediation in these longitudinal studies. However, it is well established that lordosis reflex intensity is estrogen dependent (7,24). Moreover, group living decreases plasma estradiol levels (16). Thus, our findings suggest that group living may decrease the lordosis reflex intensity
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by reducing estrogen levels. It may also affect receptor levels, other hormones that act synergistically with estrogen, or even the neural circuitry involved in the lordosis reflex, without any associated endocrine changes. Finally, our findings indicate that the lordosis reflex, as measured by manual palpation, is a biomarker for other aspects of behavioral reproductive senescence. Specifically, this measure was associated with sexual receptivity to a male in both old and young rats. Nonetheless, the relationship between these two measures was different in the two age groups. In young rats, the lordosis reflex elicited by manual palpation and by a male were temporally coupled, cooccurring only on proestrus. In old rats, the magnitude and frequency of the two behaviors over the whole vaginal-smear cycle were associated, but the behaviors were no longer temporally coupled. Other behavioral components of the estrous cycle (e.g., activity, feeding behavior) may also decouple during aging~ If so, a decline in the temporal coordination of estrous cycle components may be an important feature of reproductive senescence in rats. Since successful reproduction depends on the temporal coordination of behavioral and physiological components of the
estrous cycle (22), social signals may play a critical role in maintaining fertility. In the wild, most female rats live in stable groups with other females, sleeping together and interacting socially within a burrow system (3,8). However, some females live alone in an isolated part of the burrow and have decreased fertility and fecundity (3). Social signals may extend the reproductive life span, by delaying senescence in those components which age first and preventing their decoupling from other estrous cycle components. Conversely, the early behavioral reproductive senescence and decoupling of behavioral and neuroendocrine components that occurs in isolated rats may contribute to their early loss of fertility and fecundity.
ACKNOWLEDGEMENTS Research supported by NIA grant PHS 5 R23 AG02408 and NIMH grant PHS 2 R37 MH41788 to M.K.M. and PHS S07 RR07029 to The University of Chicago. Fellowship support to J.L. from the Brookdale National Foundation. Many thanks to S. Cogswell and T. L. Butler for animal colony management and to C. Gerrish for data collection.
REFERENCES 1. Aron, C. Mechanisms of control of the reproductive function by olfactory stimuli in female mammals. Physiol. Rev. 59:229-284; 1979. 2. Butcher, R. L.; Page, R. D. Role of the aging ovary in cessation of reproduction. In: Schwartz, N. B.; Hunzicker-Dunn, M., eds. Dynamics of ovarian function. New York: Raven Press; 1981:253271. 3. Calhoun, J..B. The ecology and sociology of the Norway rat. Bethesda, MD: U.S. Dept. of Health, Education, and Welfare; 1962. 4, Chambers, K. C.; Phoenix, C. H. Sexual behaviors of aging female rats: Influence of age and hormonal state of male partners. Neurobiol. Aging 7:165-171; 1986. 5, Cooper, R. L. Sexual receptivity in aged female rats. Behavioral evidence for increased sensitivity to estrogen. Horm. Behav, 9:321333; 1977. 6, Cowley, J. J. Olfaction and the development of sexual behavior. In: Hutchinson, J. B., ed. Biological determinants of sexual behavior. New York: John Wiley and Sons; 1978:87-126. 7. Davidson, J. M.; Smith, E. R.; Rodgers, C. H.; Bloch, G. J. Relative threshold of behavioral and somatic responses to estrogen. Physiol. Behav. 3:227-229; 1968. 8. Davis, D. E. Social interactions of rats as indicated by trapping procedures. Behavior 8:335-344; 1955. 9. Finch, C. E.; Felicio, L. S.; Mobbs, C. V.; Nelson, J. F. Ovarian and steroidal influences in neuroendocrine aging processes in female rodents, Endocr. Rev. 5:467-497; 1984. 10. Harlan, R. E.; Shivers, B. D.; Shivers, R. L.; Moss, R. L.; Shryne, J. F.; Aarski, R. A. Sexual performance as a function of time of day in male and female rats. Biol. Reprod. 23:64-71; 1980. 1t. Hays, W. L. Statistics for the social sciences. New York: Holt, Rinehart and Winston; 1973. 12. Hendricks, S. E.; Lehman, J. R.; Oswalt, G. L. Effects of copulation on reproductive function in aged female rats. Physiol. Behav. 23: 267-272; 1979. 13. LeFevre, J. A.; McClintock, M. K. Reproductive senescence in female rats: A longitudinal study of individual differences in estrous cycles and behavior. Biol. Reprod. 38:780-789; 1988.
14. LeFevre, J. A.; McClintock, M. K. Isolation accelerates reproductive senescence and alters its predictors in female rats. Horm. Behav. 25: 258-272; 1991. 15. Long, J. A.; Evans, H. M. The oestrous cycle in the rat and its associated phenomena. Berkeley, CA: The University of California Press; 1922. 16. Lupo Di Prisco, C.; Lucarini, N.; Dessi-Fulgheri, F. Testosterone aromatization in rat brain is modulated by social environment. Physiol. Behav. 20;345-348; 1978. 17. McClintock, M. K. Socialcontrol ofthe ovarian cycle andthe function of estrous synchrony. Am. Zool. 2:243-256; 1981. 18. McClintock, M. K. Pheromonal regulation of the ovarian cycle: Enhancement, suppression and synchrony. In: Vandenbergh, J. H., ed. Pheromones and reproduction in mammals. New York: Academic Press; 1983:113-149. 19. McClintock, M. K. Modulation of the estrous cycle by pheromones from pregnant and lactating rats. Biol. Reprod. 28:823-829; 1983. 20. Nelson, J. F.; Felicio, L. S. Reproductive aging in the female: An etiological perspective. In: Rothstein, M., ed. Review of biological research in aging, vol. 2. New York: Alan R. Liss, Inc.; 1985:251-314. 21. Nelson, J. F.; Felicio, L. S.; Randall, P. K.; Sims, C.; Finch, C. E. A longitudinal study ofestrous cyclicity in aging C57B1/6J mice: 1. Cycle frequency, length, and vaginal cytology. Biol, Reprod. 27: 327-339; 1982. 22. Nequin, L. G.; Alvarez, J.; Schwartz, N. B. Measurement of serum steroid and gonadotropin levels and uterine and ovarian variables throughout 4 day and 5 day estrous cycles in the rat. Biol. Reprod. 20:659-670; 1979. 23. Peng, M. T.; Mu, S. C. Difference in responsiveness of old female rats to estrogen to secrete sex attractants as a function of different reproductive states. Gerontology 34:110-114; 1988. 24. Pfaff, D. W. Estrogen and brain function. New York: SpringerVerlag; 1980. 25. SAS Institute. SAS user's guide: Statistics. Version 5 edition. Cary, NC: SAS Institute, Inc.; 1985. 26. Siegel, S. Nonparametric statistics for the behavioral sciences. New York: McGraw Hill Book Company; 1956,
0031-9384/92 $5.00 + .00 Copyright © 1992PergamonPressLtd.
Printed in the USA.
Social Modulation of Behavioral Reproductive Senescence in Female Rats JUDITH
A. L E F E V R E ~ A N D M A R T H A
K. M c C L I N T O C K 2
Department of Psychology, The University of Chicago, Chicago, I L 60637 R e c e i v e d l l F e b r u a r y 1991 LEFEVRE, J. A. AND M. K. McCLINTOCK. Social modulation of behavioral reproductivesenescence infemale rats. PHYSIOL BEHAV 52(3) 603-608, 1992.--During aging, the lordosis reflex of female rats changes from a state of cyclic lordosis, when the lordosis reflex can be elicited by manual palpation only on proestrus, to a state of constant lordosis (CL), when it can be elicited daily. Social signals from other females altered this pattern of behavioral senescence. Group living decreased the lordosis reflex intensity in both old and young rats and delayed the onset of CL. Long-term group living delayed CL until late in the life span, while temporary group living delayed it only until rats returned to isolation. Long-term group living also changed the temporal relationship between CL and the acyclic, anovulatory state of constant estrus. Finally, the lordosis reflex intensity, measured by manual palpation, predicted the old rats' receptivity to a male, indicating that constant lordosis is a biomarker for other aspects of behavioral reproductive senescence. Aging
Sexual behavior
Rats
Social environment
R E P R O D U C T I V E senescence has typically been described in terms of changes in vaginal cytology as well as hypothalamic, pituitary, and ovarian function. These neuroendocrine changes are associated with a sequence of reproductive states in the female rat: regular estrous cycles, irregular cycles, constant estrus (the acyclic state which only half of females enter), persistent diestrus, and finally anestrus (2,9,13,20). Less frequently, reproductive senescence has been described in terms of female sexual behavior. During aging, the temporal coordination between sexual receptivity and the ovarian cycle changes. Receptivity is characterized by a lordosis reflex in response to male mounts, a behavioral posture which facilitates intromission (24). Young rats are receptive only on proestrus, just prior to ovulation, maximizing the likelihood of conception (15,22). Middle-aged rats that are in constant estrus, as defined by vaginal cytology, are often receptive repeatedly on consecutive days (5,12). Finally, old rats that are in persistent diestrus (e.g., repetitive pseudopregnancies) are rarely receptive, if at all (5). A similar pattern, of increase and then decrease, emerges when the lordosis reflex is elicited by manual palpation and measured longitudinally throughout reproductive senescence (13). The frequency of the lordosis reflex increases until the reflex can be elicited on every day of the cycle, the state of constant lordosis. Then the frequency of the lordosis reflex decreases, until the reflex can no longer be elicited. In isolated rats, this process of behavioral reproductive senescence precedes and predicts changes in the other components
Social isolation
Lordosis reflex
of the estrous cycle. As the lordosis reflex increases in frequency, it decouples from the other estrous-cycle components that are still cycling. For example, when the lordosis reflex becomes acyclic, rats still have regular or irregular cycles in vaginal cytology (13). In fact, the onset of constant lordosis predicts when rats will stop cycling and enter constant estrus, a n acyclic, anovulatory state (13,14). In young rats, social signals from other females enhance the temporal coordination of the lordosis reflex with other estrous cycle components. They modulate the frequency and intensity of the lordosis reflex during the estrous cycle, as well as modulating cycle length, regularity, and the frequency of ovulation ( 1,6,17,18). However, their effect on behavioral reproductive senescence is unknown. There is some evidence that the effect of social signals on behavior declines during aging. For example, social signals from males have less effect on the approach behavior of old than young females (4). It is u n k n o w n , however, whether social signals from other females continue to modulate the lordosis reflex of old rats, just as they modulate that of young rats (14,17). Thus, one purpose of this research was to determine whether living in groups with other females affects the lordosis reflex of old rats to the same extent that it affects that of young rats. If social signals continue to modulate the lordosis reflex during aging, they may delay the onset of constant lordosis, the acyclic behavioral state. However, there may be a limit to how long social signals can delay constant lordosis, just as there is a limit
1Present address: Andrus Gerontology Center, Leonard Davis School of Gerontology, University of Southern California, University Park, MC0191, Los Angeles, CA 90089-0191. 2 Requests for reprints should be addressed to Dr. Martha K. McClintock, Department of Psychology, The University of Chicago, 5730 South Woodlawn Ave., Chicago, IL 60637.
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to their effect on another biomarker of aging, the acyclic state of constant estrus (14). To first determine whether social signals can delay the onset of constant lordosis, we examined the effect of group living during the period when isolated animals begin to enter the state. Next, we determined how long social signals can delay constant lordosis, by maintaining group living throughout the lifespan. Then, we determined whether differences in the effect of group living on constant lordosis and constant estrus alters the temporal relationship or prevalence of the two biomarkers of reproductive aging during the lifespan. Finally, we investigated whether the lordosis reflex measured by manual palpation is a biomarker for other aspects of behavioral reproductive senescence. Because our longitudinal studies necessitated daily assessment of the lordosis reflex, it was measured by manual palpation. However, it is unknown whether this measure is associated with receptivity to a male in old rats, as it is in young rats (17,24). Thus, a final purpose was to determine whether the relationship between the lordosis reflex measured by manual palpation and receptivity is different in old and young rats. GENERAL METHOD
Animals Female Sprague-Dawley rats (n = 172) were received from Charles River Breeding Laboratories at 1-1/2to 2 months of age (n = 34) or bred from Charles River-derived females (n = 138). Rats were maintained in the laboratory throughout the life span [note that these cohorts of rats are different from those used in previous studies (13,14,17)]. Females were group housed five per cage in stainless steel cages (18 X 24 X 14") or isolated in plastic cages with wire bottoms ( 10 × 9 × 8-1/2"),as required, for each study. Light intensity and the amount of space per rat were the same in the isolation and group cages. None of the rats were ever mated, except as specified in Study 3. Colony rooms were lit with a reversed light cycle (14L: 10D; middark at 1330 h with dim red illumination during the dark phase) and maintained at 22 _+ 2°C, with relative humidity 3555%. Food (Teklad rat chow, 4% fat) and tap water were supplied ad lib. Animal health was monitored during daily data collection and biweekly health checks. Individuals were sacrificed at the first sign of respiratory or other disease.
Daily Measures and Coding The lordosis reflex and vaginal smear were measured once daily from 2 or 4 months of age throughout the life span. The intensity of the lordosis reflex was measured at middark by manual palpation. Intensity was scored on a ten-point scale based on two measures: strength and habituation. Each level of strength (0-4, ranging from a hunched posture to intense dorsiflexion of the back) was combined with one of two levels of habituation (0-1, reflex habituates vs. does not habituate with repeated stimulation) (13,19). Interrater reliability was 91%. These daily measures were then analyzed to determine the lordosis state of the animal (13). The daily scores were averaged to obtain a mean for each vaginal-smear cycle. From the record of mean cycle scores, the state of constant lordosis (CL) was identified when the mean was >5 for at least three vaginal-smear cycles spanning at least 16 days. In our laboratory, this criterion corresponds to a condition in which the lordosis reflex can be elicited on every day. A mean of <5 indicated the state of cyclic lordosis. The vaginal smears were made from vaginal lavages taken daily at middark. They were examined immediately and classified
according to the stage of the estrous cycle (2,13,15). Interrater reliability was 87%. The daily vaginal smear records were then used to code vaginal smear cycles and reproductive states as previously described: regular cycles, irregular cycles, constant estrus, and persistent diestrus (13).
Analysis Frequencies were analyzed with the Pearson chi-square test and Fisher's Exact test (11,26). The onset, duration, and termination of constant lordosis and constant estrus were analyzed with the Log-Rank test in the SAS Life Test procedure for survival analysis (25). Changes in the lordosis reflex intensity were analyzed with a three-way analysis of variance (2 × 2 X 2 factorial model: age and social environment, with time period as a repeated measure) and univariate analyses for each time period, using the SAS General Linear Models Procedure (25). Associations between the lordosis reflex elicited by manual palpation and that elicited by male mounts were analyzed with the SAS linear regression procedure (25). Summary statistics are presented as the means and SEM. STUDY 1: SOCIAL MODULATIONOF THE LORDOSIS REFLEX INTENSITY IN OLD RATS Although social signals modulate the lordosis reflex during the estrous cycle of young rats, it is unknown whether they continue to do so during aging. This study determined whether group living affects the lordosis reflex intensity of old rats to the same extent that it affects that of young females.
Method All females were housed alone at 4 months and were maintained in isolation until the start of the study. Their vaginalsmear cycles were continuously monitored beginning at 3-1h months of age. The old rats (15-L/2 months of age; n = 40) all had irregular vaginal-smear cycles (of varying lengths), although many had passed through the acyclic state of constant estrus (63%). The young rats (4-Vz months of age; n = 53) had regular cycles, 4 days in length (42% of rats) or irregular cycles of varying lengths (58% of rats). Rats remained alone during a baseline period (28 days). Females were then divided into experimental and control groups. In the young rats, the two groups were matched by cycle length during the baseline period in order to standardize the time during which the average lordosis reflex intensity was calculated. However, we did not include a detailed analysis of vaginal-smear cycle length in this behavioral study because a preliminary analysis of these data confirmed our previous reports (17,19) that the effect of isolation on the lordosis reflex is independent of any change in vaginal-smear cycle length. During the experimental period (28 days), the experimental females (young rats, n = 25; old rats, n = 22) were moved to group housing. The control rats (young rats, n = 28; old rats, n = 18) remained isolated to control for the effects of aging during the course of the experiment. Each rat's average lordosis reflex intensity was calculated separately for the baseline and experimental periods, using data from complete vaginal-smear cycles
(20). Results As expected, there were age differences in the lordosis reflex during the baseline isolation period. Almost half of the old rats (48%) were in CL, but none of the young rats were (Pearson chisquare test, p _< 0.001). In general, the old rats had an elevated
SOCIAL M O D U L A T I O N OF B E H A V I O R A L A G I N G average lordosis reflex intensity which was higher than that in the young rats [old rats = 5.1 _+ 0.3; young rats = 3.0 + 0.2; main effect of age during the baseline period, F(1, 89) = 36.86, p ~< 0.0001]. Despite these initial age differences, group living had the same effect on the behavior of old and young females, decreasing their lordosis reflex intensity to a similar extent from baseline intensity (see Fig. 1). In contrast, the lordosis reflex intensity of the control rats maintained in isolation did not change. STUDY 2: SOCIAL MODULATION OF THE BIOMARKER CONSTANT LORDOSIS
In Study 1, group living decreased the lordosis reflex intensity in old rats to the same extent that it decreased it in young rats. This finding suggested that living in groups for a long time would delay the onset of the biomarker constant lordosis (CL) during aging, particularly because it is a biomarker for constant estrus and other aspects of the reproductive aging process (13,14). To test this hypothesis, we first grouped rats at the age when isolated females begin to enter CL. Then we determined how long social signals could delay the onset of CL, by grouping animals throughout their whole life span. Finally, we assessed the effects of long-term group living on the temporal relationship of CL and constant estrus (CE), to determine whether social signals alter the sequence or prevalence of these two biomarkers o f reproductive aging.
Method In part 1 of this study, the young rats that were grouped in Study 1 continued living in groups until the age when isolated rats typically enter CL (temporary group living; 5.5-7 months of age; n = 25). The young females that were controls in Study 1 continued in isolation (n = 28). In the part 2 of the study, females from a second cohort were either grouped throughout the whole life span (long-term group living; n = 25) or isOlated from 2 months of age on (n = 24), when data collection began.
Resu#s Part 1. Temporary group living. Although temporary group living had no effect on the incidence of CL (percent of rats entering CL: grouped rats = 56%, isolated rats - 64%, Pearson chi-square test, NS), it did delay the onset of CL among rats that entered the state (age of CL onset: grouped rats = 9.6 + 0.5 months; isolated rats = 8.0 + 0.4 months; L o g - R a n k test, p _< 0.04; see Fig. 2A). The onset of CL was delayed until rats returned to isolation. Then rats entered CL at a rate comparable to that seen in females that were always isolated (as indicated by the parallel slopes of the two curves in Fig. 2A; the rate of entry into CL is proportional to the slope of the log survivor curve). Part 2. Long-term group living. In contrast to temporary group living, group living throughout the whole life span decreased the incidence of CL (incidence of CL: grouped rats, 24%; isolated rats, 79%; Pearson chi-square test, p < 0.001). A m o n g rats that entered CL, long-term group living dramatically delayed its onset (onset of CL: grouped rats - 23 _+ 0.8 months; isolated rats = 8.6 + 0.9 months; L o g - R a n k test, p _< 0.0002; see Fig. 2B). This delay was much greater than the delay in temporary group living (Log-Rank test, p _< 0.0001; there was no difference in CL onset in isolation in the two studies, L o g - R a n k test, NS). Long-term group living also delayed the termination of CL (termination of CL: grouped rats = 27.0 _+ 1.0 months; isolated rats = 15.8 _+ 1.1 months; L o g - R a n k test, p < 0.0002). However, it did not
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FIG. 1. The effect of the social environment on the lordosis reflex intensity. (A) Effect on young rats. (B) Effect on old rats. In both graphs, the lordosis reflex intensity represents the mean intensity during complete vaginal-smear cycles. Main effect of social environment, F(1, 89) = 15.77, p -< 0.0001; main effect of age, F( 1, 89) = 35.15, p _<0.0001; interaction of social environment and age, F(1, 89) = 0.23, NS; period (baseline vs. experimental), F(1, 89) - 32.88, p _< 0.0001; interaction of period and social environment, F(I, 89) = 70.01, p _< 0.0001; interaction of period and age, F(1, 89) = 5.19, p _< 0.0252; interaction of period and social environment and age, F(1, 89) = 1.21, NS. alter the duration of the state (duration of CL: grouped rats = 3.9 _+ 1.6 months; isolated rats = 5.8 + 0.9 months; L o g - R a n k test, NS). Group living throughout the whole life span also changed the temporal relationship between CL and CE. Although group living delayed CE (onset of CE: grouped rats = 12.9 _+ 1.3 months; isolated rats = 8.8 + 0.9 months; L o g - R a n k test, p _< 0.05), the delay was less than for CL. Consequently, the onset of CL actually followed that of CE in rats that entered both states, reversing the temporal relationship that occurred in isolated rats (CL follows CE: grouped rats = 100%; isolated rats = 31%; Fisher's Exact test, p -< 0.05). G r o u p living also shortened the duration of CE, in contrast to its effects on CL (duration of CE: grouped rats = 2.4 _+ 0.4 months; isolated rats = 5.0 + 1.2 months; L o g - R a n k test, p -< 0.008), perhaps as a consequence of the delayed onset reported above (20,21). Consequently, group living altered the prevalence of both states during the life span (see Fig. 3). STUDY 3: LORDOSIS REFLEX AND RECEPTIVITY TO A MALE IN OLD RATS
The previous longitudinal studies focused on the lordosis reflex measured noninvasively by manual palpation, independent
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SOCIAL MODULATION OF BEHAVIORAL AGING lordosis reflex pattern similar to that manifested by all young rats, these old rats were less likely to have both their maximum lordosis reflex intensity to manual palpation and their maximum receptivity to a male occur on the same day (temporal coordination of the two responses: old rats with a cyclic reflex = 33%; young rats = 100%; Pearson chi-square test, p -< 0.01). Furthermore, the rest of the old rats did not have a cyclic lordosis reflex to manual palpation and instead maintained the same intensity across the cycle, which was either tonically low (maximum intensity _< 1.5; range _<2; 38% of all old rats) or tonically high (maximum intensity ~ 7, range _< 2; 10% of all old rats). Thus, these old rats did not have an identifiable day of a maximum lordosis reflex intensity. Consequently, their maximum lordosis reflex elicited by a male and manual palpation were also no longer temporally coordinated. Despite this loss of temporal coordination in the old rats, the average intensity of the lordosis reflex to manual palpation predicted the magnitude of their receptivity to a male. The mean lordosis reflex intensity to manual palpation, averaged over the vaginal-smear cycles of each old rat, predicted both the percentage of cycle days that the rats were receptive (r 2 - 0.45, linear regression, n - 21, p _< 0.0008; see Fig. 4) and the mean lordosis quotient, averaged over cycle days (r 2 = 0.35, linear regression, n = 21, p _< 0.0047). Comparable correlations could not be calculated for young rats because they did not have the variance that old females had in these measures of mating with the male: all young females were receptive to the male only on proestrus and had a high lordosis quotient. Thus, the two behaviors remained associated in old rats, although they were no longer temporally coordinated within a vaginal-smear cycle, as they were in young rats. GENERAL DISCUSSION
Social signals from other females dramatically alter behavioral reproductive senescence in female rats. Group living throughout the whole life span decreased the incidence of the acyclic behavioral state, constant lordosis (CL), by almost 70% compared to isolation. It also greatly delayed its onset and termination in rats that entered the state. Consequently, the state began at the end of the life span, rather than during the first half. Thus, social signals continued to modulate the lordosis reflex intensity of rats as they aged. In fact, group living had as strong an effect on the behavior of old rats as on that of young rats. These findings in old rats extend previous reports on young rats that social signals from females modulate the lordosis reflex and enhance its coordination with other components of the estrous cycle (14,17). They also replicate our earlier findings in different cohorts of rats that the lordosis reflex intensity increases during aging, particularly in isolation (13,14). Social signals delayed behavioral acyclicity for almost as long as rats lived in groups, even when females stopped having estrous cycles and entered constant estrus (CE). Conversely, isolating rats after temporary group living initiated the onset of CL even when rats were still cycling. Thus, the social environment had a greater effect on behavior than on vaginal cyclicity, although it affected both components of the estrous cycle. Since group living delayed behavioral acyclicity more than vaginal acyclicity, it consequently reversed the temporal relationship between CL and CE. Group living also altered their prevalence during the lifespan. These findings demonstrate that both of these biomarkers of aging are highly responsive to the social environment, rather than developmentally fixed. The two components of the estrous cycle may be affected by different social signals. Olfactory signals alter vaginal cyclicity,
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1 2 3 4 5 6 7 8 9 10 Mean Lordosis Reflex Intensity to Manual Palpation
FIG. 4. The relationship between the lordosis reflex elicited by manual palpation (mean intensity during the vaginal-smear cycle of each old rat) and by a male, as measured by either (A) the percentage of cycle days rats displayed lordosisreflexes in responseto a male or (B) the mean lordosis quotient during the vaginal-smearcycle. but do not affect the lordosis component of the estrous cycle (17). Instead, preliminary results suggest that body contact modulates the lordosis reflex (17). The exact nature of the signals provided by body contact, as well as their transduction pathways, need to be further investigated. However, it is clear that the lordosis reflex and cyclicity are affected by different social signals when animals are young, and presumably this difference continues during aging. If body contact is the social signal that affects the lordosis reflex, rather than pheromonal or other signals, it would explain why old and young rats are equally affected by group living. The potency of body contact is not likely to be reduced by the aging of the source rats, as is the potency of pheromones (23). Likewise, our data suggest that response to body contact does not depend on age or the endocrine state of the recipient animal. Thus, social modulation of the lordosis reflex was age independent, rather than age dependent. This distinction between age-independent and age-dependent effects is important for understanding when during the life span the social environment modulates reproductive function. The effect of social signals on the lordosis reflex may be mediated by ovarian hormones. It is not possible to measure levels of ovarian hormone daily over a long period of time, as would be required to demonstrate endocrine mediation in these longitudinal studies. However, it is well established that lordosis reflex intensity is estrogen dependent (7,24). Moreover, group living decreases plasma estradiol levels (16). Thus, our findings suggest that group living may decrease the lordosis reflex intensity
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by reducing estrogen levels. It may also affect receptor levels, other hormones that act synergistically with estrogen, or even the neural circuitry involved in the lordosis reflex, without any associated endocrine changes. Finally, our findings indicate that the lordosis reflex, as measured by manual palpation, is a biomarker for other aspects of behavioral reproductive senescence. Specifically, this measure was associated with sexual receptivity to a male in both old and young rats. Nonetheless, the relationship between these two measures was different in the two age groups. In young rats, the lordosis reflex elicited by manual palpation and by a male were temporally coupled, cooccurring only on proestrus. In old rats, the magnitude and frequency of the two behaviors over the whole vaginal-smear cycle were associated, but the behaviors were no longer temporally coupled. Other behavioral components of the estrous cycle (e.g., activity, feeding behavior) may also decouple during aging~ If so, a decline in the temporal coordination of estrous cycle components may be an important feature of reproductive senescence in rats. Since successful reproduction depends on the temporal coordination of behavioral and physiological components of the
estrous cycle (22), social signals may play a critical role in maintaining fertility. In the wild, most female rats live in stable groups with other females, sleeping together and interacting socially within a burrow system (3,8). However, some females live alone in an isolated part of the burrow and have decreased fertility and fecundity (3). Social signals may extend the reproductive life span, by delaying senescence in those components which age first and preventing their decoupling from other estrous cycle components. Conversely, the early behavioral reproductive senescence and decoupling of behavioral and neuroendocrine components that occurs in isolated rats may contribute to their early loss of fertility and fecundity.
ACKNOWLEDGEMENTS Research supported by NIA grant PHS 5 R23 AG02408 and NIMH grant PHS 2 R37 MH41788 to M.K.M. and PHS S07 RR07029 to The University of Chicago. Fellowship support to J.L. from the Brookdale National Foundation. Many thanks to S. Cogswell and T. L. Butler for animal colony management and to C. Gerrish for data collection.
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