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Postnatal Experiences and Genetic Effects on Squirrel Monkey Social Affinities and Emotional Distress
Hormones and Behavior 36, 266 –275 (1999) Article ID hbeh.1999.1547, available online at http://www.idealibrary.com on
Postnatal Experiences and Genetic Effects on Squirrel Monkey Social Affinities and Emotional Distress David M. Lyons,* ,1 Frances L. Martel,* Seymour Levine,† Neil J. Risch,‡ and Alan F. Schatzberg* *Department of Psychiatry and Behavioral Science, Stanford University School of Medicine, Stanford, California; †Department of Psychology, University of Delaware, Newark, Delaware; and ‡Department of Genetics, Stanford University School of Medicine, Stanford, California Received February 26, 1999; revised July 12, 1999; accepted July 21, 1999
Most nonhuman primate research on risk factors underlying vulnerability to stress has focused on early psychosocial experiences in various species of macaques. To test for genetic and experiential effects on emotional vulnerability in randomly bred squirrel monkeys, here we combined a paternal half-sibling analysis with three postnatal rearing protocols that altered aspects of maternal availability. In one condition offspring were periodically removed from natal groups, whereas differences in maternal availability were produced in two other conditions by manipulating the effort required of lactating mothers to successfully locate food. After completion of these protocols at 21 weeks of age, social affinities, maternal separation induced peep-calls, and plasma levels of cortisol were assessed from 29 to 37 weeks of age. Significant postnatal rearing effects and the lowest heritabilities were detected in peak elevations of cortisol measured 1 day after the removal of mothers from otherwise undisturbed groups. Individual differences in cortisol 3–7 days later revealed negligible postnatal rearing effects and the highest heritabilities (h 2 ; .70), as offspring sired by certain fathers failed to return to the preseparation level found in undisturbed natal groups. Paternal half-siblings that responded with long lasting increases in cortisol spent more time near their mother in undisturbed groups and exhibited long-lasting increases in separation induced peep-calls. These findings concur with human twin studies that suggest genetic and experiential factors contribute to individual differences in vulnerability to emotional distress. © 1999 Academic Press
1 To whom correspondence and reprint requests should be addressed at Department of Psychiatry and Behavioral Science, Stanford University School of Medicine, MSLS P111, Stanford, CA 94305-5485. Fax: (650) 498-7761. E-mail: [email protected].
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Key Words: mother–infant relationships; psychosocial development; hypothalamic-pituitary-adrenal physiology; affective disorders; behavioral genetics.
Apart from reports of heritable differences in stress induced anxiety during development in studies of rhesus macaques (Suomi, 1987; Suomi, Kraemer, Baysinger, and DeLizio, 1981), most primate research on risk factors underlying vulnerability to stress has focused on early maternal availability. Various species of macaques raised in the absence of mothers subsequently exhibit atypical hypothalamic-pituitary-adrenal responses to stress, abnormal levels of brain dopamine and norepinephrine, altered autonomic activity, fragmented patterns of sleep, and unusual behavioral propensities (Clarke, Kraemer, and Kupfer, 1998; Kaemingk and Reite, 1987; Kraemer, 1997; Martin, Sackett, Gunderson, and Goodlin-Jones, 1988; Mason, Mendoza, and Moberg, 1991; Shannon, Champoux, and Suomi, 1998). Maternal deprivation induced changes in dendritic branching have been characterized in cortical tissue (Struble and Riesen, 1978), and changes in tyrosine hydroxylase activity are evident in the striatum, substantia nigra, and ventral tegmental area, but not the paraventricular nucleus of the hypothalamus (Ginsberg, Hof, McKinney, and Morrison, 1993; Martin, Spicer, Lewis, Gluck, and Cork, 1991). Ecologically informed research on early maternal availability has likewise begun to identify untoward effects on primate postnatal development. Stressful foraging demands alter aspects of maternal care and 0018-506X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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development in vervet monkeys, baboons, and macaques (Altmann, 1980; Barnett, Dunbar, and Dunbar, 1995; Boccia, Laundenslager, and Reite, 1994; Hauser and Fairbanks, 1988; Karssemeijer, Vos, and Van Hooff, 1990; Rosenblum and Andrews, 1994). Squirrel monkey mothers in high demand foraging conditions stop carrying their infants at earlier ages and maintain elevated plasma levels of cortisol (Levine, Champoux, and Wiener, 1991; Lyons, Kim, Schatzberg, and Levine, 1998a). Mother reared bonnet macaques exposed as infants to variable foraging demands show increased emotional vulnerability when separated from their mother, are more reluctant to leave their mother in unfamiliar situations, and demonstate deficits in affiliative behavior during interactions with peers (Andrews and Rosenblum, 1991; Andrews and Rosenblum, 1994; Rosenblum and Andrews, 1994). These same monkeys exhibit in early adulthood elevated cerebrospinal fluid levels of biogenic amines, somatostatin, and corticotropin-releasing factor (CRF) (Coplan, Andrews, Rosenblum, Owens, Friedman, Gorman, and Nemeroff, 1996; Coplan, Trost, Owens, Cooper, Gorman, Nemeroff, and Rosenblum, 1998). Hypersecretion of CRF and elevated levels of cortisol have been implicated in the pathophysiology of human affective disorders which are often triggered or exacerbated by stressful events (Chrousos and Gold, 1998; McEwen, 1998; Schatzberg and Nemeroff, 1998). Recent twin studies suggest that genetic factors play an equally influential role in the etiology of anxiety and depression (Kendler and Karkowski-Shuman, 1997; Kendler, Walters, Neale, Kessler, Heath, and Eaves, 1995; Lyons, Eisen, Goldberg, True, Lin, Meyer, Toomey, Faraone, Merla-Ramos, and Tsuang, 1998b; Silove, Manicavasagar, O’Connell, and Morris-Yates, 1995). Based on results from the human twin study approach, genetic factors may also contribute to individual differences in the elicitation of parental care (Kendler, 1996; O’Connor, Hetherington, Reiss, and Plomin, 1995) and reliance on supportive social relationships as a strategy for coping with stress (Kendler, 1997; Mellins, Gatz, and Baker, 1996). Although twinning is uncommon in most monkeys and apes, a suitable alternative for primate genetic research is the paternal half-sibling analysis (Clarke, Kammerer, George, Kupfer, McKinney, Spence, and Kraemer, 1995; Higley, Thompson, Champoux, Goldman, Hasert, Kraemer, Scanlan, Suomi, and Linnoila, 1993; Suomi et al., 1981). A number of males are each randomly mated with several different females, and the offspring of each female is measured on the phenotypic characters of interest. Under the null hypoth-
esis of no hereditary effect, within- and between-father components of phenotypic variance are equivalent, and F ratios approximate 1. As the between-father component of variance increases F ratios grow larger in the analysis of variance and the null hypothesis is rejected (Falconer, 1960). A strength of the paternal half-sibling approach is that phenotypic similarities found within groupings of paternal halfsiblings cannot be attributed to shared family environments when paternal half-siblings are raised by different mothers in the absence of fathers. To test for genetic and experiential effects on emotional vulnerability in randomly bred squirrel monkeys, here we combined a paternal half-sibling analysis with three postnatal rearing protocols that altered aspects of maternal availability. In one condition offspring were periodically removed from natal groups, whereas differences in maternal availability were produced in the other two conditions by manipulating the effort required of lactating mothers to successfully locate food. After completion of these protocols at 21 weeks of age, all offspring were treated in identical fashion under standard laboratory conditions. Social affinities, maternal separation induced peep-calls, and plasma levels of cortisol were assessed from 29 to 37 weeks of age before and after the removal of mothers. Offspring were well past weaning at this stage of development and feeding on solid foods (Lyons et al., 1998a).
METHODS Subjects Forty infants sired by 13 fathers who had no form of contact with their offspring were randomly distributed in 12 small groups each composed of 3 or 4 mother–infant pairs. All monkeys were of Guyanese origin (Saimiri sciureus) and were born and raised at the Stanford University Primate Facility. Groups were housed indoors in 1.8 3 1.2 3 1.8 m wire-mesh cages adjacent to large windows and maintained on natural light– dark cycles. Solid partitions between wire-mesh cages prevented visual and physical contact between monkeys in different groups. A sliding door in the front panel of each cage provided access to a portable transport cage. Monkeys were pretrained to quickly enter the transport cage on command to facilitate the sampling procedures described below. All procedures were conducted in accordance with and as required by the Animal Welfare Act and approved by Stanford
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FIG. 1.
Schematic representation of the experimental design.
University’s Administrative Panel on Laboratory Animal Care. Experimental Design and Procedures As part of ongoing research on the consequences of postnatal stress, four natal groups were randomly assigned to each of the following three rearing conditions when infants were 10 weeks of age (range, 8 –13 weeks). 1. Low foraging demands. A total of 14 infants (8 males, 6 females) and their 14 mothers were maintained from 10 to 21 weeks postpartum in low demand conditions where food was easy to find. Each group received 600% by weight of their normal daily food intake buried in foraging boards (for details, see Lyons et al., 1998a). All 80 holes in the foraging boards contained abundant amounts of food, so for these mothers and their infants food was not difficult to find. 2. High foraging demands. A total of 13 infants (7 males, 6 females) and 13 mothers were maintained from 10 to 21 weeks postpartum in high-demand conditions where each group was provisioned with 120% of their daily food intake buried in foraging boards. Since many holes contained little or no food, more time and effort was required in the high demand condition to successfully locate food. Throughout the 12-week foraging protocols body weights and amounts of food consumed by high- and low-demand monkeys were not significantly different (Lyons et al., 1998a). 3. Intermittent social deprivations. A total of 13 infants (6 males, 7 females) and 13 mothers fed from standard food hoppers were intermittently separated for five
sessions, each lasting 5 h. Every other week from 13 to 21 weeks postpartum each intermittently separated infant was removed one at a time, placed in a cage adjacent to unfamiliar monkeys, and temporarily deprived of all forms of contact with members of the natal group. Upon completion of these protocols at 21 weeks of age, all monkeys were treated in identical fashion under standard laboratory conditions. At 29 weeks of age, a blood sample was collected from each offspring in undisturbed natal groups. Seven days later a second blood sample was obtained. Thereafter, one mother was removed from each otherwise undisturbed group and temporarily housed in a different building. The removed mother’s offspring was observed immediately before, 1 h after, and 1 day after removal of the mother using 10-min focal animal observation methods described below. A blood sample from the focal offspring was obtained immediately after the last (1day) observation, and the mother was returned to the group. These procedures were repeated with a different mother removed at weekly intervals, until each mother was removed and returned 1 day later within each of the natal groups (Fig. 1). After temporarily removing each mother one at a time, two focal animal observations were conducted and a blood sample was collected at 35 weeks of age from offspring in undisturbed groups. Focal animal observations and blood sample collections were performed on different days. All mothers were then simultaneously removed at 36 weeks postpartum and returned to the breeding facility. Focal animal observations of each offspring were conducted 1 h, 2 days, and 6 days later, and blood samples were collected 1,
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TABLE 1 Interaction of Distribution of Offspring with Rearing Condition Fathers Postnatal rearing conditions
Jon
a
High demands Low demands
Don
?
b
? ?
Ben i
Intermittent separations
? ? /
c f
Ric
?
Dan
Jed
?
/
?
/
a
/
Lee d
h
/ /
?
Sid g
?
Gil e
Ted
Ken
/ /
? /
/
/
/ /
Jun
? /
h
? /
Ray b g
? ?
? / d ? c f
/
e
? /
?
i
? / ?
Note. Male (?) and female (/) offspring in each postnatal rearing condition were sired by 13 fathers that had no form of contact with their offspring. Twenty-two mothers each produced a single offspring and nine mothers produced with different fathers on separate occasions two offspring (designated as italicized letter pairs).
3, and 7 days later in undisturbed juvenile peer groups. Focal animal observations were conducted between 08:00 and 10:00 h with individuals easily identified by dye marks and number tags on necklaces. Using a time-ruled check sheet and countdown timer, trained observers scored on-the-signal at 15-s intervals scan samples of time spent near (,10 cm) the focal animal’s own mother, near other mothers, and near peers. Peep-calls were scored using one-zero sampling at 15-s intervals as a behavioral index of maternal separation induced anxiety (Weerts and Miczek, 1996; Wiener, Bayart, Faull, and Levine, 1990). Interobserver correlations for time spent near own mother (r 5 0.96), near other mothers (r 5 0.83), near peers (r 5 0.78), and peep-calls (r 5 0.78) were all statistically significant (P , 0.01). Interobserver agreements corrected for chance (Cohen’s kappa) ranged from .78 to .95. Blood samples (1 ml) were collected at 09:00 h from manually restrained monkeys by femoral venipuncture. Blood was transferred to glass tubes on ice and centrifuged at 4°C, and the plasma fraction was stored in polypropylene tubes at 280°C. Cortisol was measured in unextracted plasma using a previously described radioimmunoassay (Lyons, Ha, and Levine, 1995). Within- and between-assay coefficients of variation were, respectively, 4.8 and 6.1%. Assay sensitivity was 3 mg/dl. Most blood samples (89%) were collected within 3 min from cage entry (median latency to sample collection 5 100 s; range, 47–358 s), and all but three samples were collected within 4 min. In keeping with reports that cortisol levels sampled within these time limits using these procedures do not
reflect capture and handling effects (Lyons et al., 1995; Lyons, Wang, Lindley, Levine, Kalin, and Schatzberg, 1999), sample collection latencies accounted for less than 2% of the variance in levels of plasma cortisol. Data Analysis Postnatal rearing experiences and hereditary effects were assessed with repeated measures analysis of variance (ANOVA) using least squares estimates from general linear models in the MGLH module of Systat. In each three-factor ANOVA, the postnatal rearing condition was considered a between-subjects factor, paternity (offspring grouped by father) was considered a between-subjects factor, and postseparation time course was considered a within-subjects factor. Gender differences were not significant and were excluded from analysis. The paternity 3 postnatal rearing condition interaction was likewise excluded from ANOVAs due to the incomplete distribution of offspring sired by each father in the unbalanced factorial design (Table 1). A total of 13 fathers and 31 mothers produced the 40 offspring that composed the study cohort. Twenty-two mothers each contributed one offspring, and nine mothers produced with different fathers on separate occasions two offspring. No more than one offspring from a given mother was included in each paternal half-sibling group. To identify the proportion of total variance in each phenotypic measure that was attributable to additive genetic variance, estimates of heritability were derived from paternal half-sibling groupings using the standard ANOVA approach (Falconer, 1960). For each
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TABLE 2 Estimates of Heritability (h 2) for Social Affinities, Peep-calls, and Plasma Cortisol Levels Measured in Three Different Social Settings Phenotypic measure
h2
Undisturbed natal group Near own mother Near other mothers Near peers Peep-calls Cortisol levels
.56* .50* .34 .01 .22
Own mother removed Near other mothers Neer peers Peep calls at 1-hr Peep calls at 1-day Cortisol levels at 1-day
.58* .32 .16 .48* .06
All mothers removed Near peers Peep calls at 1 hr Peep calls at 2 days Peep-calls at 6 days Cortisol levels at 1 day Cortisol levels at 3 days Cortisol levels at 7 days
After the removal of each mother one at a time, the removed mother’s offspring spent more time near other mothers [F(2,50) 5 5.74, P , 0.01] and more time near peers [F(2,50) 5 6.58, P , 0.01]. Postnatal rearing effects were not evident in time spent near other mothers, but a paternity main effect was discerned [F(12,25) 5 2.34, P , 0.04]. Paternal half-siblings that spent more time near other mothers when their own
,.01 .22 ,.01 ,.01 .05 .68* .70*
* Significantly greater than zero, P , .05.
phenotypic measure a one-way ANOVA was performed with paternal half-sibling grouping (paternity) considered a between-subjects factor. The betweenfather mean-square minus the within-father meansquare was divided by 3.1 (average number of offspring per father), multiplied by 4 (half-siblings share, on average, 25% of their genome by common descent), and divided by the total variance. Heritability estimates and all ANOVA test statistics were evaluated with two-tailed probabilities, and descriptive statistics are presented as mean 6 SEM.
RESULTS In undisturbed natal groups, measures of time spent by offspring near their own mother, near other mothers, and near peers were not significantly different. Postnatal rearing effects likewise failed to achieve statistical significance. The only significant finding was the paternity main effect [F(12,25) 5 2.38, P , 0.04]. For social affinities in undisturbed groups the highest heritability (Table 2) and most salient differences between groupings of paternal half-siblings were in time spent by offspring near their own mother (Fig. 2A).
FIG. 2. Differences between offspring grouped by father on (A) scan sample measures of time spent by each offspring near its own mother, (B) time spent near other mothers after the removal of each offspring’s own mother, and (C) peep-calls emitted 1 day after the removal of each mother one at a time from otherwise undisturbed groups (mean 6 SEM).
Stress, Heredity, and Hypercortisolism
FIG. 3. Postnatal rearing effects on (A) maternal separation induced peep-calls and (B) plasma cortisol levels measured before and after the removal of each mother one at a time from otherwise undisturbed groups (mean 6 SEM).
mother was removed (Fig. 2B) spent more time near their own mother in undisturbed natal groups (r 5 0.32, df 38, P , 0.05). Paternal half-sibling effects were not evident in time spent near peers, and postnatal rearing effects fell short of significance. After the removal of each mother one at a time, the removed mother’s offspring exhibited increases in peep-calls and increases in plasma levels of cortisol (Fig. 3). Relative to monkeys from the postnatal foraging conditions, the intermittently separated monkeys responded with smaller maternal separation induced increases in peep-calls [rearing condition 3 time course interaction, F(4,50) 5 5.22, P , 0.01] and smaller increases in cortisol [rearing condition 3 time course interaction, F(2,25) 5 9.32, P , 0.01]. Paternal half-sibling effects were also expressed in the production of maternal separation induced peep-calls [paternity 3 time course interaction, F(24,50) 5 1.80, P , 0.05]. Peep-calls were seldom emitted in undisturbed groups, whereas peak levels of peep-calls were produced 1 h after the removal of each mother. Paternal half-sibling effects were not evident in either of these measures, nor in the maternal separation induced adrenocortical response. But 1 day after the removal of each mother more peep-calls (Fig. 2C) were emitted by paternal half-siblings that spent more time near their own mother in undisturbed natal groups (r 5 0.48, df 38, P , 0.01).
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When all mothers were simultaneously removed their offspring spent more time near peers (Fig. 4A). The rearing condition 3 postseparation time course interaction was not significant, but a rearing condition main effect was discerned [F(2,25) 5 14.36, P , 0.001]. Relative to offspring from the postnatal foraging conditions, the intermittently separated offspring spent more time near peers. Significant rearing effects were likewise expressed in the production of maternal separation induced peep-calls [rearing condition 3 time course interaction, F(6,75) 5 3.88, P , 0.01]. Offspring from the postnatal foraging conditions emitted the most peep-calls, whereas the intermittently separated offspring emitted the fewest peep-calls (Fig. 4B). Comparable effects were also detected in acute increases in plasma levels of cortisol. Offspring from the postnatal foraging conditions initially produced greater increases in cortisol relative to the intermittently separated offspring (Fig. 4C). But early rearing effects on cortisol levels were diminished 3 and 7 days after the removal of all mothers, as reflected in the rearing condition 3 time course interaction [F(6,75) 5 6.65, P , 0.001]. From 1 to 7 days after the removal of all mothers decreases in cortisol were most clearly expressed in offspring from the foraging conditions. Postnatal rearing effects on cortisol levels rapidly subsided over a short period of time, while differences between groupings of paternal half-siblings emerged 3–7 days after the removal of all mothers [paternity 3 time course interaction, [F(36,75) 5 2.00, P , 0.01]. A few fathers sired offspring whose cortisol levels re-
FIG. 4. Postnatal rearing effects on (A) scan sample measures of time spent near peers, (B) maternal separation induced peep-calls, and (C) plasma cortisol levels measured before and after the simultaneous removal of all mothers (mean 6 SEM).
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turned to the preseparation level within 3 days after all mothers were removed, whereas cortisol levels in the offspring sired by other fathers remained elevated for more than 7 days (Fig. 5). The lowest heritabilities (Table 2) and consistent postnatal rearing effects (Fig. 3B and 4C) were evident in peak elevations of cortisol measured 1 day after the removal of mothers simultaneously or one at a time. Cortisol levels 3 and 7 days after the simultaneous removal of all mothers showed negligible postnatal rearing effects (Fig. 4C) and the highest heritabilities (Table 2), as offspring sired by certain fathers failed to return to the preseparation level found in undisturbed natal groups (Fig. 5). Paternal half-siblings that spent more time near their mother in undisturbed groups exhibited higher levels of cortisol 3 and 7 days after the removal of all mothers (3 days; r 5 0.40, df 38, P , 0.05; 7 days; r 5 0.58, df 38, P , 0.01). Individual differences in peep-calls emitted 1 day after the removal of each mother one at a time (Fig. 2C) were correlated with chronic but not acute increases in cortisol measured after the removal of all mothers from groups (1 day: r 5 0.15, df 38, P 5 0.35; 3 days: r 5 0.28, df 38, P 5 0.08; 7 days, r 5 0.39, df 38, P , 0.02).
DISCUSSION We previously reported that squirrel monkey mothers randomly assigned to the high demand condition consistently exhibit elevated cortisol levels relative to mothers maintained in the low-demand condition where food is easy to find (Levine et al., 1991; Lyons et al., 1998a). Although squirrel monkey mothers in the high demand condition stop carrying their offspring at earlier ages, high- and low-demand offspring spend similar amounts of time nursing on their mother and feeding on solid foods. Squirrel monkey mothers direct little aggression toward their offspring in either postnatal foraging condition, and cortisol levels during the foraging protocols are not consistently different in high- and low-demand squirrel monkey infants (Lyons et al., 1998a). Evidence presented in this study suggests that by accelerating some transitions in development (carry 3 self-transport) and not others (nursing 3 self-feeding), squirrel monkey mothers in the high demand condition spared their offspring from the deficits discovered in experiments with bonnet macaques. Squirrel monkeys exposed as infants to the high demand condition were not significantly different from the low demand offspring in social affinities, maternal separa-
FIG. 5. Differences between offspring grouped by father in plasma cortisol levels measured 1, 3, and 7 days after the simultaneous removal of all mothers (mean 6 SEM).
tion induced peep-calls, and serial assessments of the adrenocortical response. Variable demands composed of alternating 2-weeks blocks of high- and low-demand foraging may be required to produce deviant outcomes in development as reported for bonnet macaques (Coplan et al., 1996; Coplan et al., 1998; Rosenblum and Andrews, 1994). Relative to squirrel monkey offspring from the postnatal foraging conditions, the intermittently separated offspring responded with blunted increases in cortisol
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1 day after the removal of mothers simultaneously or one at a time. Postnatally handled rats periodically separated from their mother for short periods of time likewise exhibit blunted hypothalamic-pituitary-adrenal (HPA) responses to mildly stressful events (Levine, 1967; Meaney, Diorio, Francis, Widdowson, LaPlante, Caldji, Sharma, Seckl, and Plotsky, 1996; Plotsky and Meaney, 1993). Blunted HPA responses to stress in postnatally handled rats are mediated, in part, by enhanced glucocorticoid negative-feedback sensitivity resulting from increased glucocorticoid receptor gene expression in the hippocampus (O’Donnell, Larocque, Seckl, and Meaney, 1994). Conversely, prolonged intermittent periods of maternal separation decrease glucocorticoid receptor binding sites in hippocampus and impair glucocorticoid negative-feedback sensitivity in rats studied as adults (Meaney et al., 1996; Plotsky and Meaney, 1993). Despite these findings in laboratory rodents, it is not yet clear that early environmental regulation of glucocorticoid feedback accounts for the blunted adrenocortical responses observed in our intermittently separated squirrel monkeys. Relative to offspring from the postnatal foraging conditions, the intermittently separated offspring also emitted fewer maternal separation-induced peep-calls and spent significantly more time near peers. These results concur with numerous reports that nonmaternal forms of social support eliminate or reduce stress induced anxiety and adrenocortical responses in primates (Coe, Rosenberg, Fischer, and Levine, 1987; Gust, Gordon, Brodie, and McClure, 1996; Smith, McGreer-Whitworth, and French, 1998; Stanton, Patterson, and Levine, 1985). Negligible postnatal rearing effects and significant heritabilities were evident in time spent by offspring near their own mother in natal groups. High heritabilities were also expressed in measures of maternal separation induced anxiety that reflect aspects of filial attachment (Mason and Mendoza, 1998). Paternal halfsiblings that spent more time near their mother in undisturbed groups exhibited longer lasting increases in maternal separation induced peep-calls and prolonged adrenocortical responses. Similar effects evident among pairs of offspring produced from the same mother in different groupings of paternal halfsiblings do not account for high heritabilities in the paternal half-sibling analysis. Similarities within maternal half-sibling pairs randomized across different groupings of paternal half-siblings reduce the between-father component of variance. This decreases the magnitude of heritability estimates derived using the paternal half-sibling approach.
Estimates of heritability are of course specific to the population and the circumstance in which they are assessed. Genetically diverse populations studied in uniform conditions show larger heritabilities than genetically homogeneous populations assessed in diverse environments (Falconer, 1960). Few fathers or mothers that produced the study cohort shared a common parent, but not all grandparents could be identified with absolute certainty from long standing breeding colony records. Nevertheless, it is intriguing find in this cohort of monkeys evidence consistent with human twin studies that indicate genetic and experiential factors contribute to individual differences in vulnerability to social loss (Goldsmith and Gottesman, 1981; Kendler and Karkowski-Shuman, 1997; Kendler et al., 1995; Silove et al., 1995). Whereas certain individuals quickly disengage and move on, demonstrating little emotional distress, others take much longer and experience sustained negative affect.
ACKNOWLEDGMENTS The authors thank C. Chen, K. Fong, B. Gavino, G. Ha, S. Kim, N. Shrieken, and L. Steffey for assistance with sample collections, E. Lowe, S. Weiner, and L. Steffey for meticulously maintaining 24 years of breeding colony records, and the Pritzker Network for constructive comments on this research. Supported by the Pritzker Network and Grant MH47573 from the National Institute of Mental Health.
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rearing condition on HPA axis response to fluoxetine and desipramine treatment over repeated social separations in young rhesus monkeys. Psychiat. Res. 79, 91–104. Coe, C. L., Rosenberg, L. T., Fischer, M., and Levine, S. (1987). Psychological factors capable of preventing the inhibition of antibody responses in separated infant monkeys. Child Dev. 58, 1420 –1430. Coplan, J. D., Andrews, M. W., Rosenblum, L. A., Owens, M. J., Friedman, S., Gorman, J. M., and Nemeroff, C. B. (1996). Persistent elevations of cerebrospinal fluid concentrations of corticotropinreleasing factor in adult nonhuman primates exposed to early-life stressors: Implications for the pathophysiology of mood and anxiety disorders. Proc. Natl. Acad. Sci. USA 93, 1619 –1623. Coplan, J. D., Trost, R. C., Owens, M. J., Cooper, T. B., Gorman, J. M., Nemeroff, C. B., and Rosenblum, L. A. (1998). Cerebrospinal fluid concentrations of somatostatin and biogenic amines in grown primates reared by mothers exposed to manipulated foraging conditions. Arch. Gen. Psychiat. 55, 473– 477. Falconer, D. S. (1960). Introduction to Quantitative Genetics. Ronald Press, New York. Ginsberg, S. D., Hof, P. R., McKinney, W. T., and Morrison, J. H. (1993). The noradrenergic innervation density of the monkey paraventricular nucleus is not altered by early social deprivation. Neurosci. Lett. 158, 130 –134. Goldsmith, H. H., and Gottesman, II (1981). Origins of variation in behavioral style: A longitudinal study of temperament in young twins. Child Dev. 52, 91–103. Gust, D. A., Gordon, T. P., Brodie, A. R., and McClure, H. M. (1996). Effect of companions in modulating stress associated with new group formation in juvenile rhesus macaques. Physiol. Behav. 59, 941–945. Hauser, M. D., and Fairbanks, L. A. (1988). Mother–infant conflict in vervet monkeys: Variation in response to ecological conditions. Anim. Behav. 36, 802– 813. Higley, J. D., Thompson, W. W., Champoux, M., Goldman, D., Hasert, M. F., Kraemer, G. W., Scanlan, J. M., Suomi, S. J., and Linnoila, M. (1993). Paternal and maternal genetic and environmental contributions to cerebrospinal fluid monoamine metabolites in rhesus monkeys (Macaca mulatta). Arch. Gen. Psychiat. 50, 615– 623. Kaemingk, K., and Reite, M. (1987). Social environment and nocturnal sleep: Studies in peer-reared monkeys. Sleep 10, 542–550. Karssemeijer, G. J., Vos, D. R., and Van Hooff, J. A. R. A. M. (1990). The effect of some nonsocial factors on mother–infant contact in long-tailed macaques (Macaca fascicularis). Behaviour 113, 273–291. Kendler, K. S. (1996). Parenting: A genetic-epidemiologic perspective. Am. J. Psychiat. 153, 11–20. Kendler, K. S. (1997). Social support: A genetic-epidemiologic analysis. Am. J. Psychiat. 154, 1398 –1404. Kendler, K. S., and Karkowski-Shuman, L. (1997). Stressful life events and genetic liability to major depression: Genetic control of exposure to the environment? Psychol. Med. 27, 539 –547. Kendler, K. S., Walters, E. E., Neale, M. C., Kessler, R. C., Heath, A. C., and Eaves, L. J. (1995). The structure of the genetic and environmental risk factors for six major psychiatric disorders in women: Phobia, generalized anxiety disorder, panic disorder, bulimia, major depression, and alcoholism. Arch. Gen. Psychiat. 52, 374 –383. Kraemer, G. W. (1997). Psychobiology of early social attachment in rhesus monkeys: Clinical implications. Ann. N.Y. Acad. Sci. 807, 401– 418. Levine, S. (1967). Maternal and environmental influences on the
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Shannon, C., Champoux, M., and Suomi, S. J. (1998). Rearing condition and plasma cortisol in rhesus monkey infants. Am. J. Primatol. 46, 311–321. Silove, D., Manicavasagar, V., O’Connell, D., and Morris-Yates, A. (1995). Genetic factors in early separation anxiety: Implications for the genesis of adult anxiety disorders. Acta Psychiatr. Scand. 92, 17–24. Smith, T. E., McGreer-Whitworth, B., and French, J. A. (1998). Close proximity of the heterosexual partner reduces the physiological and behavioral consequences of novel-cage housing in black tufted-ear marmosets (Callithrix kuhli). Horm. Behav. 34, 211–222. Stanton, M. E., Patterson, J. M., and Levine, S. (1985). Social influences on conditioned cortisol secretion in the squirrel monkey. Psychoneuroendocrinology 10, 125–134. Struble, R. G., and Riesen, A. H. (1978). Changes in cortical dendritic branching subsequent to partial social isolation in stumptailed monkeys. Dev. Psychobiol. 11, 479 – 486.
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Suomi, S. J. (1987). Genetic and maternal contributions to individual differences in rhesus monkey biobehavioral development. In N. A. Krasnegor, E. M. Blass, M. A. Hofer, and W. P. Smotherman (Eds.), Perinatal Development: A Psychobiological Perspective, pp. 397– 419. Academic Press, New York. Suomi, S. J., Kraemer, G. W., Baysinger, C. M., and DeLizio, R. D. (1981). Inherited and experiential factors associated with individual differences in anxious behavior displayed by rhesus monkeys. In D. F. KLein and J. Rabkin (Eds.), Anxiety: New Research and Changing Concepts, pp. 179 –199. Raven, New York. Weerts, E. M., and Miczek, K. A. (1996). Primate vocalizations during social separation and aggression: Effects of alcohol and benzodiazepines. Psychopharmacology (Berl) 127, 255–264. Wiener, S. G., Bayart, F., Faull, K. F., and Levine, S. (1990). Behavioral and physiological responses to maternal separation in squirrel monkeys (Saimiri sciureus). Behav. Neurosci. 104, 108 –115.
Postnatal Experiences and Genetic Effects on Squirrel Monkey Social Affinities and Emotional Distress David M. Lyons,* ,1 Frances L. Martel,* Seymour Levine,† Neil J. Risch,‡ and Alan F. Schatzberg* *Department of Psychiatry and Behavioral Science, Stanford University School of Medicine, Stanford, California; †Department of Psychology, University of Delaware, Newark, Delaware; and ‡Department of Genetics, Stanford University School of Medicine, Stanford, California Received February 26, 1999; revised July 12, 1999; accepted July 21, 1999
Most nonhuman primate research on risk factors underlying vulnerability to stress has focused on early psychosocial experiences in various species of macaques. To test for genetic and experiential effects on emotional vulnerability in randomly bred squirrel monkeys, here we combined a paternal half-sibling analysis with three postnatal rearing protocols that altered aspects of maternal availability. In one condition offspring were periodically removed from natal groups, whereas differences in maternal availability were produced in two other conditions by manipulating the effort required of lactating mothers to successfully locate food. After completion of these protocols at 21 weeks of age, social affinities, maternal separation induced peep-calls, and plasma levels of cortisol were assessed from 29 to 37 weeks of age. Significant postnatal rearing effects and the lowest heritabilities were detected in peak elevations of cortisol measured 1 day after the removal of mothers from otherwise undisturbed groups. Individual differences in cortisol 3–7 days later revealed negligible postnatal rearing effects and the highest heritabilities (h 2 ; .70), as offspring sired by certain fathers failed to return to the preseparation level found in undisturbed natal groups. Paternal half-siblings that responded with long lasting increases in cortisol spent more time near their mother in undisturbed groups and exhibited long-lasting increases in separation induced peep-calls. These findings concur with human twin studies that suggest genetic and experiential factors contribute to individual differences in vulnerability to emotional distress. © 1999 Academic Press
1 To whom correspondence and reprint requests should be addressed at Department of Psychiatry and Behavioral Science, Stanford University School of Medicine, MSLS P111, Stanford, CA 94305-5485. Fax: (650) 498-7761. E-mail: [email protected].
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Key Words: mother–infant relationships; psychosocial development; hypothalamic-pituitary-adrenal physiology; affective disorders; behavioral genetics.
Apart from reports of heritable differences in stress induced anxiety during development in studies of rhesus macaques (Suomi, 1987; Suomi, Kraemer, Baysinger, and DeLizio, 1981), most primate research on risk factors underlying vulnerability to stress has focused on early maternal availability. Various species of macaques raised in the absence of mothers subsequently exhibit atypical hypothalamic-pituitary-adrenal responses to stress, abnormal levels of brain dopamine and norepinephrine, altered autonomic activity, fragmented patterns of sleep, and unusual behavioral propensities (Clarke, Kraemer, and Kupfer, 1998; Kaemingk and Reite, 1987; Kraemer, 1997; Martin, Sackett, Gunderson, and Goodlin-Jones, 1988; Mason, Mendoza, and Moberg, 1991; Shannon, Champoux, and Suomi, 1998). Maternal deprivation induced changes in dendritic branching have been characterized in cortical tissue (Struble and Riesen, 1978), and changes in tyrosine hydroxylase activity are evident in the striatum, substantia nigra, and ventral tegmental area, but not the paraventricular nucleus of the hypothalamus (Ginsberg, Hof, McKinney, and Morrison, 1993; Martin, Spicer, Lewis, Gluck, and Cork, 1991). Ecologically informed research on early maternal availability has likewise begun to identify untoward effects on primate postnatal development. Stressful foraging demands alter aspects of maternal care and 0018-506X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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development in vervet monkeys, baboons, and macaques (Altmann, 1980; Barnett, Dunbar, and Dunbar, 1995; Boccia, Laundenslager, and Reite, 1994; Hauser and Fairbanks, 1988; Karssemeijer, Vos, and Van Hooff, 1990; Rosenblum and Andrews, 1994). Squirrel monkey mothers in high demand foraging conditions stop carrying their infants at earlier ages and maintain elevated plasma levels of cortisol (Levine, Champoux, and Wiener, 1991; Lyons, Kim, Schatzberg, and Levine, 1998a). Mother reared bonnet macaques exposed as infants to variable foraging demands show increased emotional vulnerability when separated from their mother, are more reluctant to leave their mother in unfamiliar situations, and demonstate deficits in affiliative behavior during interactions with peers (Andrews and Rosenblum, 1991; Andrews and Rosenblum, 1994; Rosenblum and Andrews, 1994). These same monkeys exhibit in early adulthood elevated cerebrospinal fluid levels of biogenic amines, somatostatin, and corticotropin-releasing factor (CRF) (Coplan, Andrews, Rosenblum, Owens, Friedman, Gorman, and Nemeroff, 1996; Coplan, Trost, Owens, Cooper, Gorman, Nemeroff, and Rosenblum, 1998). Hypersecretion of CRF and elevated levels of cortisol have been implicated in the pathophysiology of human affective disorders which are often triggered or exacerbated by stressful events (Chrousos and Gold, 1998; McEwen, 1998; Schatzberg and Nemeroff, 1998). Recent twin studies suggest that genetic factors play an equally influential role in the etiology of anxiety and depression (Kendler and Karkowski-Shuman, 1997; Kendler, Walters, Neale, Kessler, Heath, and Eaves, 1995; Lyons, Eisen, Goldberg, True, Lin, Meyer, Toomey, Faraone, Merla-Ramos, and Tsuang, 1998b; Silove, Manicavasagar, O’Connell, and Morris-Yates, 1995). Based on results from the human twin study approach, genetic factors may also contribute to individual differences in the elicitation of parental care (Kendler, 1996; O’Connor, Hetherington, Reiss, and Plomin, 1995) and reliance on supportive social relationships as a strategy for coping with stress (Kendler, 1997; Mellins, Gatz, and Baker, 1996). Although twinning is uncommon in most monkeys and apes, a suitable alternative for primate genetic research is the paternal half-sibling analysis (Clarke, Kammerer, George, Kupfer, McKinney, Spence, and Kraemer, 1995; Higley, Thompson, Champoux, Goldman, Hasert, Kraemer, Scanlan, Suomi, and Linnoila, 1993; Suomi et al., 1981). A number of males are each randomly mated with several different females, and the offspring of each female is measured on the phenotypic characters of interest. Under the null hypoth-
esis of no hereditary effect, within- and between-father components of phenotypic variance are equivalent, and F ratios approximate 1. As the between-father component of variance increases F ratios grow larger in the analysis of variance and the null hypothesis is rejected (Falconer, 1960). A strength of the paternal half-sibling approach is that phenotypic similarities found within groupings of paternal halfsiblings cannot be attributed to shared family environments when paternal half-siblings are raised by different mothers in the absence of fathers. To test for genetic and experiential effects on emotional vulnerability in randomly bred squirrel monkeys, here we combined a paternal half-sibling analysis with three postnatal rearing protocols that altered aspects of maternal availability. In one condition offspring were periodically removed from natal groups, whereas differences in maternal availability were produced in the other two conditions by manipulating the effort required of lactating mothers to successfully locate food. After completion of these protocols at 21 weeks of age, all offspring were treated in identical fashion under standard laboratory conditions. Social affinities, maternal separation induced peep-calls, and plasma levels of cortisol were assessed from 29 to 37 weeks of age before and after the removal of mothers. Offspring were well past weaning at this stage of development and feeding on solid foods (Lyons et al., 1998a).
METHODS Subjects Forty infants sired by 13 fathers who had no form of contact with their offspring were randomly distributed in 12 small groups each composed of 3 or 4 mother–infant pairs. All monkeys were of Guyanese origin (Saimiri sciureus) and were born and raised at the Stanford University Primate Facility. Groups were housed indoors in 1.8 3 1.2 3 1.8 m wire-mesh cages adjacent to large windows and maintained on natural light– dark cycles. Solid partitions between wire-mesh cages prevented visual and physical contact between monkeys in different groups. A sliding door in the front panel of each cage provided access to a portable transport cage. Monkeys were pretrained to quickly enter the transport cage on command to facilitate the sampling procedures described below. All procedures were conducted in accordance with and as required by the Animal Welfare Act and approved by Stanford
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FIG. 1.
Schematic representation of the experimental design.
University’s Administrative Panel on Laboratory Animal Care. Experimental Design and Procedures As part of ongoing research on the consequences of postnatal stress, four natal groups were randomly assigned to each of the following three rearing conditions when infants were 10 weeks of age (range, 8 –13 weeks). 1. Low foraging demands. A total of 14 infants (8 males, 6 females) and their 14 mothers were maintained from 10 to 21 weeks postpartum in low demand conditions where food was easy to find. Each group received 600% by weight of their normal daily food intake buried in foraging boards (for details, see Lyons et al., 1998a). All 80 holes in the foraging boards contained abundant amounts of food, so for these mothers and their infants food was not difficult to find. 2. High foraging demands. A total of 13 infants (7 males, 6 females) and 13 mothers were maintained from 10 to 21 weeks postpartum in high-demand conditions where each group was provisioned with 120% of their daily food intake buried in foraging boards. Since many holes contained little or no food, more time and effort was required in the high demand condition to successfully locate food. Throughout the 12-week foraging protocols body weights and amounts of food consumed by high- and low-demand monkeys were not significantly different (Lyons et al., 1998a). 3. Intermittent social deprivations. A total of 13 infants (6 males, 7 females) and 13 mothers fed from standard food hoppers were intermittently separated for five
sessions, each lasting 5 h. Every other week from 13 to 21 weeks postpartum each intermittently separated infant was removed one at a time, placed in a cage adjacent to unfamiliar monkeys, and temporarily deprived of all forms of contact with members of the natal group. Upon completion of these protocols at 21 weeks of age, all monkeys were treated in identical fashion under standard laboratory conditions. At 29 weeks of age, a blood sample was collected from each offspring in undisturbed natal groups. Seven days later a second blood sample was obtained. Thereafter, one mother was removed from each otherwise undisturbed group and temporarily housed in a different building. The removed mother’s offspring was observed immediately before, 1 h after, and 1 day after removal of the mother using 10-min focal animal observation methods described below. A blood sample from the focal offspring was obtained immediately after the last (1day) observation, and the mother was returned to the group. These procedures were repeated with a different mother removed at weekly intervals, until each mother was removed and returned 1 day later within each of the natal groups (Fig. 1). After temporarily removing each mother one at a time, two focal animal observations were conducted and a blood sample was collected at 35 weeks of age from offspring in undisturbed groups. Focal animal observations and blood sample collections were performed on different days. All mothers were then simultaneously removed at 36 weeks postpartum and returned to the breeding facility. Focal animal observations of each offspring were conducted 1 h, 2 days, and 6 days later, and blood samples were collected 1,
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TABLE 1 Interaction of Distribution of Offspring with Rearing Condition Fathers Postnatal rearing conditions
Jon
a
High demands Low demands
Don
?
b
? ?
Ben i
Intermittent separations
? ? /
c f
Ric
?
Dan
Jed
?
/
?
/
a
/
Lee d
h
/ /
?
Sid g
?
Gil e
Ted
Ken
/ /
? /
/
/
/ /
Jun
? /
h
? /
Ray b g
? ?
? / d ? c f
/
e
? /
?
i
? / ?
Note. Male (?) and female (/) offspring in each postnatal rearing condition were sired by 13 fathers that had no form of contact with their offspring. Twenty-two mothers each produced a single offspring and nine mothers produced with different fathers on separate occasions two offspring (designated as italicized letter pairs).
3, and 7 days later in undisturbed juvenile peer groups. Focal animal observations were conducted between 08:00 and 10:00 h with individuals easily identified by dye marks and number tags on necklaces. Using a time-ruled check sheet and countdown timer, trained observers scored on-the-signal at 15-s intervals scan samples of time spent near (,10 cm) the focal animal’s own mother, near other mothers, and near peers. Peep-calls were scored using one-zero sampling at 15-s intervals as a behavioral index of maternal separation induced anxiety (Weerts and Miczek, 1996; Wiener, Bayart, Faull, and Levine, 1990). Interobserver correlations for time spent near own mother (r 5 0.96), near other mothers (r 5 0.83), near peers (r 5 0.78), and peep-calls (r 5 0.78) were all statistically significant (P , 0.01). Interobserver agreements corrected for chance (Cohen’s kappa) ranged from .78 to .95. Blood samples (1 ml) were collected at 09:00 h from manually restrained monkeys by femoral venipuncture. Blood was transferred to glass tubes on ice and centrifuged at 4°C, and the plasma fraction was stored in polypropylene tubes at 280°C. Cortisol was measured in unextracted plasma using a previously described radioimmunoassay (Lyons, Ha, and Levine, 1995). Within- and between-assay coefficients of variation were, respectively, 4.8 and 6.1%. Assay sensitivity was 3 mg/dl. Most blood samples (89%) were collected within 3 min from cage entry (median latency to sample collection 5 100 s; range, 47–358 s), and all but three samples were collected within 4 min. In keeping with reports that cortisol levels sampled within these time limits using these procedures do not
reflect capture and handling effects (Lyons et al., 1995; Lyons, Wang, Lindley, Levine, Kalin, and Schatzberg, 1999), sample collection latencies accounted for less than 2% of the variance in levels of plasma cortisol. Data Analysis Postnatal rearing experiences and hereditary effects were assessed with repeated measures analysis of variance (ANOVA) using least squares estimates from general linear models in the MGLH module of Systat. In each three-factor ANOVA, the postnatal rearing condition was considered a between-subjects factor, paternity (offspring grouped by father) was considered a between-subjects factor, and postseparation time course was considered a within-subjects factor. Gender differences were not significant and were excluded from analysis. The paternity 3 postnatal rearing condition interaction was likewise excluded from ANOVAs due to the incomplete distribution of offspring sired by each father in the unbalanced factorial design (Table 1). A total of 13 fathers and 31 mothers produced the 40 offspring that composed the study cohort. Twenty-two mothers each contributed one offspring, and nine mothers produced with different fathers on separate occasions two offspring. No more than one offspring from a given mother was included in each paternal half-sibling group. To identify the proportion of total variance in each phenotypic measure that was attributable to additive genetic variance, estimates of heritability were derived from paternal half-sibling groupings using the standard ANOVA approach (Falconer, 1960). For each
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TABLE 2 Estimates of Heritability (h 2) for Social Affinities, Peep-calls, and Plasma Cortisol Levels Measured in Three Different Social Settings Phenotypic measure
h2
Undisturbed natal group Near own mother Near other mothers Near peers Peep-calls Cortisol levels
.56* .50* .34 .01 .22
Own mother removed Near other mothers Neer peers Peep calls at 1-hr Peep calls at 1-day Cortisol levels at 1-day
.58* .32 .16 .48* .06
All mothers removed Near peers Peep calls at 1 hr Peep calls at 2 days Peep-calls at 6 days Cortisol levels at 1 day Cortisol levels at 3 days Cortisol levels at 7 days
After the removal of each mother one at a time, the removed mother’s offspring spent more time near other mothers [F(2,50) 5 5.74, P , 0.01] and more time near peers [F(2,50) 5 6.58, P , 0.01]. Postnatal rearing effects were not evident in time spent near other mothers, but a paternity main effect was discerned [F(12,25) 5 2.34, P , 0.04]. Paternal half-siblings that spent more time near other mothers when their own
,.01 .22 ,.01 ,.01 .05 .68* .70*
* Significantly greater than zero, P , .05.
phenotypic measure a one-way ANOVA was performed with paternal half-sibling grouping (paternity) considered a between-subjects factor. The betweenfather mean-square minus the within-father meansquare was divided by 3.1 (average number of offspring per father), multiplied by 4 (half-siblings share, on average, 25% of their genome by common descent), and divided by the total variance. Heritability estimates and all ANOVA test statistics were evaluated with two-tailed probabilities, and descriptive statistics are presented as mean 6 SEM.
RESULTS In undisturbed natal groups, measures of time spent by offspring near their own mother, near other mothers, and near peers were not significantly different. Postnatal rearing effects likewise failed to achieve statistical significance. The only significant finding was the paternity main effect [F(12,25) 5 2.38, P , 0.04]. For social affinities in undisturbed groups the highest heritability (Table 2) and most salient differences between groupings of paternal half-siblings were in time spent by offspring near their own mother (Fig. 2A).
FIG. 2. Differences between offspring grouped by father on (A) scan sample measures of time spent by each offspring near its own mother, (B) time spent near other mothers after the removal of each offspring’s own mother, and (C) peep-calls emitted 1 day after the removal of each mother one at a time from otherwise undisturbed groups (mean 6 SEM).
Stress, Heredity, and Hypercortisolism
FIG. 3. Postnatal rearing effects on (A) maternal separation induced peep-calls and (B) plasma cortisol levels measured before and after the removal of each mother one at a time from otherwise undisturbed groups (mean 6 SEM).
mother was removed (Fig. 2B) spent more time near their own mother in undisturbed natal groups (r 5 0.32, df 38, P , 0.05). Paternal half-sibling effects were not evident in time spent near peers, and postnatal rearing effects fell short of significance. After the removal of each mother one at a time, the removed mother’s offspring exhibited increases in peep-calls and increases in plasma levels of cortisol (Fig. 3). Relative to monkeys from the postnatal foraging conditions, the intermittently separated monkeys responded with smaller maternal separation induced increases in peep-calls [rearing condition 3 time course interaction, F(4,50) 5 5.22, P , 0.01] and smaller increases in cortisol [rearing condition 3 time course interaction, F(2,25) 5 9.32, P , 0.01]. Paternal half-sibling effects were also expressed in the production of maternal separation induced peep-calls [paternity 3 time course interaction, F(24,50) 5 1.80, P , 0.05]. Peep-calls were seldom emitted in undisturbed groups, whereas peak levels of peep-calls were produced 1 h after the removal of each mother. Paternal half-sibling effects were not evident in either of these measures, nor in the maternal separation induced adrenocortical response. But 1 day after the removal of each mother more peep-calls (Fig. 2C) were emitted by paternal half-siblings that spent more time near their own mother in undisturbed natal groups (r 5 0.48, df 38, P , 0.01).
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When all mothers were simultaneously removed their offspring spent more time near peers (Fig. 4A). The rearing condition 3 postseparation time course interaction was not significant, but a rearing condition main effect was discerned [F(2,25) 5 14.36, P , 0.001]. Relative to offspring from the postnatal foraging conditions, the intermittently separated offspring spent more time near peers. Significant rearing effects were likewise expressed in the production of maternal separation induced peep-calls [rearing condition 3 time course interaction, F(6,75) 5 3.88, P , 0.01]. Offspring from the postnatal foraging conditions emitted the most peep-calls, whereas the intermittently separated offspring emitted the fewest peep-calls (Fig. 4B). Comparable effects were also detected in acute increases in plasma levels of cortisol. Offspring from the postnatal foraging conditions initially produced greater increases in cortisol relative to the intermittently separated offspring (Fig. 4C). But early rearing effects on cortisol levels were diminished 3 and 7 days after the removal of all mothers, as reflected in the rearing condition 3 time course interaction [F(6,75) 5 6.65, P , 0.001]. From 1 to 7 days after the removal of all mothers decreases in cortisol were most clearly expressed in offspring from the foraging conditions. Postnatal rearing effects on cortisol levels rapidly subsided over a short period of time, while differences between groupings of paternal half-siblings emerged 3–7 days after the removal of all mothers [paternity 3 time course interaction, [F(36,75) 5 2.00, P , 0.01]. A few fathers sired offspring whose cortisol levels re-
FIG. 4. Postnatal rearing effects on (A) scan sample measures of time spent near peers, (B) maternal separation induced peep-calls, and (C) plasma cortisol levels measured before and after the simultaneous removal of all mothers (mean 6 SEM).
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turned to the preseparation level within 3 days after all mothers were removed, whereas cortisol levels in the offspring sired by other fathers remained elevated for more than 7 days (Fig. 5). The lowest heritabilities (Table 2) and consistent postnatal rearing effects (Fig. 3B and 4C) were evident in peak elevations of cortisol measured 1 day after the removal of mothers simultaneously or one at a time. Cortisol levels 3 and 7 days after the simultaneous removal of all mothers showed negligible postnatal rearing effects (Fig. 4C) and the highest heritabilities (Table 2), as offspring sired by certain fathers failed to return to the preseparation level found in undisturbed natal groups (Fig. 5). Paternal half-siblings that spent more time near their mother in undisturbed groups exhibited higher levels of cortisol 3 and 7 days after the removal of all mothers (3 days; r 5 0.40, df 38, P , 0.05; 7 days; r 5 0.58, df 38, P , 0.01). Individual differences in peep-calls emitted 1 day after the removal of each mother one at a time (Fig. 2C) were correlated with chronic but not acute increases in cortisol measured after the removal of all mothers from groups (1 day: r 5 0.15, df 38, P 5 0.35; 3 days: r 5 0.28, df 38, P 5 0.08; 7 days, r 5 0.39, df 38, P , 0.02).
DISCUSSION We previously reported that squirrel monkey mothers randomly assigned to the high demand condition consistently exhibit elevated cortisol levels relative to mothers maintained in the low-demand condition where food is easy to find (Levine et al., 1991; Lyons et al., 1998a). Although squirrel monkey mothers in the high demand condition stop carrying their offspring at earlier ages, high- and low-demand offspring spend similar amounts of time nursing on their mother and feeding on solid foods. Squirrel monkey mothers direct little aggression toward their offspring in either postnatal foraging condition, and cortisol levels during the foraging protocols are not consistently different in high- and low-demand squirrel monkey infants (Lyons et al., 1998a). Evidence presented in this study suggests that by accelerating some transitions in development (carry 3 self-transport) and not others (nursing 3 self-feeding), squirrel monkey mothers in the high demand condition spared their offspring from the deficits discovered in experiments with bonnet macaques. Squirrel monkeys exposed as infants to the high demand condition were not significantly different from the low demand offspring in social affinities, maternal separa-
FIG. 5. Differences between offspring grouped by father in plasma cortisol levels measured 1, 3, and 7 days after the simultaneous removal of all mothers (mean 6 SEM).
tion induced peep-calls, and serial assessments of the adrenocortical response. Variable demands composed of alternating 2-weeks blocks of high- and low-demand foraging may be required to produce deviant outcomes in development as reported for bonnet macaques (Coplan et al., 1996; Coplan et al., 1998; Rosenblum and Andrews, 1994). Relative to squirrel monkey offspring from the postnatal foraging conditions, the intermittently separated offspring responded with blunted increases in cortisol
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1 day after the removal of mothers simultaneously or one at a time. Postnatally handled rats periodically separated from their mother for short periods of time likewise exhibit blunted hypothalamic-pituitary-adrenal (HPA) responses to mildly stressful events (Levine, 1967; Meaney, Diorio, Francis, Widdowson, LaPlante, Caldji, Sharma, Seckl, and Plotsky, 1996; Plotsky and Meaney, 1993). Blunted HPA responses to stress in postnatally handled rats are mediated, in part, by enhanced glucocorticoid negative-feedback sensitivity resulting from increased glucocorticoid receptor gene expression in the hippocampus (O’Donnell, Larocque, Seckl, and Meaney, 1994). Conversely, prolonged intermittent periods of maternal separation decrease glucocorticoid receptor binding sites in hippocampus and impair glucocorticoid negative-feedback sensitivity in rats studied as adults (Meaney et al., 1996; Plotsky and Meaney, 1993). Despite these findings in laboratory rodents, it is not yet clear that early environmental regulation of glucocorticoid feedback accounts for the blunted adrenocortical responses observed in our intermittently separated squirrel monkeys. Relative to offspring from the postnatal foraging conditions, the intermittently separated offspring also emitted fewer maternal separation-induced peep-calls and spent significantly more time near peers. These results concur with numerous reports that nonmaternal forms of social support eliminate or reduce stress induced anxiety and adrenocortical responses in primates (Coe, Rosenberg, Fischer, and Levine, 1987; Gust, Gordon, Brodie, and McClure, 1996; Smith, McGreer-Whitworth, and French, 1998; Stanton, Patterson, and Levine, 1985). Negligible postnatal rearing effects and significant heritabilities were evident in time spent by offspring near their own mother in natal groups. High heritabilities were also expressed in measures of maternal separation induced anxiety that reflect aspects of filial attachment (Mason and Mendoza, 1998). Paternal halfsiblings that spent more time near their mother in undisturbed groups exhibited longer lasting increases in maternal separation induced peep-calls and prolonged adrenocortical responses. Similar effects evident among pairs of offspring produced from the same mother in different groupings of paternal halfsiblings do not account for high heritabilities in the paternal half-sibling analysis. Similarities within maternal half-sibling pairs randomized across different groupings of paternal half-siblings reduce the between-father component of variance. This decreases the magnitude of heritability estimates derived using the paternal half-sibling approach.
Estimates of heritability are of course specific to the population and the circumstance in which they are assessed. Genetically diverse populations studied in uniform conditions show larger heritabilities than genetically homogeneous populations assessed in diverse environments (Falconer, 1960). Few fathers or mothers that produced the study cohort shared a common parent, but not all grandparents could be identified with absolute certainty from long standing breeding colony records. Nevertheless, it is intriguing find in this cohort of monkeys evidence consistent with human twin studies that indicate genetic and experiential factors contribute to individual differences in vulnerability to social loss (Goldsmith and Gottesman, 1981; Kendler and Karkowski-Shuman, 1997; Kendler et al., 1995; Silove et al., 1995). Whereas certain individuals quickly disengage and move on, demonstrating little emotional distress, others take much longer and experience sustained negative affect.
ACKNOWLEDGMENTS The authors thank C. Chen, K. Fong, B. Gavino, G. Ha, S. Kim, N. Shrieken, and L. Steffey for assistance with sample collections, E. Lowe, S. Weiner, and L. Steffey for meticulously maintaining 24 years of breeding colony records, and the Pritzker Network for constructive comments on this research. Supported by the Pritzker Network and Grant MH47573 from the National Institute of Mental Health.
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