Kristine Erickson Mood and Anxiety Disorders Program National Institute of Mental Health Bethesda, MD E-mail:
[email protected]
K. Eddie Gabry Jay Schulkin Philip Gold Clinical Neuroendocrinology Branch National Institute of Mental Health Bethesda, MD E-mail:
[email protected]
Stephen Lindell J. Dee Higley Section of Primate Models of Psychopathology National Institute of Alcohol Abuse and Alcoholism Poolesville, MD E-mail:
[email protected]
Maribeth Champoux Stephen J. Suomi Laboratory of Comparative Ethology National Institute of Child Health and Human Development Poolesville, MD E-mail:
[email protected]
Social Withdrawal Behaviors in Nonhuman Primates and Changes in Neuroendocrine and Monoamine Concentrations During a Separation Paradigm ABSTRACT: This study investigated relationships between withdrawal behaviors in rhesus macaques and changes in monoamine metabolite and endocrine concentrations during repeated psychosocial stress. Rhesus monkeys (N ¼ 71) experienced maternal separation in which four separations took place during four consecutive weeks. Behavioral observations were made, as well as plasma concentrations of cortisol and cerebrospinal fluid concentrations of the serotonin, dopamine, and norepinephrine metabolites were obtained. Animals were assigned to high, moderate, and low withdrawal groups, defined using baseline durations of withdrawal behaviors. Highly withdrawn animals showed less reduction than nonwithdrawn animals in serotonin metabolite concentrations over repeated separations. Highly withdrawn macaques also failed to significantly reduce cortisol concentrations across separation weeks. More adaptation in central serotonin functioning and cortisol concentrations was seen in nonwithdrawn primates than in highly withdrawn primates; these findings have implications for increased risk of developing anxiety disorders in highly inhibited children. ß 2005 Wiley Periodicals, Inc. Dev Psychobiol 46: 331–339, 2005. Keywords: cortisol; temperament; development; anxiety; serotonin; dopamine; norepinephrine
Nonhuman primates provide an opportunity to study responses to stressful situations in some detail under controlled conditions, and these animals display variability of temperament as humans do (Byrne & Suomi, 2002; Higley & Suomi, 1989). For example, macaques’ time spent freezing in response to a human entering the cage can be used as an analog to extreme behavioral inhibition in children (Kalin et al., 1998a,b). Additionally, maternal separation in nonhuman primates elicits behaviors characteristic of anxiety responses, including Received 5 May 2004; Accepted 5 December 2004 P. Gold, S. J. Suomi, and J. D. Higley contributed equally and should be considered interchangeable in order of sequence. Correspondence to: K. Erickson Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/dev.20061 ß 2005 Wiley Periodicals, Inc.
increased distress vocalizations, behavioral withdrawal, and agitated locomotion around the cage (Suomi, 1991). In nonhuman primates, separation from the mother increases infants’ time spent freezing, and the duration of freezing behavior is correlated with increased cortisol concentrations (Kalin et al., 1998b). Behavioral responses to aversive events are associated with various neuroendocrine and neurotransmitter actions. Cortisol levels increased in rhesus infants when they were separated from their mothers and peers (Higley et al., 1992; Shannon et al., 1998). More specifically, in a separation paradigm similar to that of the present study, cortisol and adrenocorticotropic hormone (ACTH) were elevated during Day 1 of each separation, but subsequently these concentrations decreased over time (Higley et al., 1992). Primates characterized as having inhibited temperament also have higher cortisol reactivity (Byrne & Suomi, 2002). Neurotransmitters, such as serotonin
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(Higley et al., 1996a), dopamine (Brake et al., 1997; Higley et al., 1991), and norepinephrine (Higley et al., 1992; Schneider et al., 1998), are also linked to various types of acute and chronic stressors or to stressful situations, including maternal separation (Bayart et al., 1990; Breese et al., 1973). As with other mammals, rhesus macaques have a wellcharacterized developmental sequence. At birth through the first 30 days, infants seldom leave their mother. From Day 30 onwards, the infant begins to explore its environment using mother as a secure base. The bulk of time is spent in interactions with other infants and manipulating the environment. By 4–6 of age, weaning begins and by the 8th–9th month of life, weaning is almost complete. There are, however, large interindividual differences both in the timing of these patterns, as well as in how uninhibited they are in such explorations (Higley & Suomi, 1989). In this study, withdrawal was defined more broadly than freezing behavior, which allowed observations of baseline withdrawal characteristics. In a large sample of infant preweanling rhesus macaques (the infants are still very dependent on their mothers at this point), baseline duration of withdrawal behaviors in the home cage was used to categorically separate the animals into low, moderate, and high withdrawal groups; these withdrawal behaviors were used to describe the temperaments of rhesus macaques. In the relatively stress-free home cage environment, withdrawal was defined using baseline rates of nonlocomotive, withdrawal behaviors. Additionally, plasma levels of cortisol and ACTH, and cerebrospinal fluid concentrations of the dopamine (HVA), serotonin (5-HIAA), and norepinephrine (MHPG) metabolites were measured in these macaques at baseline and during each of the four separation periods.
METHODS Subjects Seventy-one (44 male, 27 female) 6- to 7-month-old rhesus macaques housed at the National Institutes of Health Animal Center in Poolesville, MD were included in the study. The study received approval from the NIAAA and NICHD ACUC (DH013 and 02-008). These subjects were reared by their mothers in social groups with 10–15 other females, two adult males, and other infants, circumstances that approximated natural conditions within the limits of the study. They lived in indoor-outdoor pens with their mothers and fathers in mixed-sex social groups containing two adult males and six to eight adult females with other same-aged infants. No siblings or older juveniles were present. All of the social groups had been together for at least 2 years at the time when maternal separations were performed, allowing the animals to form natural bonds and establish stable social conditions.
Separation Procedures The separations took place over a period of 4 years. Each social group included multiple infants of appropriate age for the separation procedures. Each infant received four sequential mother-infant separations lasting 4 days. They were reunited with their mother for the weekend. The separations went as follows: At 6 months of age, mothers were removed from the home cage, as the subjects underwent four sequential 4-day maternal separations (Monday to Friday morning). All subjects were separated in their home cage by removing the mother from the infant’s home cage. The mother– infant pair was separated from the rest of the group members, the animals were caught with nets and gloves, and blood samples were taken from the infant as described below. Animals were then reunited with their mothers for the weekend following each of the 4-day separation periods. The baseline period was defined as the 2-week period prior to the initial separation. Anticipatory periods refer to the ‘blood draws’ taken 1 hr prior to the start of each 4-day separation. These blood draws are referred to as ‘‘anticipatory’’ because, following the first separation, the infant presumably anticipates the subsequent separations during capture. Acute separation periods were defined as Day 1 of the 4-day separation periods. Chronic separation periods were defined as the average of Days 2–4 of the separation periods. Behavioral Observations Observations of behaviors were made during the 2 weeks of baseline and the four 4-day separation periods. Behavioral observations were performed with the animals in the outdoor section of the home cage, which was 10 15 10 feet in size. Observers were stationed outside of the cage. Inter-rater reliability for behavioral coding was .85. During baseline, there were ten separate behavioral observations, and during each week of separation, there were nine observation periods, making a total of 36 separation observations. Each observation period was 300 s in duration. All observations were recorded as durations in seconds, except vocalizations that were recorded as frequencies. Withdrawal was characterized as time spent in nonlocomotive, withdrawn behaviors, defined as an absence of directed movement, social behaviors, and manipulation of objects in the environment. The only other behaviors that could also be scored during withdrawal were vocalizations, bouncing in place, and self-directed behaviors. Other exclusive behaviors scored were environmental exploration, grooming, other social behaviors (social play, social contact), and locomotion. Self-directed behaviors were selfclasping, self-grooming, and self-mouthing. Table 1 describes the behavioral definitions in more detail. Biological Samples Baseline CSF and blood samples were obtained at 1300 hours 1-week prior to the first separation. Separation CSF samples were obtained on the fourth day of each separation, also at 1300 hours. CSF samples were obtained while the animals were anesthetized with ketamine hydrochloride (15 mg/kg body
Social Withdrawal Behaviors in Nonhuman Primates Table 1.
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Definitions for Behavioral Observations
Term
Measure
Withdrawal
Duration (s)
Self-directed behaviors
Duration (s)
Locomotion Vocalizations
Duration (s) Frequency (#)
Social behaviors
Duration (s)
Grooming
Duration (s)
Stereotypic & stypic behaviors
Duration (s)
Definition Absence of directed movement, social behaviors, and environmental manipulation; vocalizations, bouncing in place, and self-directed behaviors may be scored simultaneously Self-groom: Grooming of one’s own body, self-scratching, biting, or cleaning nails Self-clasp: Firm manual or pedal gripping of self, which is not a component of an ongoing behavior Self-mouth: Sucking (not biting) at any bodily appendage or own fur Any self-induced change in location of self Any vocal sound emitted by the subject; includes coo, bark, screech, squeal, etc. Ventral cling: Focal animal ventral contact with any other animal; focal animal must be touching its ventral surface to the other animal with at least one arm or leg wrapped around the other animal Social contact: Includes proximity; also includes any exploration, oral, pedal, or manual, of other animal; and receiving any such exploration from other monkey Social play: Performance of any play behaviors including: initiating play by ‘‘play face,’’ wrestling, chasing, etc. Cleaning/grooming of another animal, including scratching, biting, licking, and rubbing Stereotypic: Any repetitive, patterned, and rhythmic locomotive movement Stypic: Idiosyncratic nonlocomotor stereotyped actions such as repetitively saluting, picking the teeth, or strumming the mesh
weight, IM) as described previously (Anderson et al., 2002). Blood was also obtained during each CSF sample. An unanesthetized baseline blood sample was obtained just prior to each of the separations, and the Day 4 separation blood samples were obtained from the femoral veins while the juvenile was manually restrained (see below, the Day 4 samples were obtained under ketamine anesthesia). Both our own observations as well as the behavioral and HPA data indicated that the monkeys were anticipating the separations. Hence, the samples taken just prior to separation are called the anticipation period. Additional samples were taken 1, 2 hr, and 4 days after the initiation of separation procedures. All blood samples on Day 1 of separation were obtained while the monkey was awake. Blood samples for the fourth day of separation were obtained about 5 min following anesthesia, and CSF samples were obtained from the cisterna magna within 30 min following the blood samples. As has been shown in other studies, time to obtain the CSF sample is rapid enough such that variation with time to sample is not observed (Higley et al., 1991, 1992, 1996a,b). Cisternal CSF samples were immediately aliquoted into polypropylene tubes and frozen in liquid nitrogen. Blood samples were placed on wet ice and centrifuged at 4 C for 20 min. Then the plasma was aliquoted and frozen in liquid nitrogen. CSF and plasma samples were stored at 70 C until assay. Plasma was assayed for cortisol and ACTH concentrations by Hazleton Biotechnologies using standard radioimmunoassays (Scheinin et al., 1983). Simultaneous determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) in
cerebrospinal fluid were performed with high-performance liquid chromatography using electrochemical detection (Scheinin et al., 1983; Seppala et al., 1984). Statistical Analyses StatView 5.0.1 (SAS Institute, Inc., Cary, NC) was used for all statistical analyses. To create low, mid, and high withdrawal categories for use as independent variables in an analysis of variance (ANOVA), time spent in nonlocomotive, withdrawn behaviors during the baseline period was converted to standard scores. The same procedure was performed for Day 1 separation withdrawal behaviors. Mixed design, repeated measures ANOVAs were performed for each experiment to determine whether behaviors, neurotransmitter metabolites, or endocrine concentrations changed over time, and whether group differences (low, mid, or high withdrawal) or group by time interactions was present. Scheffe’s test was used for post hoc comparisons. Alpha level was set at .05.
RESULTS A high withdrawal (N ¼ 14), a moderate withdrawal (N ¼ 47), and a low withdrawal group (N ¼ 10) were identified by converting withdrawal during the baseline observation period into standard scores. Standard scores below 1 for withdrawal defined the low withdrawal
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baseline group; values between 1 and þ1 defined the moderate baseline withdrawal group, and scores above þ1 defined the high baseline withdrawal group.
Behavioral Characteristics of Animals During Maternal Separation Behavioral observations of the animals were made during baseline (2 weeks prior to initial separation) and maternal separation. The mean baseline, mean Day 1 of separation, and mean chronic (Days 2–4) separation scores were the repeated measures in the behavioral analyses. Analyses of nonsocial behaviors (locomotion, vocalizations, and selfdirected behaviors) of the low, moderate, and high baseline withdrawal groups showed differences in rates or durations of these behaviors. Repeated measures ANOVA for the amount of time spent moving around the cage in nonstereotypic behavior (locomotion) indicated a significant time (F ¼ 31.489, p < .0001), group (F ¼ 11.46, p < .0001), and group by time interaction (F ¼ 3.57, p ¼ .002). There was a significant increase in locomotion during Day 1 from baseline levels (p < .0001), a significant decrease from Day 1 to chronic separation (p ¼ .0002), and another significant decrease from chronic separation to reunion (p < .0001). In addition, the high withdrawal group showed more time engaged in locomotion than the mid (p ¼ .0037) and low withdrawal (p < .0001) groups. For number of vocalizations, there was a significant effect for time (F ¼ 40.87, p < .0001), but no group differences were noted. All animals vocalized more during Day 1 of the separation periods than during baseline (p < .0001) or chronic separation (p < .0001). A significant time effect emerged for selfdirected behaviors (F ¼ 6.71, p ¼ .0002), with animals spending more time engaged in self-directed behaviors during Day 1 (p ¼ .004) and chronic (p ¼ .003) separation periods than during baseline. Also, there was a trend for the low withdrawal group to spend less time engaged in self-directed behaviors than the high withdrawal group (p ¼ .07). Social behaviors (grooming and other social behaviors) were also observed and analyzed. Amount of time spent grooming showed a significant effect for time (F ¼ 5.94, p ¼ .0007), with animals spending more time grooming during the chronic separation than during baseline (p ¼ .017) or Day 1 (p < .0001) of the separations, and grooming decreasing from chronic separation levels during reunion (p ¼ .0001). Social behaviors other than grooming also showed a significant time effect (F ¼ 52.50, p < .0001) and group effect (F ¼ 10.04, p ¼ .0002). All groups spent less time engaging in social behaviors during Day 1 of the separations than during baseline (p < .0001), chronic separation (p < .0001), and reunion periods (p < .0001); and the high withdrawal
animals spent less time than the low withdrawal animals (p ¼ .0001) and moderate withdrawal animals (p ¼ .007) overall in social behaviors. For each separation week, observations of durations for each behavior category were collapsed to investigate whether these behaviors changed over the four separations. Repeated measure ANOVAs indicated that locomotion increased significantly (F ¼ 4.75, p ¼ .003), with the significant increases taking place between separation weeks 1–3 (p ¼ .0009) and weeks 1–4 (p ¼ .015). Grooming behavior also changed significantly across separations (F ¼ 3.05, p ¼ 03), with post hoc tests indicating an increase in grooming between separation weeks 2 and 4 (p ¼ .04).
Withdrawal and CSF Measures CSF measures were collected during baseline and Day 4 of each separation period. Therefore, repeated measures for the ANOVAs were baseline, Separations 1, 2, 3, and 4. Figure 1 shows the mean concentrations at each time period of 5-HIAA, HVA, MHPG for the baseline withdrawal groups. For 5-HIAA, repeated measures ANOVA indicated significant differences over time (F ¼ 27.19, p < .0001) and for group by time interaction (F ¼ 2.14, p ¼ .03) (see Figure 2A). Scheffe’s test indicated that mean 5-HIAA concentrations decreased significantly from baseline to Separation 1 (p < .0001), from Separations 1 to 2 (p < .0001), and remained significantly below baseline concentrations during Separations 3 and 4 (p < .0001). Additionally, 5-HIAA concentrations in high withdrawal animals did not decrease as dramatically as those in the moderate and low withdrawal groups; 5-HIAA levels were significantly higher for the high withdrawal animals during separation 2 (p ¼ .03) (see Figure 2A). Significant differences emerged for HVA concentrations over repeated measures (F ¼ 12.40, p < .0001). Concentrations of HVA were significantly lower for the four separation weeks than for the baseline period (p < .0001 for Separations 1–3; p ¼ .0002 for Separation 4). Following the first two separations, mean HVA levels increased slightly. For MHPG, repeated measures ANOVA revealed significant change over time (F ¼ 4.53, p ¼ .0015). Overall, MHPG levels were significantly lower during Separation 3 than during baseline (p ¼ .012) and Separation 1 (p ¼ .0004). Subsequently, mean concentrations of MHPG rose nonsignificantly during Separation 4. There were no group differences for CSF HVA or MHPG.
Withdrawal, Cortisol, and ACTH For the endocrine measures, the mean concentrations of cortisol and ACTH during anticipatory (immediately after
Social Withdrawal Behaviors in Nonhuman Primates
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FIGURE 1 Groupings by baseline behavioral withdrawal and associated social and nonsocial behaviors. (A) Locomotion, (B) vocalizations, (C) self-directed behaviors, (D) grooming, and (E) other social behaviors. Error bars indicate standard error scores.
capture, at the beginning of each separation procedure), Day 1, and Day 4 of each separation were analyzed by group, with mean anticipatory, Day 1, and Day 4 concentrations as the repeated measures. Repeated measures ANOVA of the cortisol concentrations indicated a significant time effect (F ¼ 254.57, p < .0001). Concentrations increased from the anticipatory periods to Day 1 (p < .0001) and decreased again from Day 1 to Day 4, to anticipatory levels (p < .0001). For ACTH, a significant
time effect was detected (F ¼ 93.85, p < .0001), with concentrations decreasing by Day 4 (p < .0001), but no increase in ACTH from the anticipatory period to Day 4. To investigate possible group differences within the anticipatory periods, Day 1 measures, and Day 4 measures, additional separate repeated measure analyses were performed for each of these periods, broken down by separation week. For the anticipatory cortisol measures, a significant group by time interaction occurred (F ¼ 2.162,
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cortisol levels decreased across the four separations. ACTH also decreased significantly between Separations 2 and 3 (F ¼ 44.05, p < .0001). ACTH concentrations decreased over separations for the anticipatory (F ¼ 8.582, p < .0001), Day 1 measures (F ¼ 30.21, p < .0001), and Day 4 separation measures (F ¼ 4.58, p ¼ .004), but no group effects were found.
FIGURE 2 Groupings by baseline behavioral withdrawal and changes in (A) 5HIAA, (B) homovanillic acid, and (C) MHPG concentrations at baseline and during the four separation periods. CSF was collected during baseline (2 weeks prior to initial separation) and on Day 4 of each of the four separation periods.
p ¼ .05), with the low withdrawal animals showing a gradual decrease in anticipatory cortisol over the separation periods, whereas the high withdrawal animals showed relatively constant cortisol concentrations (see Figure 3A). During Day 1 measures (F ¼ 14.55, p < .0001) and Day 4 measures (F ¼ 17.76, p < .0001),
FIGURE 3 Groupings by baseline behavioral withdrawal and changes in cortisol concentrations during (A) anticipatory phase (1 hr prior to each of the four separations); (B) Day 1 of the four separations; (C) Day 4 of the four separations.
Social Withdrawal Behaviors in Nonhuman Primates
DISCUSSION In general, our results showed that the macaques displayed increases in nonsocial behaviors (locomotion, vocalizations, self-directed behaviors) and decreases in grooming and other social behaviors during Day 1 of the four maternal separations. Increases in vocalizations and locomotion during Day 1 are characteristic of the ‘‘protest phase’’ (Harlow et al., 1971), which serves to increase the likelihood that the separated juvenile will be located by the mother. Concurrently, grooming and other social behaviors that are present during relaxed, friendly interactions were reduced. Long-term, chronic separation periods (Days 2–4) were associated with decreases in locomotion and vocalizations, and increases in grooming and other social behaviors from the rates observed during Day 1 of the separations. CSF 5-HIAA and HVA concentrations did not decrease as much in the highly withdrawn animals over time as in the other two groups. These highly withdrawn animals may be experiencing more anxiety than the less withdrawn animals, and this is reflected in the functioning of the serotonergic system. Previously, impaired CNS serotonin functioning has been linked to anxiety disorders that show a complex relationship with the serotonergic system (for review, see Charney & Drevets, 2002), and variations in the serotonin transporter gene have been associated with shy temperament in children (Arbelle et al., 2003). Behavioral inhibition describes children with extremely shy temperament and, although the stressor used in the current study was qualitatively different from the social stressors often used in human behavioral inhibition research, our serotonergic and glucocorticoid findings may be relevant. For example, in young children, behavioral inhibition is associated with an increased risk for developing anxiety disorders (Biederman et al., 1990, 2001), which are often successfully treated using SSRIs or other serotonergic-acting medications (Blanco et al., 2003). Perhaps sustained serotonin activity in highly withdrawn macaques contributes to the behavioral manifestations of withdrawal and decreased social behaviors. Additionally, anticipatory measures of cortisol in the low withdrawal animals decreased dramatically during consecutive separations, whereas cortisol in the high withdrawal group remained relatively constant throughout the separations. Such habituation in the low withdrawal animals can be interpreted as evidence for reduced aversive responses and better coping (Clarke, 1993; Eriksen et al., 1999). This suggests that these animals may be able to cope more effectively with the expectation of maternal separation than the highly withdrawn animals. The high withdrawal group showed no significant decrease in cortisol across the anticipatory and Day 1
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samples of successive separation periods. Conversely, the low withdrawal animals did exhibit decreases in cortisol concentrations during these periods, suggesting an adaptation of the HPA axis with repeated stressors. Behaviorally, the high withdrawal animals vacillated between periods of withdrawal and high activity. For example, to the extent that locomotion represents increased arousal and agitation, the greater increase in locomotion by the highly withdrawn animals during Day 1 of separation also supports the hypothesis that these animals are less able to skillfully cope with the separation procedures. This may be analogous to human infant profiles at 4 months of age, which predicted the later emergence of inhibited and uninhibited behaviors. Highly reactive, vigorous motor activity and displays of distress to unfamiliarity at this early age were associated with inhibited behavior at a later age, while low reactive human infants were more likely to become uninhibited (Kagan et al., 1998). Elevated concentrations of cortisol are recognized as part of the physiological response to aversive events, and have been described in behaviorally inhibited children (Bayart et al., 1990; Clarke, 1993; Dettling et al., 1999; Kagan et al., 1988; Kalin et al., 1998a). However, our study revealed that the initial cortisol concentrations during the separation stressor are at least as high in the low withdrawal animals as in the high withdrawal animals. The substantial reduction in cortisol seen in the low withdrawal animals was observed following the initial separation procedure. Elevated cortisol is particularly important in the experience of aversive events because glucocorticoids facilitate emotional memory consolidation and attention toward emotional information (Buchanan & Lovallo, 2001; McGaugh, 2000). The failure in reducing cortisol concentrations seen in the highly withdrawn animals may not only reflect suboptimal coping strategies, but also lead to enhanced memory of the aversive events. The cortisol concentrations of the low withdrawal group are diminished over multiple separations, suggesting the intriguing possibility that the aversive memories may be reduced in these animals and, therefore, less pervasive. In addition, this finding illustrates the importance of longitudinal studies when investigating the effects of stress on dynamic physiological systems as they relate to variables such as temperament. Chronically elevated cortisol concentrations, as the highly withdrawn animals in our study demonstrated, can lead to changes in neurobiology such as dendritic reshaping in the hippocampus and medial prefrontal cortex (Leverenz et al., 1999; Sapolsky et al., 1990; Wellman, 2001), changes in 5-HT1A receptor mRNA expression (Meijer & de Kloet, 1994), and altered corticotropin releasing hormone mRNA expression in the amygdala (Makino et al., 1994; Watts & Sanchez-Watts, 1995). Therefore, those individuals who spend more time
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in heightened aversive psychological states, such as behaviorally inhibited children, may be at risk for altered neural development, which may contribute to their higher risk for developing anxiety disorders.
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