Amanda M. Dettmer

Physiological and Behavioral Adaptation to Relocation Stress in Differentially Reared Rhesus Monkeys: Hair Cortisol as a Biomarker for Anxiety-related Responses

Increased hair cortisol concentrations have been associated with stress exposure in both human and nonhuman primates, and hair cortisol is now gaining attention as a biomarker for stress-related health problems. The present study examined the behavioral and physiological reactions of rhesus monkey (Macaca mulatta) infants reared in three different rearing environments to the major stressor of relocation. Infant monkeys (n=61) were studied from birth through 2 years of age. For the first 8 months of life, infants were either with their mothers and peers (MPR, n=21) or reared in a nursery using either peer-rearing (PR, n=20) or surrogate-peer-rearing (SPR, n=20). At approximately 8 months of age, infants were removed from their rearing group, simultaneously placed into a large social environment consisting of infants from all three rearing conditions, and observed for the next 16 months. Behavior was recorded twice per week from 1 to 24 months, and composite anxiety scores were calculated for each monkey. Monkeys were initially shaved at the nape of the neck on day 14 to remove any prenatal effects on hair cortisol deposition. Hair samples were then collected by re-shaving at 6, 12, 18 and 24 months and analyzed for cortisol content. MPR monkeys were the least affected by the stressor, showing smaller increases in anxious behavior than the other groups and more rapid physiological adaptation as assessed using hair cortisol. PR monkeys showed heightened and prolonged anxious behavior, had the highest cortisol levels prior to relocation, and their cortisol levels did not decline until more than a year later. SPR monkeys exhibited more rapid behavioral adaptation than PR monkeys, showing heightened but not prolonged anxious behavior. However, the SPR group showed a marked increase in cortisol in response to the relocation, and like the PR group, their physiological adaptation was slower than that of the MPR group as indicated by elevated cortisol levels at 18 months. By 24 months of age (16 months after relocation), all rearing groups were indistinguishable from one another physiologically and behaviorally. Spearman rank correlation revealed that hair cortisol taken at month 6 was not correlated with composite anxiety scores from months 6 to 8 (just before the relocation), but was positively correlated with composite anxiety scores between months 8 and 12 (immediately after relocation) for PR infants only (r(s)=0.75, p<0.001). Month 6-hair cortisol tended to positively correlate with composite anxiety scores for the following 6 months (months 12-18) for PR monkeys only (r(s)=0.47, p=0.037), which exhibited more anxious behavior than MPR and SPR infants during this period (ANOVA: F((2,60))=14.761, p<0.001) This is the first study to show that elevated hair cortisol early in life is a biomarker for the later development of anxious behavior in response to a major life stressor, particularly for infant monkeys exposed to early life adversity in the form of peer-rearing.

Effects of shampoo and water washing on hair cortisol concentrations.

BACKGROUND Measurement of cortisol in hair is an emerging biomarker for chronic stress in human and nonhuman primates. Currently unknown, however, is the extent of potential cortisol loss from hair that has been repeatedly exposed to shampoo and/or water. METHODS Pooled hair samples from 20 rhesus monkeys were subjected to five treatment conditions: 10, 20, or 30 shampoo washes, 20 water-only washes, or a no-wash control. For each wash, hair was exposed to a dilute shampoo solution or tap water for 45 s, rinsed 4 times with tap water, and rapidly dried. Samples were then processed for cortisol extraction and analysis using previously published methods. RESULTS Hair cortisol levels were significantly reduced by washing, with an inverse relationship between number of shampoo washes and the cortisol concentration. This effect was mainly due to water exposure, as cortisol levels following 20 water-only washes were similar to those following 20 shampoo treatments. CONCLUSIONS Repeated exposure to water with or without shampoo appears to leach cortisol from hair, yielding values that underestimate the amount of chronic hormone deposition within the shaft. Collecting samples proximal to the scalp and obtaining hair washing frequency data may be valuable when conducting human hair cortisol studies.

Population density-dependent hair cortisol concentrations in rhesus monkeys (Macaca mulatta).

Population density is known to influence acute measures of hypothalamic-pituitary-adrenal (HPA) axis activity in a variety of species, including fish, deer, birds, and humans. However, the effects of population density on levels of chronic stress are unknown. Given the fact that exposure to chronically elevated levels of circulating glucocorticoids results in a host of health disparities in animals and humans alike, it is important to understand how population density may impact chronic stress. We assessed hair cortisol concentrations (HCCs), which are reliable indicators of chronic HPA axis activity, in rhesus monkeys (Macaca mulatta) to determine the influence of population density on these values. In Experiment 1, we compared HCCs of monkeys living in high-density (HD; 1 monkey/0.87m(2)) and low-density (LD; 1 monkey/63.37m(2)) environments (N=236 hair samples) and found that HD monkeys exhibited higher hair cortisol across all age categories (infant, juvenile, young adult, adult, and aged) except infancy and aged (F(5)=4.240, p=0.001), for which differences were nearly significant. HD monkeys also received more severe fight wounds than LD monkeys (χ(2)=26.053, p<0.001), though no effects of dominance status emerged. In Experiment 2, we examined how HCCs change with fluctuating population levels across 5 years in the adult LD monkeys (N=155 hair samples) and found that increased population density was significantly positively correlated with HCCs in this semi-naturalistic population (r(s)=0.975, p=0.005). These are the first findings to demonstrate that increased population density is associated with increased chronic, endogenous glucocorticoid exposure in a nonhuman primate species. We discuss the implications of these findings with respect to laboratory research, population ecology, and human epidemiology.

Neonatal face-to-face interactions promote later social behaviour in infant rhesus monkeys

In primates, including humans, mothers engage in face-to-face interactions with their infants, with frequencies varying both within and across species. However, the impact of this variation in face-to-face interactions on infant social development is unclear. Here we report that infant monkeys (Macaca mulatta) who engaged in more neonatal face-to-face interactions with mothers have increased social interactions at 2 and 5 months. In a controlled experiment, we show that this effect is not due to physical contact alone: monkeys randomly assigned to receive additional neonatal face-to-face interactions (mutual gaze and intermittent lip-smacking) with human caregivers display increased social interest at 2 months, compared with monkeys who received only additional handling. These studies suggest that face-to-face interactions from birth promote young primate social interest and competency.

Hair cortisol predicts object permanence performance in infant rhesus macaques (Macaca mulatta)

Although high circulating levels of glucocorticoids are associated with impaired cognitive performance in adults, less is known about this relationship in infancy. Furthermore, because studies have relied on acute cortisol measures in blood plasma or saliva, interpretation of the results may be difficult as acute measures may in part reflect emotional responses to testing procedures. In this study we examined whether hair cortisol, an integrated measure of hypothalamic-pituitary-adrenal (HPA) axis functioning, predicted performance of nursery-reared (NR) infant rhesus monkeys (n = 32) on Piagetian object permanence tasks. Testing of NR infants began at 19.8 +/- 2.2 (mean +/- SE) days of age and continued for the next several months. Hair cortisol concentrations from the 32 NR monkeys were compared to those of 20 mother-peer-reared (MPR) infants. Hair was shaved at Day 14, allowed to regrow, and obtained again at month 6, thus representing integrated cortisol over a 5.5-month period of time. NR and MPR infants did not differ in month 6 hair cortisol values (t((50)) = 0.02, p = 0.98). Linear regression revealed that hair cortisol predicted object permanence performance in the NR infants. Infants with higher hair cortisol reached criterion at later ages on the well (p < 0.01), screen (p < 0.05), and A-not-B (p < 0.05) tasks and required more test sessions to complete the well (p < 0.01) and screen tasks (p < 0.05). These data are the first to implicate hair cortisol as a reliable predictor of early cognitive performance in infant macaque monkeys.

Associations between Parity, Hair Hormone Profiles during Pregnancy and Lactation, and Infant Development in Rhesus Monkeys (Macaca mulatta).

Studies examining hormones throughout pregnancy and lactation in women have been limited to single, or a few repeated, short-term measures of endocrine activity. Furthermore, potential differences in chronic hormonal changes across pregnancy/lactation between first-time and experienced mothers are not well understood, especially as they relate to infant development. Hormone concentrations in hair provide long-term assessments of hormone production, and studying these measures in non-human primates allows for repeated sampling under controlled conditions that are difficult to achieve in humans. We studied hormonal profiles in the hair of 26 female rhesus monkeys (Macaca mulatta, n=12 primiparous), to determine the influences of parity on chronic levels of cortisol (hair cortisol concentration, HCC) and progesterone (hair progesterone concentration, HPC) during early- to mid-pregnancy (PREG1), in late pregnancy/early lactation (PREG2/LACT1), and in peak lactation (LACT2). We also assessed infants’ neurobehavioral development across the first month of life. After controlling for age and stage of pregnancy at the first hair sampling period, we found that HCCs overall peaked in PREG2/LACT1 (p=0.02), but only in primiparous monkeys (p<0.001). HPCs declined across pregnancy and lactation for all monkeys (p<0.01), and primiparous monkeys had higher HPCs overall than multiparous monkeys (p=0.02). Infants of primiparous mothers had lower sensorimotor reflex scores (p=0.02) and tended to be more irritable (p=0.05) and less consolable (p=0.08) in the first month of life. Moreover, across all subjects, HCCs in PREG2/LACT1 were positively correlated with irritability (r(s)=0.43, p=0.03) and negatively correlated with sensorimotor scores (r(s)=-0.41, p=0.04). Together, the present results indicate that primiparity influences both chronic maternal hormonal profiles and infant development. These effects may, in part, reflect differential reproductive and maternal effort in mothers with varied caretaking experience. In addition, infant exposure to relatively higher levels of maternal cortisol during the late fetal and early postnatal periods is predictive of poorer developmental outcomes.

Nonhuman Primate Models of Neuropsychiatric Disorders: Influences of Early Rearing, Genetics, and Epigenetics

This report reviews the scientific literature from the past several decades that focuses on nonhuman primates (NHPs) as models of neuropsychiatric disorders, including anxiety, and alcoholism. In particular, we highlight the approaches, advantages, and disadvantages of the rearing, genetic, and epigenetic methodologies behind these studies as a means of evaluating the application of these methods in assessing disorders in NHPs as models of human disease. Finally, we describe the contributions the NHP studies have made to neuropsychiatric research and areas for future research.

First-time rhesus monkey mothers, and mothers of sons, preferentially engage in face-to-face interactions with their infants

Face‐to‐face interactions between mothers and infants occur in both human and non‐human primates, but there is large variability in the occurrence of these behaviors and the reason for this variability remains largely unexplored. Other types of maternal investment have been shown to be dependent on infant sex (e.g. milk production and maternal responsiveness) and maternal experience (e.g. symmetrical communication). Thus, we sought to determine whether variability in face‐to‐face interactions, that is, mutual gazing (MG), which are hypothesized to be important for later socio‐cognitive development, could be explained by these variables. We studied 28 semi‐free ranging rhesus monkey (Macaca mulatta) mother‐infant dyads (6 primiparous; 12 male infants) born and reared at the Laboratory of Comparative Ethology field station at the NIH Animal Center in Poolesville, MD, across the first 90 postnatal days. Infant sex (i.e. male) was a significant predictor of maternal grooming (β ± SE = 0.359 ± 0.164, Z = 2.19, P = 0.029) whereas both parity (i.e. first time mothers) and infant sex (i.e. male) significantly predicted MG (parity: β ± SE = −0.735 ± 0.223, Z = −3.30, P < 0.001; infant sex: β ± SE = 0.436 ± 0.201, Z = 2.17, P = 0.029). Separation from the mother (outside of arms reach) was not influenced by parity or infant sex. Together with existing literature, these findings point toward differential maternal investment for sons versus daughters. Mothers may be investing differentially in sons, behaviorally, to ensure their future social competence and thus later reproductive success. Collectively, our findings add to the literature that is beginning to identify early life experiences that may lead to sex differences in neurological and behavioral development. Am. J. Primatol. 78:238–246, 2016.

Surrogate Mobility and Orientation Affect the Early Neurobehavioral Development of Infant Rhesus Macaques (Macaca mulatta)

A biological mothers movement appears necessary for optimal development in infant monkeys. However, nursery-reared monkeys are typically provided with inanimate surrogate mothers that move very little. The purpose of this study was to evaluate the effects of a novel, highly mobile surrogate mother on motor development, exploration, and reactions to novelty. Six infant rhesus macaques (Macaca mulatta) were reared on mobile hanging surrogates (MS) and compared to six infants reared on standard stationary rocking surrogates (RS) and to 9-15 infants reared with their biological mothers (MR) for early developmental outcome. We predicted that MS infants would develop more similarly to MR infants than RS infants. In neonatal assessments conducted at Day 30, both MS and MR infants showed more highly developed motor activity than RS infants on measures of grasping (p = .009), coordination (p = .038), spontaneous crawl (p = .009), and balance (p = .003). At 2-3 months of age, both MS and MR infants displayed higher levels of exploration in the home cage than RS infants (p = .016). In a novel situation in which only MS and RS infants were tested, MS infants spent less time near their surrogates in the first five minutes of the test session than RS infants (p = .05), indicating a higher level of comfort. Collectively, these results suggest that when nursery-rearing of infant monkeys is necessary, a mobile hanging surrogate may encourage more normative development of gross motor skills and exploratory behavior and may serve as a useful alternative to stationary or rocking surrogates.

Reproductive consequences of a matrilineal overthrow in rhesus monkeys

Matrilineal overthrows in macaque societies are rare but devastating events, often resulting in severe morbidity, mortality, and loss of individual and group fitness. The handful of documented macaque overthrows provides scant evidence to reveal the severity or longevity of reproductive consequences resulting from such violent events. We analyzed archival records from semi‐free ranging rhesus monkeys, Macaca mulatta, across 6 years (55 ≤ N ≤ 107, from 2007 to 2012) during which time a matrilineal overthrow occurred (in 2009) to test the hypothesis that extremely violent interactions such as a matrilineal overthrow would significantly reduce reproductive fitness for the involved matrilines and for the troop collectively. The matrilineal overthrow resulted in a significant increase in infant loss for the population from the previous year (χ2 = 8.117, df = 1, P = 0.004), as evidenced by the fact that in 2009, but not in other years, the proportion of infants lost was greater than the proportion of viable infants (χ2 = 4.55, df = 1, P = 0.03). Moreover, the deposed matriline suffered 100% infant loss in 2009, a significant change from the previous year (χ2 = 7.87, df = 1, P = 0.005) while the attacking matriline suffered 50% infant loss (also a significant change from the previous year; χ2 = 4.44, df = 1, P = 0.035), with the uninvolved, lowest‐ranking matriline showing no change in infant loss from the previous year (χ2 = 0.008, df = 1, P = 0.93). The deposed matriline did not produce viable offspring again until 3 years later. We further found that rates of severe fighting (as indicated by the number of fight wounds requiring medical treatment) were positively correlated with infant loss across the 6 years of the study (r[s] = 0.943, P = 0.005). Our data indicate that extreme periods of intra‐group conflict, such as the matrilineal overthrow, have marked short‐term consequences for individual fitness, and may be extreme examples of the long‐term influences that group violence exerts on the mean fitness within a primate troop. Am. J. Primatol. 77:346–352, 2015. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.