3.1. Parental separation models
It has long been known that the responses of monkey infants to separation from their mothers share striking similarities with children’s responses to maternal separation (Bowlby 1969
). Early mother-infant separation studies in monkeys were designed to evaluate the mechanisms underlying mother-infant attachment, such as the importance of the mother as a source of contact comfort versus nutrition (Harlow 1959
). Interest in separation as a paradigm to investigate the nature of attachment has persisted over the years under the assumption that investigating the mechanisms underlying the disruption of the mother-infant bond could also provide useful information to understand the formation and maintenance of such a bond (Kraemer 1992
; Reite 1987
). Some researchers, however, have challenged these assumptions and argued that responses to maternal separation are better viewed within the framework of responsiveness to stress than within the framework of attachment (Insel 1992
; Levine and Wiener 1988
). Consequently, physiological responses to separation have attracted a great deal of interest as a prototype of the organism’s response to stress (e.g. Levine and Wiener 1988
). Finally, investigation of responses to maternal separation has also been stimulated by the prospect that they could provide a primate model for human major depression (McKinney and Bunney 1969
; Reite 1977
). Whether the separation response was investigated from the perspective of attachment, stress, or as a model for major depression, reviewing the studies investigating the acute responses to maternal separation is beyond the scope of this article because such studies generally were not conducted within a clear developmental framework (see Mineka and Suomi, 1978
, for a review) and have been reviewed elsewhere in detail (Levine and Wiener 1988
). More relevant to the focus of this article, however, is the research that has investigated the long-term developmental effects of maternal separation. This research has developed along two independent lines: one investigating the long-term developmental effects of permanent maternal separation and social deprivation rearing, and the other investigating the long-term developmental effects of repeated, short parental separation-reunion experiences.
3.1.1. Long-term effects of early maternal and social deprivation
Many studies conducted in the 1960s and 1970s showed that separating primate infants permanently from their mothers and rearing them under conditions of total isolation can have devastating effects on subsequent development and behavior (see Suomi 1997
for review). These effects, usually subsumed under the label of “isolation syndrome”, or “isolate-rearing”, have been best studied in rhesus monkeys. Rhesus monkey infants typically spend the first few days of life in continuous physical contact with their mothers, and maintain close proximity to them for 1–2 years or longer. At the behavioral level, isolation-reared rhesus monkeys include displays of abnormal self-directed and stereotypic behavior and gross deficits in all aspects of social interactions, including affiliation, aggression, communication, mating, and parenting. Isolation effects are generally more pronounced when infants are isolated early in life and for at least 6 months, and when infants are denied access to any sensory stimuli from conspecifics. Denial of tactile contact appears to have more dramatic effects on infant development than denial of visual, auditory, and olfactory stimuli. Isolation-rearing, however, generally involves the simultaneous deprivation of social and nonsocial sensory stimuli as well as the deprivation of different types of social stimuli.
The behavioral effects of isolation-rearing generally persist into adulthood. Although some procedures, such as pairing a previously isolated infant with a non-isolated same-aged monkey “therapist” can reverse some of the social deficits displayed by isolates (Novak and Harlow 1975
; Suomi and Harlow 1972
), these individuals can rarely be successfully reintegrated into a complex social group with mother-reared individuals (Anderson and Mason 1974
). Other environmental or pharmacological treatments may temporarily reduce abnormal behavior but the isolation syndrome usually returns if the treatment is withdrawn (Kraemer 1992
). Recovery from isolation-rearing is considerably more effective in some species of monkeys and apes than in others (Dienske & de Jonge 1982
). Although in some cases the differential effects of early isolation in different species can be accounted for by differences in life-history and social organization (e.g., solitary orangutans appear to be affected by maternal and social deprivation to a lesser extent than highly social chimpanzees), in other cases the causes of interspecific differences in vulnerability to early maternal and social deprivation are less clear (e.g., in the case of closely related macaque species; Sackett et al 1976
Over time, research on the long-term effects of isolation-rearing has shifted its focus from behavior to physiology and neuroanatomy. Pioneering studies by Kraemer and colleagues reported that previously isolated rhesus monkeys showed considerable developmental variation in CSF concentrations of norepinephrine, dopamine, and serotonin metabolites relative to non-isolated control monkeys, as well as a lack of correlation between the different monoamine metabolites (Kraemer and McKinney 1979
; Kraemer et al 1989
). These effects persisted years after the isolation experience. Previously isolated and subsequently “rehabilitated” rhesus monkeys were also behaviorally and neurochemically hypersensitive to d-amphetamine. After the pharmacological treatment, they showed striking increases in aggressive/submissive behavior and in CSF concentrations of norepinephrine and the serotonin metabolite 5-hydroxy-indoleacetic acid (5-HIAA) (but not of the dopamine metabolite homovanilic acid, HVA; Kraemer et al 1983
). On the basis of these findings, Kraemer (1992)
hypothesized that isolation-rearing may produce cytoarchitectural changes in brain monoamine systems that result in a functional dysregulation of these systems.
Neuroanatomical alterations of isolation-reared monkeys have subsequently been reported in such brain areas as basal ganglia, hippocampus, cerebellum, corpus callosum, and neocortex. For example, isolates showed changes in dendritic branching in the neocortex (Struble and Riesen 1978
), modifications in Purkinje cell structure in the cerebellum (Floeter and Greenough 1979
), fewer tyrosine hydroxylase-immunoreactive fibers in the dopaminergic neurons of the neocortex (Morrison et al 1990
), altered chemoarchitecture of some basal ganglia regions (but not of the amygdala and other basal forebrain regions; Martin et al 1991
), cytoskeletal changes in the dentate gyrus cells of the hippocampus (Siegel et al 1993
), and reduced size of the corpus callosum (Sanchez et al 1998
). Several studies, however, reported lack of significant differences between isolates and controls in monoamine metabolite concentrations (Lewis et al 1990
), tyrosine hydroxylase- and CRF- immunoreactive neurons in the paraventricular nucleus and arcuate nucleus of the hypothalamus (Ginsberg et al 1993a
), noradrenergic innervation density of the paraventricular nucleus of the hypothalamus (Ginsberg et al 1993b
), and overall brain volume (Sanchez et al 1998
). In some cases, long-term developmental effects of isolation-rearing on brain structure or neurotransmitter systems were not directly observed but inferred from altered responses to pharmacological challenges. For example, Lewis et al. (1990)
reported changes in dopamine receptor function following apomorphine challenge in previously isolated rhesus monkeys. Similarly, Coplan et al. (1992)
reported differential effects of a low dose (but not of a high dose) of yohimbine in isolation-reared bonnet macaques and controls.
Further information on the long-term consequences of early maternal and social deprivation for behavioral, neuroendocrine, and neuroanatomical development has been provided by studies using the peer-rearing paradigm (Chamove et al 1973
; Harlow 1969
). In this paradigm, rhesus infants are permanently separated from their mothers within hours after birth, hand-raised in a nursery, and housed in small groups of same-aged infants, typically three, for the first six months of life. A longitudinal study of peer-reared versus mother-reared rhesus infants showed that, as neonates, the former were more awake, active, and irritable than the latter (Champoux et al 1991
). From 1 to 5 months of age, the peer-reared infants exhibited a greater variety of behaviors relative to the mother-reared infants, which spent most of their time in contact with their mothers. As juveniles, the two groups of individuals were indistinguishable with the exception that the peer-reared individuals showed more self-directed behaviors. Other studies have found that peer-reared juveniles showed more distress in a novel environment than mother-reared juveniles (Chamove et al 1973
; Higley et al 1992a
; Kraemer and McKinney 1979
; but see Shannon et al., 1998
). Peer-reared monkeys also exhibit greater fear and anxiety as measured by acoustic startle response paradigms (Parr et al 2002
Research investigating the long-term consequences of peer-rearing for physiological development, particularly the activity of the HPA axis, has produced some conflicting results. In various studies comparing cortisol levels, peer-reared individuals have been reported to have higher basal cortisol levels than mother-reared individuals (first month of life; Champoux et al 1989
; first 2 years of life: Higley et al 1992b
), lower basal cortisol levels (14–30 days of age: Shannon et al 1998
), or lower basal ACTH but similar cortisol levels (1–6 months of age: Clarke 1993
). Studies comparing peer-reared and isolation-reared animals have reported higher basal cortisol levels in peer-reared animals (19 months of age: Sackett et al 1973
) or no differences in cortisol levels (4 years of age: Meyer and Bowman 1972
). When physiological responses to stressful challenges were assessed, some studies found no differences in cortisol levels between peer-reared, mother-reared, and isolation-reared monkeys (Champoux et al 1989
; Meyer and Bowman 1972
; Sackett et al 1973
), one study found that peer-reared monkeys had lower increases in both ACTH and cortisol than mother-reared monkeys (Clarke 1993
), another study found that peer-reared infants had lower increases in cortisol but not in ACTH, when compared to mother-reared infants (Barr et al 2004b
), and yet another study reported that the cortisol levels of peer-reared monkeys were higher than those of isolation-reared monkeys but similar to those of mother-reared monkeys (Shannon et al 1998
). Shannon et al. (1998)
suggested that these discrepancies in the activity of the HPA axis in relation to early rearing may have resulted from differences in the housing environment, type of stressful experiences, feeding regimen, or diurnal variation in HPA axis activity across studies.
More consistent findings have been obtained by studies investigating the consequences of peer-rearing on CSF concentrations of monoamine metabolites. In a study comparing rhesus monkeys that were peer-reared or mother-reared in the first 6 months of life, Higley et al (1991)
reported that peer-reared males but not females had higher levels of 5-HIAA at 6 and 18 months than mother-reared males. Peer-reared subjects also had higher values of the norepinephrine metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG; see also Higley et al 1992b
). No rearing effects on the dopamine metabolite HVA levels were found. In a related longitudinal study, no differences in CSF concentrations of 5-HIAA were found between peer-reared and mother-reared monkeys in the first 1.5 years of life (Higley et al 1992b
). The higher levels of MHPG in peer-reared monkeys were consistent with previous findings by Kraemer et al. (1989)
suggesting that rearing under conditions of maternal and social deprivation results in enhanced noradrenergic activity and responsivity to stress. In follow-up studies, peer-reared subjects exhibited lower CSF 5-HIAA concentrations at 50 months of age than mother-reared individuals (Higley et al 1996a
). When peer-reared and mother-reared juveniles were observed in social groups, individuals with low CSF 5-HIAA and MHPG concentrations from both rearing groups exhibited reduced rates of social interaction and low dominance rank (Higley et al 1996b
). In addition, peer-reared subjects with low CSF 5-HIAA concentrations exhibited inept social behaviors and were frequently removed from their social groups for excessive aggression and deviant social behaviors. Based on these findings, Higley (2003)
has suggested that the peer-rearing paradigm exacerbates the negative social consequences associated with low CSF 5-HIAA such as high behavioral reactivity to the environment, high impulsivity, and high aggressiveness (see also Shannon et al 2005
Consistent with this hypothesis, there is some evidence that lower serotonergic function in rhesus monkeys carrying the short allele of the serotonin transporter (SERT) gene is associated with differential responsiveness to stress, higher impulsivity, and higher aggressiveness in peer-reared than in mother-reared individuals. Specifically, Barr et al (2004a
) reported that in males, ACTH responses to social separation were higher in individuals carrying the short (s
) SERT allele (l/s
) than in individuals carrying two copies of the long (l
) SERT allele (l/l
) – both in mother-reared and in peer-reared infants. In females, however, higher ACTH responses to separation in l/s
relative to l/l
individuals were only observed in peer-reared and not in mother-reared individuals. Furthermore, peer-reared rhesus monkeys, in particular females, carrying the s
allele showed lower cortisol responses to stress than mother-reared infants. Finally, the aggressiveness toward an unfamiliar conspecific of rhesus males carrying the s
allele was greater in peer-reared than in mother-reared individuals (Schwandt et al 2010
Behavioral and neurobiological differences between mother-reared and peer-reared infants have also been documented for other genotypes. For example, mother-reared male infants with the low-activity-associated allele of the MAO-A gene promoter were more aggressive than peer-reared infants (Newman et al 2005
). Furthermore, monkey infants carrying the −248 T allele of the CRF gene showed reduced exploration and greater ACTH and cortisol responses to social separation than carriers of the −248 C allele, and more so in peer-reared than in mother-reared monkeys (Barr et al 2009
). Another single nucleotide CRF gene polymorphism (−2232 C, −2232 G) was also associated with behavioral and physiological differences in response to a social challenge presented by an unfamiliar male: carriers of the G allele had lower CSF levels of CRF, higher levels of ACTH, and showed bolder and more exploratory behavior than carriers of the C allele, independent of rearing condition (Barr et al 2008
). Differences in stress reactivity, and also in alcohol consumption, between peer-reared and mother-reared monkeys have been reported in relation to polymorphisms in the DRD1 receptor gene (Newman et al 2009
) and in the Neuropeptide Y (NPY) gene (Lindell et al 2010
Similar to isolate-rearing, research investigating the biology of peer-rearing has also been extended to examine structural brain differences. One recent study has reported neuroanatomical differences between peer-reared and mother-reared monkeys. Specifically, peer-reared monkeys showed reduced vermis, dorsomedial prefrontal cortex, and dorsal anterior cingulated cortex, but no differences in corpus callosum and hippocampus, when compared to mother-reared individuals (Spinelli et al 2009
It is clear from the studies reviewed above that maternal and social deprivation in early life can result in abnormalities and deficits in behavioral development accompanied by alterations in neural structures and neurochemical/neuroendocrine function. These alterations may reflect a combination of the effects of the early maternal separation experience with those of the subsequent rearing environment. The neurobiological and endocrine effects of isolation-rearing and peer-rearing, however, have been variable among different studies. Variability in developmental outcomes probably reflects variation in the degree of sensory, social, and motor deprivation experienced by socially deprived infants (part of which is intentionally manipulated by researchers and part of which is unintentionally caused by them though variation in animal housing conditions and laboratory protocols) and also variation in the type and magnitude of stimulation provided by the human caretakers with whom the socially deprived monkeys necessarily interact. The alterations induced by long-term maternal and social deprivation are generally greater the earlier the mother’s removal and the longer and the more complete the deprivation of stimuli. Since the peer-reared infants experience a lower degree of sensory, social, and motor deprivation than the isolation-reared infants, peer-rearing is considered a less intense form of social deprivation than isolation-rearing. Thus, when peer-reared infants are compared to isolation-reared and mother-reared infants, the assumption is made that these three groups of infants differ in the “quantity” of early social experience and that, as a result, the peer-reared infants should exhibit behavioral and physiological parameters that are intermediate between those of mother-reared and isolation-reared infants. In reality, both the quantity and the quality of early experience of each group of infants differ in multiple and complex ways from those of the other two groups. For example, it is almost impossible to assess whether peer-reared monkeys differ from mother-reared ones because they lack a mother, because they extensively interact with their human caretakers, because they drink formula instead of monkey milk, or because they have the opportunity to interact with peers much earlier and more extensively than the mother-reared monkeys.
The fact that the maternal and social deprivation paradigm combines and confounds different forms of deprivation (e.g., motor, sensory, and social) and stress (physical and psychosocial) makes it difficult to assess the specific mechanisms and pathways through which early adverse experience alters the course of normal biobehavioral development. Primate research conducted with maternal and social deprivation paradigms has undoubtedly contributed some general principles of the relationship between early stress and development such as: (1) early experiences that deviate greatly from the norm are those most likely to result in long-term effects, (2) the most dramatic long-term effects are observed after stressful challenges (i.e., not at baseline), and (3) there may be considerable interindividual variability in the extent to which early experiences affect biobehavioral development (Suomi 1991
). We believe, however, that research with primate models can and should, not only contribute general principles, but also elucidate the specific mechanisms and pathways through which early stress affects development in humans. In this regard, however, the maternal and social deprivation paradigm has already shown its limitations.
Since the type of environmental deprivation experienced by isolation-reared and peer-reared monkeys is rarely, if ever, experienced by primates or humans under natural conditions, the ecological validity of such paradigms is limited. The functioning of stress-sensitive physiological systems is also typically impaired by these drastic alterations of early development and it cannot be expected that these alterations have adaptive consequences, i.e. result in later resilience to stress. Since we believe that the relationship between early stress exposure and later resilience is a natural phenomenon, rather than an artifact of experimental manipulations, this relationship should be investigated using models of early experience that are within the norm for a particular species, i.e. models with greater ecological validity than the maternal and social deprivation paradigms.
3.1.2. Long-term effects of short mother-infant separations in macaques
An alternate approach to isolate-rearing and peer-rearing models of early life stress has been to expose infant macaques to short mother-infant separation paradigms. Although macaque mother-infant separation protocols have varied across laboratories, in general, infants are separated from their mothers for brief periods ranging from a few days to a few weeks during the infant’s first months of life (Caine and Reite, 1983
; Hinde and Spencer-Booth, 1971
; Hinde et al 1978
; Suomi et al 1983
). In some cases, the infant is removed from its group and housed alone in a cage (e.g., Suomi et al 1983
), while in others the mother is removed (e.g., Caine and Reite, 1983
), or both mothers and infants are removed, separated, and individually housed (e.g., Hinde et al 1978
). When sufficient detail is available from published reports, it is included in the findings from the mother-infant separation paradigms reviewed below.
The earliest studies of the long-term effects of short mother-infant separation in macaques were independently conducted in Hinde’s and Harlow’s laboratories in the early 1970s. In one study of rhesus monkeys, Spencer-Booth and Hinde (1971)
separated infants from their mothers for 6 or 13 days at the age of 21–32 weeks. Infant activity and performance in a variety of tests were assessed when infants were 12 and 30 months old and compared to those of infants that had never been separated from their mothers. There were few or no behavioral differences in infants’ interactions with their mothers or in their tendency to approach novel objects in the home cage. When tested in a novel environment, however, the previously separated infants showed significantly greater disturbance of behavior, lower exploration, and lower manipulation of novel objects than controls. Such differences were evident at both 12 and 30 months of age, although they were less marked when the animals were older. Hinde et al (1978)
subsequently reported some other effects of a brief maternal separation of 13 days on subsequent mother-infant interactions 5 months after the separation, and Stevenson-Hinde et al (1980)
reported some differences in subjectively assessed personality measures in infants previously separated from their mothers.
In Harlow’s laboratory, the long-term effects of brief separations from the mother were investigated by Suomi et al (1983)
. In this study, four infants were subjected to repeated 4-day maternal separations conducted between the 3rd
month of life. The separated infants spent more time in contact with their mothers following reunion, although the relative role played by mother and infant in maintaining contact was not assessed. Separated infants and non-separated control infants showed few or no differences in response to permanent separation from their mothers, which occurred at the age of 43 weeks, and during the subsequent 7 months of peer housing. When separated infants and controls were exposed to their mothers, 7 months after permanent separation, previously separated infants showed less interest in interacting with them than did non-separated control infants. Thus, although the long-term effects of early separations seemed to disappear in some situations (e.g., peer housing), they were apparent in others (e.g., in the presence of mothers). The mechanisms underlying the long-term effects of peer or maternal separations remained unclear.
Studies of the long-term effects of brief maternal separations that lasted 10 days in duration were also conducted with pigtail macaques by Reite and colleagues. Caine et al (1983)
reported that 6 pigtail infants who experienced a 10-day maternal separation at 4–7 months of age did not differ in dominance rank but were rated as less sociable than controls at the age of 40.7 months. Capitanio and Reite (1984)
reported that such previously separated infants had fewer social contact preferences, and Capitanio et al (1986)
reported that they showed more disturbance behavior and longer latency to retrieve food (but no difference in cortisol levels) in a novel environment than controls when tested 2.5–4.9 years after the separation experience. Finally, Laudenslager et al (1985)
found that when individuals previously separated from their mothers or peers for 10–14 days in infancy were later tested at 4–7 years of age, they showed a suppression of the in vitro lymphocyte proliferation in response to B cell and T cell mitogens, suggesting that the early separation experience resulted in altered immune function. In reviewing these studies, Reite (1987)
argued that possible alterations in maternal behavior following early separation and reunion (as is known to be the case in rodents; e.g. Liu et al 1997
) may have been the mechanism underlying the developmental effects of early separation on infant behavior and physiology. Due to lack of further research using this paradigm in macaques, the mechanisms underlying the developmental effects of brief separation experiences have remained unclear and the question of whether these effects should be viewed as the direct consequence of an acute stressful experience or as mediated by subsequent alterations in maternal behavior or maternal physiology (e.g., cortisol in mother’s milk) remains unanswered.
More recently, however, some of these questions have been addressed by studies of New World monkeys such as marmosets and squirrel monkeys, which have used repeated mother-infant separations as a model for studying the relationship between early stress and development. Some of these studies have not only examined how exposure to early stress results in increased vulnerability to stress later in life, but also how early life stress exposure may enhance later resilience under certain circumstances.
3.1.3. Long-term effects of short parent-infant separations in common marmosets
The parental separation paradigm in common marmosets was first employed in the early 2000s, and therefore is comparatively new amongst the various primate models considered herein. This paradigm is notable for involving both parents, the only such primate separation paradigm to do so. Common marmoset social ecology is characterized by monogamous breeding pairs, twin births, and intensive biparental care of young (Pryce 1996
). Infants are in constant (i.e., 24 hours a day) contact with their parents throughout the first 2–3 weeks of life (Ingram 1977
; Pryce 1993
To examine the long-term consequences of early stressful experience, Pryce and colleagues exposed marmoset infants to daily parental separations on postnatal days 2–28 (Dettling et al 2002a
; Dettling et al 2002b
; Law et al 2009
; Pryce et al 2004a
; Pryce et al 2004b
; Pryce et al 2002
). During each separation session, marmoset monkeys are socially isolated in a plastic mouse cage which is placed in an isolation chamber with 4 W light (Dettling et al 2002b
). Marmoset infants have total auditory, olfactory, and visual deprivation of their families and other monkeys during this time. Each infant is exposed to a total of 9 hours of social deprivation each week, with each session being variable in duration (30–120 minutes per session) and time of day, a feature implemented to increase stressor unpredictability. The parental separation procedure is carried out consecutively on each twin pair. Control infants are briefly handled on their parents’ backs daily during postnatal days 2–28. After postnatal day 28, infants typically remain undisturbed in their family groups.
Infants exposed to repeated parental separations have 12% lower body weights than control infants at the conclusion of this repeated separation protocol. The observed body weight loss is likely due to impaired homeostasis, reduction in time available for nursing, and/or reduction in milk quality/quantity from stressed moms (Dettling et al 2002b
). These separation sessions, therefore, interfere with both the physiological and psychological needs of marmoset infants.
During the first year of life, marmosets exposed to repeated parental separations exhibited increased basal urinary NE levels, increased basal urinary dopamine levels, and increased systolic blood pressure in the absence of a significant change in heart rate compared to non-separated controls (Pryce et al 2004a
; Pryce et al 2004b
). Previously separated marmosets also perform fewer operant responses on a neuropsychological test, indicative of anhedonia (Pryce et al 2004b
). Several domains of cognitive functioning are likewise impaired in this parental separation paradigm. For example, previously separated marmosets exhibit impaired behavioral inhibition on an object retrieval/detour task as well as impaired reversal learning on a two-way discrimination task (Pryce et al 2004a
Studies of emotional and cognitive functioning in previously separated marmosets have recently been extended to include postmortem neuroanatomical and neurobiological analyses. Brains were collected when adolescent monkeys were 48 weeks of age. In the first paper from this series, genes implicated in synaptic function and plasticity were examined in the hippocampus. Previously separated marmosets exhibited a reduction in hippocampal GAP-43 mRNA and serotonin 1A receptor mRNA, findings similar to those reported for patients with stress-related mood disorders (Law et al 2009
). Although previously separated monkeys did not differ from control monkeys on hippocampal volumes (Law et al 2009
), evidence from a follow-up study indicates that repeated parental separations during the first month of life induce mild reductions in MR and GR gene expression in the hippocampus (in CA 1–2 subfield). These effects were found to be specific to this neuroanatomical region, as no differences in MR or GR gene expression were observed in the prefrontal cortex, other cortical areas, or the hypothalamus (Arabadzisz et al 2010
). These findings are consistent with human data indicating that individuals with major depression likewise exhibit mild to moderate reductions in hippocampal MR and GR gene expression.
In summary, the marmoset model of parental separation has yielded important behavioral and neurobiological findings relevant to stress vulnerability. Perhaps due to its relatively recent conception, data are not yet available from this model of parental separation beyond one year of age (i.e., marmoset adolescence). There is a great deal of evidence for long-term effects of early experience in rodents, but surprisingly few studies have examined the enduring effects of early experiences in the same cohorts of adult monkeys or humans (Schaffer 2000
). Given the growing interest in early experience effects in biological psychiatry, longitudinal studies of these behavioral, neuroendocrine, neuroanatomical, and molecular measures are critically needed in the marmoset parental separation paradigm to verify (or refute) findings from rodent research in animals with a life span of greater than 2–3 years.
In the common marmoset, as well as in the isolate-rearing and peer-rearing, paradigms, the types of early parental manipulations were all “non-biological events” (Dettling et al 2002b
), and as such, were of limited ecological validity. Moreover, in each of these paradigms, key features of early stress exposure were shown to act together to undermine the development of resilience and produce stress vulnerable phenotypes. We now turn our attention to another parental separation model involving squirrel monkeys. This laboratory model is unique in that it is employed in late infancy when free-living infants often face the challenge of coping with intermittent separations from their mothers while foraging in the forest canopy. Not coincidentally, this is the only parental separation paradigm that produces stress resilience, and likely does so because it is employed during a developmental period when the key features of early stress exposure do not overwhelm the infant’s coping capacity as discussed in greater detail below.
3.1.4. Long-term effects of short mother-infant separations in squirrel monkeys
In this maternal separation model, called “stress inoculation”, squirrel monkey infants are initially raised in undisturbed natal groups of 3–5 mother-infant pairs. At ~ 17 weeks of age, when free-living young monkeys are becoming nutritionally independent (Boinski and Fragaszy 1989
), natal groups are randomized to one of two postnatal treatment conditions. In one condition, monkeys are exposed to a 10-week stress inoculation protocol that consists of weekly 1-hour sessions of separation from the mother and natal group (Parker et al 2004
). The separation session occurs in an elevated wire-mesh cage in a colony room different from the one in which the infant lives. During the separation, the infant is surrounded by monkeys housed in adjacent cages. Infants are able to vocally communicate with their mother and natal group which are housed in a colony room on the same hallway. Control monkeys remain in their home cages throughout the same developmental period. Separation protocols for different monkey cohorts have varied slightly over the years, with separations being fewer in frequency but of greater duration (i.e., 4–6 total separation sessions, each lasting 4–6 hours in duration) (Levine and Mody 2003
) or beginning earlier in life and occurring with diminished frequency for longer periods (postnatal week 13, every other week for 5 hours) (Lyons et al 1999
), or with the mother accompanying the infant during each separation session (Parker et al 2006
). These protocols have generally produced the same developmental outcomes, and will be considered together below.
Short repeated social separations in squirrel monkeys evoke species-typical distress peep-calls, locomotor agitation, and acute elevations in plasma cortisol levels. Recovery of baseline measures is achieved after each social reunion (Coe et al 1983
; Hennessy 1986
; Stanton and Levine 1985
). Unlike previous primate paradigms, the stress inoculation protocol provides repeated opportunities for emotion regulation, while not overwhelming the juvenile monkey’s capacity for coping with stress. The protocol is administered weekly or biweekly to allow sufficiently long intervals for recovery at an age when squirrel monkeys are developing physical, emotional, and psychosocial independence.
Outcome studies of squirrel monkeys exposed to this paradigm have shown that stress inoculated monkeys better regulate negative emotional arousal and exhibit diminished HPA axis activation in response to subsequent acute stressors. For example, at 9 months of age, stress inoculated vs. non-inoculated monkeys were tested with their mothers for 30 minutes on five consecutive days in a novel environment that contained an assortment of familiar and unfamiliar foods and toys (Parker et al 2004
). The novel environment test revealed that stress inoculated vs. non-inoculated monkeys were less anxious as inferred by decreased maternal clinging, increased object exploration, and increased food consumption. Stress inoculated monkeys also more effectively used their mothers as a secure base from which to explore. Stress inoculated compared to non-inoculated monkeys also showed smaller increases in plasma cortisol and ACTH.
These experimental results indicate that stress inoculated monkeys more readily self-regulate negative emotional arousal, engage in more exploration, and exhibit diminished stress-induced HPA axis activation compared to non-inoculated monkeys. This conclusion agrees with findings from independent studies of two additional cohorts of stress inoculated monkeys. In one cohort, stress inoculated monkeys responded to the removal of mothers at weaning with fewer distress calls, more time spent in proximity to peers, and smaller increases in plasma cortisol levels compared to non-inoculated monkeys (Lyons et al 1999
). In the other cohort, stress inoculated monkeys exhibited fewer distress calls, diminished adrenocortical activation, and smaller increases in CSF levels of MHPG compared to non-inoculated monkeys at 2, and again at 3, years of age (Levine and Mody 2003
). Finally, stress inoculated monkeys exhibited enhanced sensitivity to glucocorticoid feedback in early adulthood compared to non-inoculated monkeys (Lyons et al 2000b
). These observed stress inoculation-induced differences in cortisol suppression of the CRF-stimulated ACTH response (Lyons et al 2000b
), CSF MHPG levels (Levine and Mody 2003
), and plasma ACTH levels (Parker et al 2004
) suggest that brain mechanisms above the pituitary enhance the stress inoculated monkey’s ability to diminish HPA axis activation in response to subsequent stressful life events.
Stress inoculated monkeys have also been evaluated for postnatal treatment differences in prefrontal-dependent cognitive response inhibition as juveniles (Parker et al 2005
). Inhibitory control of the reaching response in marmosets is impaired in the marmoset monkey model reviewed above (Pryce et al 2004a
) and in squirrel monkeys treated with cortisol according to a protocol that simulates a chronic HPA axis stress response (Lyons et al 2000a
). The stress inoculated and non-inoculated monkeys performed equally well on all trials except for those that required inhibitory control of the straight-reaching response. Performance improved gradually over repeated test days, but at peak levels of performance all stress inoculated monkeys successfully completed all response inhibition trials whereas fewer than half of the non-inoculated monkeys achieved similar levels of success. These findings suggest that stress inoculation may have directly altered the neural substrates involved in cognitive function, or indirectly influenced cognitive performance by primarily changing emotion regulation. The available data do not help to distinguish between these two possibilities. It should nevertheless be noted that the rearing protocol was initiated during a period of increasing offspring independence. Far from exceeding the young monkeys’ coping abilities, the mildly stressful separation experiences which constituted the stress inoculation protocol may have provided important opportunities for stress inoculated monkeys to develop the capacity for enhanced emotion regulation. Non-inoculated monkeys, which had fewer emotional challenges, failed to adequately develop this capacity. Thus, when non-inoculated monkeys were faced with the demanding cognitive test, they engaged in more impulsive and perseverative behavior than stress inoculated monkeys, who were better equipped to deal with such challenges. Magnified across repeated life challenges, the cumulative consequences of how inoculated and non-inoculated monkeys respond to stress beginning in infancy may be profound.
Stress inoculation-induced resilience in monkeys resembles the effects of early intermittent “postnatal handling” in rats exposed to repeated, brief maternal separations. When studied as adults, “handled” rats display diminished emotionality, increased exploration, improved learning, and diminished HPA axis activation compared to rats raised in standard undisturbed laboratory conditions (Fernandez-Teruel et al 2002
; Levine 1957
; Levine 2000
; Meaney et al 1996
). In rats, these outcomes appear to reflect the effects of maternal behavior directed toward pups that are briefly separated and then returned to the nest (Denenberg 1999
; Smotherman and Bell 1980
). Importantly, the neuroendocrine indications of resilience observed in rats exposed to intermittent separations can be replicated in non-manipulated offspring that naturally receive high levels of maternal care (Liu et al 1997
To determine whether resilience in monkeys exposed to early intermittent separations is maternally mediated, maternal behavior and subsequent neuroendocrine measures of resilience were examined in monkeys randomized to three postnatal conditions (Parker et al 2006
). In one condition, each monkey was separated from its mother and the natal group for 10 weekly 1-hr sessions. In the second condition, each monkey and its mother were removed together as a pair and separated from the natal group for 10 weekly 1-hr sessions. In the third treatment condition, non-separated monkeys remained undisturbed in their natal groups. Neither separation condition induced long-lasting changes in maternal behavior (Parker et al 2006
). Moreover, the transient changes observed in maternal behavior did not correspond with differences in the development of arousal regulation and resilience. Monkeys exposed along with their mother to intermittent separations received less maternal care in the home cage, as mothers direct their attention toward re-integration with the group. Yet both intermittent separation conditions induced neuroendocrine indications of resilience compared to control monkeys not exposed to intermittent separations. Specifically, monkeys separated alone or separated along with their mother exhibited diminished cortisol responses to a subsequent novel environment stress test compared to monkeys not exposed to either separation condition (Parker et al 2006
). These and related findings suggest that arousal regulation and resilience correspond more closely to prior stress exposure than to separation-induced changes in maternal care. These findings also suggest that the mechanisms involved in fostering stress resilience may differ considerably between rodents and primates. However, it should be noted that recent investigations of developmental plasticity and early life stress in rats suggest that the rodent story of stress resilience may be more complicated than previously thought (Macri and Wurbel 2006
Very little is known about the neuroanatomical basis of stress resilience. However, because prefrontal corticolimbic brain circuits play a role in cognitive control of behavior in humans and monkeys, regulate the HPA axis stress response in various species, and have been implicated in emotional resilience in humans (Diorio et al 1993
; Garavan et al 1999
; Herman et al 2003
; Kern et al 2008
; Konishi et al 1998
; Milad et al 2007
; Miller 2000
; Ochsner and Gross 2005
; Sullivan and Gratton 2002
; Urry et al 2006
), stress inoculated and non-inoculated squirrel monkeys were tested for differences in prefrontal cortical and/or hippocampal structural volumes at 3 years of age (Katz et al 2009
). Early life stress inoculation promoted the growth of larger prefrontal cortical gray but not white matter volumes. This effect was primarily driven by differences in ventromedial prefrontal cortex, as dorsolateral prefrontal cortical volumes did not differ between stress inoculated and non-inoculated monkeys. These larger ventromedial prefrontal volumes do not reflect increased cortical thickness but instead represent surface area expansion (Katz et al 2009
Similar to findings from the marmoset model reviewed above, exposure to early life stress inoculation did not affect hippocampal volumes. These data are in keeping with evidence that mildly stressful early experiences promote the development of larger prefrontal cortical volumes without affecting hippocampal volumes as shown for 5 year old adult male and female squirrel monkeys exposed to slightly different postnatal conditions (Lyons et al 2002
; Lyons et al 2001
). Experiential effects on brain structure are most potent when the neural circuit is maturing and before it is stabilized (Knudsen 2004
). Hippocampal growth and development occurs primarily in utero
(Eckenhoff and Rakic 1991
; Khazipov et al 2001
; Rakic and Nowakowski 1981
), and so it is perhaps not surprising that structural stress inoculation effects are most evident in prefrontal cortex, the growth and development of which extends across childhood into early adulthood (Giedd et al 1999
; Rakic 1995
The absence of stress inoculation effects on hippocampal structure, however, does not preclude the existence of postnatal treatment-related differences in hippocampal molecular biology. Recent evidence from rodents (Kaffman and Meaney 2007
) suggests that stress resilience may be mediated by increased hippocampal glucocorticoid receptor (GR) expression due to diminished methylation of a cytosine residue in the GR promoter. It is not known whether early life stress inoculation increases hippocampal GR expression in adulthood, protects against stress-induced GR downregulation, and whether changes in GR promoter methylation similar to those observed in stress resilient rodents underlie these effects in monkeys. Follow-up studies are required to address these important questions to better understand the molecular mechanisms underlying the etiology and maintenance of stress resilience in monkeys.
Similar to many of the primate models reviewed above, very little is known about the scope, extent, or enduring effects of early life stress inoculation in squirrel monkeys. For instance, it is not known whether early stress inoculation effects persist across the lifespan, as the latest assessments of stress inoculated monkeys occurred three years after completion of postnatal treatment protocols (Katz et al 2009
; Levine and Mody 2003
). The persistence of stress inoculation effects, therefore, has only been documented across 14% of the squirrel monkey lifespan. Longitudinal studies, which rigorously evaluate the enduring consequences of early life stress inoculation on behavioral, neuroendocrine, neuroanatomical, and molecular measures throughout adulthood, are critically needed to address this gap in knowledge.
In concluding our review of the various Parental Separation Models, we believe that the balance of evidence suggests that primate studies employing short parent-infant separations are more ecologically valid and therefore more promising models for research on stress vulnerability and resilience than those involving extended social deprivation. Even though wild primates may not experience mother-infant separations for the duration of those used in some laboratory paradigms, a short mother-infant separation is a naturally occurring event that is likely to occur in the lifetime of every primate and for which individuals are likely to have pre-programmed adaptive coping responses. In the next section we review the Maternal Behavior Models, which are likewise well positioned to provide critical insights into the role early experiences play in shaping developmental outcomes in humans.