Serotonin and neuropeptides in affiliative behaviors

Serotonin and Neuropeptides in Affiliative Behaviors Thomas R. Insel and James T. Winslow The neuropharmacological study of serotonin and behavior has followed two fundamentally different strategies. One approach has used behavior as a dependent variable for assaying drug effects. To characterize serotonergic drugs, most studies have used relatively simple behaviors, such as locomotor activity, startle, exploration, operant responses, and sleep. A second approach has focused on behavior, with drugs used as tools to elucidate the physiological role of serotonin. These studies have increasingly focused on behaviors of ethological importance, including aggression, sexual behavior, and other forms of social interaction. Here we review studies using this approach to focus on one particular kind of social interaction: affiliation. Biol Psychiatry 1998;44: 207–219 © 1998 Society of Biological Psychiatry Key Words: Separation, attachment, oxytocin, vasopressin

Introduction

A

ffiliative behaviors are simply pro-social behaviors, that is behaviors that bring individuals into contact. For infants, affiliation can be measured as a grasping response, a tendency to huddle, or, in a negative sense, a separation cry. For juveniles, affiliation can be measured as play. And in adults, affiliation includes a variety of behaviors from side-by-side contact to grooming to pair bonding. Although some forms of social behaviors are phylogenetically ancient and may be observed across vertebrates, affiliation is only fully developed in birds and mammals, and logically may have been present in their common ancestor, the therapsids, that survived until perhaps 150 millions years ago. While behavioral ancestry is speculative, the differences in neuroanatomy and neurochemistry between reptiles and mammals can be defined within extant taxa and may be relevant to the evolution of affiliative behaviors. For instance, as suggested by MacLean (1990), the development of “paleomammalian” limbic pathways, such as the amygdala-septal-hippocampal complex with its extensive connections to the hypo-

Yerkes Regional Primate Research Center and Department of Psychiatry and Behavioral Sciences, Emory University, Atlanta, Georgia 30322 Address reprint requests to Thomas R. Insel, MD, Yerkes Primate Center, Emory University, Atlanta, GA 30322. Received January 7, 1998; accepted March 4, 1998.

© 1998 Society of Biological Psychiatry

thalamus, as well as the cingulate with its elaborate thalamic connections, may have permitted the evolution of prototypically mammalian behaviors such as nursing, separation calls, and play (MacLean 1990). If one were to design a neurotransmitter important for something as complex as affiliative behaviors, one would want a neurochemical that is ancient and yet easily modified, widely distributed in the forebrain, with abundant expression in early postnatal life. Of course, serotonin fulfills each of these requirements. Serotonin is among the oldest of mammalian neurotransmitters, with a phylogenetic history of mediating social behaviors in the primitive nervous systems of non-mammals, such as molluscs and teleost fish (Yeh et al 1996; Yeh et al 1997). While serotonin may not have changed over more than 100 million years of evolution, a family of receptors has evolved, each with independent regulatory control, to provide exquisite flexibility in response to evolutionary demands (Hoyer and Middlemiss 1989). Serotonin is widely distributed throughout the forebrain, and in mammals there are abundant receptors located within neural networks necessary for motivation, memory, sensory processing, and other integral processes for social interaction (Whitaker-Azmitia and Peroutka 1990). Finally, serotonin appears to have an important role in development, including an abundant and transient expression of terminals in specific cortical areas where serotonin may shape the developing nervous system in response to environmental change (D’Amato et al 1987). Serotonin’s role needs to be understood in a neurochemical context. The past decade has seen several excellent demonstrations that serotonin and other monoamines influence behavior by their interaction with various neuropeptide neurotransmitters. With reference to affiliation, the opioid peptides have been studied because of their presumed importance for reward and the neurohypophyseal hormones oxytocin (OT) and vasopressin (AVP) have been of interest because of their roles in reproduction. These latter hormones are descended from an ancient family of nine amino acid peptides, but OT and AVP are found exclusively in mammals (Archer 1974). It may not be a coincidence that these peptides have been implicated in prototypically mammalian functions. Oxytocin, for instance, has an important role in milk ejection during nursing and uterine contraction during labor. The tradi0006-3223/98/$19.00 PII S0006-3223(98)00133-4

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tional view of the neurohypophyseal peptides as endocrine hormones acting on peripheral organs has recently been revised to consider these peptides as neurotransmitters or neuromodulators, that is, peptides with central actions. Not only do hypothalamic cells synthesizing oxytocin or vasopressin project to diverse sites within the brain and brainstem (Sofroniew and Weindl 1981), but receptors for both peptides have been found throughout the limbic system in the forebrain and autonomic centers in the brainstem (Tribollet et al 1988). Furthermore, both peptides are released within the brain following chemical depolarization of the appropriate neurons and fibers have been demonstrated at the ultrastructural level to make synaptic contacts in the CNS (Buijs et al 1982). Thus, the evidence is quite strong that the brain is a target organ for these peptides. Do these central oxytocin and vasopressin pathways influence behavior or mental function? We now have almost 3 decades of research indicating that both peptides have important central effects (Argiolas and Gessa 1991; de Wied et al 1993). The preponderance of this literature has focused on their roles in the modulation of memory, the regulation of fluid balance, and the response to hyperthermia, but more recently, a growing body of evidence has implicated oxytocin and vasopressin in the central mediation of complex social behaviors (Carter 1992; Insel 1992; Witt 1995). This review will emphasize the “biological” in the biological psychiatry approach by focusing on the neurobiology of three behaviors: infant separation calls, grooming, and pair bonding in non-human mammals. Whereas separation calls may indicate the distress from loss of affiliative contact, grooming and pair bonding represent aspects of what Panksepp has called “social reward” (Panksepp 1992). Although there is intriguing evidence for serotonin’s involvement in separation calls and grooming, there is no evidence, at this point, for a role in pair bonding. Various neuropeptides have been implicated in all three processes. We will summarize what is known for each behavior and suggest where future studies might focus. The subtext for this research is that the study of complex social behaviors provides an important basic science for psychiatry. We assume that a detailed understanding of serotonin or oxytocin’s role in normal, biologically relevant behaviors will prove important to understanding their role in psychopathology.

Separation Calls The Paradigm Virtually all mammalian infants give calls or cries when they are initially separated from their family group.

Although primate infants may recognize that they are separated by visual or somatosensory cues and rodent infants may perceive a change in temperature or olfactory cues, the response across taxa is a high-pitched, speciestypical alarm cry that is a powerful stimulus for maternal retrieval (Newman 1988). Most rodents give birth to altricial pups, meaning that for the first week or two of postnatal life, the pups cannot see, hear, or even thermoregulate effectively. For the first 14 days postnatal, when removed from their litters, rat pups emit calls in the 37– 42 kHz range, a region of the auditory spectrum that we label ultrasonic because most humans cannot hear much above 12 kHz (Winslow and Insel 1991b). However, this range of calls is in the region of greatest auditory sensitivity for adult female rats and females exposed to these calls in the laboratory invariably respond by activation and search behavior (Winslow and Insel 1991b). Over the past decade we have learned much about the biology of rat pup ultrasounds as prototypic separation calls. The calls are laryngeal, they are elicited by changes in temperature, and they follow a predictable developmental course, fading out around day 14 when the pups become independent in terms of locomotor activity (weaning is usually at day 20 –22) (Winslow and Insel 1991b). Pups from fearful or emotionally reactive strains of rats tend to emit more calls (Insel and Hill 1987). Studies in other rodents have demonstrated that highly affiliative species call more in response to social isolation (Shapiro and Insel 1990). Thus, the isolation call, like huddling and clinging to the mother, may be a reasonable measure of affiliation in the rat pup.

Serotonin Serotonin’s influence on this behavior has been studied with both pharmacological and lesion studies. In the first such study (Figure 1), drugs that blocked 5-HT uptake (clomipramine, citalopram, paroxetine) decreased whereas NE or DA-uptake blockers (desipramine, nortriptyline, mazindol) increased the number of calls after a single administration (either 1 or 5 mg/kg) (Winslow and Insel 1990). These 5HT uptake inhibitors did not affect temperature or locomotor behavior at the doses used. Chronic administration of either desipramine or clomipramine increased calling. The surprising increase in calling rate after chronic administration of clomipramine may have resulted from the accumulation of the metabolite, desmethylclomipramine, which is a potent noradrenergic uptake inhibitor. Administration of selective (or putatively selective) 5-HT receptor ligands suggested a more complex picture (Figure 2). Injections of 5HT-1a drugs, such as buspirone and 8-OHDPAT, decreased the rate of calling at doses that

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did not affect locomotor activity or core body temperature (Winslow and Insel 1991d). These rate-reducing effects were blocked by a dose of propranolol that lacked intrinsic effects, consistent with mediation via a 5-HT1 receptor. More recent studies by Joyce and Carden replicated the quieting effects of 8-OH-DPAT, but demonstrated that the mixed 5HT1a/b-adrenergic antagonist pindolol did not alter the rate of calling and failed to block the quieting effect of a social companion (Joyce and Carden, in press). Thus, even if 5-HT1a receptor activation may reduce the distress component, this receptor is not likely to be critical to the social reward component of affiliation in the pup. In contrast to 5HT1a activation, injections of putative 5-HT1b drugs, such as CGS12066B and TFMPP, selectively increased calling (Winslow and Insel 1991d). These effects, like the 5HT1a rate reducing effects, were also blocked by propranolol. Mice in which the 5HT1b receptor gene was deleted (i.e., 5HT1b knockout mice) showed a reduced rate of calling and an attenuated response to the 5HT1b/1a agonist RU24969 relative to wild type controls (Brunner et al 1997). Although RU24969 reduced (rather than increased) calling rate in wild type mice, this effect was probably due to activation of the 5HT1a receptor as WAY100635, a 5HT1a antagonist blocked the rate reduc-

Figure 1. Data represent effects of selected doses of serotonergic and catecholaminergic reuptake inhibitors on the ultrasonic vocalizations of 9-10 day old pups during 2 min separations from mother and littermates. Columns represent means 11 s.e.m. of the rate of calling expressed as % of calling rate in a pre-injection control trial. Asterisks represent significant differences from vehicle control performance determined by Newman-Keuls tests (data from Winslow and Insel 1990a).

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Figure 2. Data represent effects of selected doses of serotonin receptor ligands with putative activity at 5HT1a (Gray Columns), 5HT1b (Black Columns) and 5HT1c/5HT2 (Hatched Columns) receptor subtypes on the ultrasonic vocalizations of 9-10 day old pups during 2 min separations from mother and littermates. Columns represent means 11 s.e.m. of the rate of calling expressed as % of calling rate in a pre-injection control trial. Asterisks represent significant differences from vehicle control performance determined by Newman-Keuls tests (data from Winslow and Insel 1991d).

ing effects of RU24969 (Brunner et al 1997). Hen and his co-workers having recently stressed the compensatory changes in other systems (e.g., increased dopaminergic activity) observed in these mice (Scearce et al 1997). The effects of 5HT1c and 5HT2 compounds were even more complex. DOI and mCPP both decreased calling, but these drugs also affected activity (mCPP increased and DOI decreased locomotor activity) (Winslow and Insel 1991d). The 5HT2 antagonist ketanserin, which by itself increased calling, appeared to augment mCPP and attenuate the effects of DOI (Winslow and Insel 1991d). It should be noted that these various effects were not unique to 5HT receptor ligands. Several other classes of drugs, including benzodiazepines, opiates, and NMDA receptor antagonists, have been described to selectively reduce the rates of ultrasonic vocalization (Winslow and Insel 1991b). In general, compounds that are effective as anxiolytics are potent for reducing calling rate. The serotonin neurotoxin, 3,4-methylenedioxymethamphetamine (MDMA) has been used to lesion 5HT pathways in rat pups (Winslow and Insel 1991b). Following MDMA administration on postnatal days 1– 4, pups show an enduring decrease of serotonin content, as well as serotonin transporter, in neocortex of roughly 50% when assayed at day 21. Remarkably, these pups grow at a

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Figure 3. Data represent effects of MDMA (repeated 10 mg/kg doses) on the subsequent ultrasonic vocalizations of 9-10 day old pups during 2 min separations from mother and littermates. The left panel represents the consequences of MDMA administration to pregnant females twice daily for 4 days beginning on gestational day 13. The right panel represents the consequences of MDMA administration to pups once or twice per day for 4 days beginning on postnatal day 1. Columns represent means 11 s.e.m. of the rate of calling during a 2 min observation. Asterisks represent significant differences from vehicle control performance determined by Newman-Keuls tests (data from Winslow and Insel 1990b).

normal rate and show normal developmental milestones (e.g., development of fur, day of eye opening, and onset of righting reflex). They also show normal thermoregulatory responses, huddling, and locomotor behavior. However, they show roughly a 75% reduction in the calling rate in response to social separation (Figure 3). The decrease in calling in the presence of reduced 5HT may seem paradoxical, as 5HT1a and 5HT2 agonists also decrease calling rate. One possible explanation is that the effect of released 5HT on vocalization depends on which of the various receptor subtypes is activated. The presynaptic 5HT1b receptors, associated with an increase in calling, might be expected to be destroyed by the lesion of 5HT terminals. Indeed, when MDMA-treated pups were injected with TFMPP, the 5HT1b agonist which increased calling in controls, no change in calling rate was observed. Presumably these receptors are critical for the normal surge in vocalization when a pup is isolated. In summary, rat pup separation calls are decreased by serotonergic lesions, 5-HT1a agonists, and a 5HT2 antagonist, and increased by 5-HT1b agonists. Although these studies cannot demonstrate conclusively that 5-HT is involved in the separation response, studies by Meaney and colleagues provide support for a physiological role. These investigators have demonstrated that daily “handling” of rat pups, defined as separation from mother and placement in a novel cage for 15 min each day, increases serotonin turnover in hippocampus (but not in amygdala or

hypothalamus) (Smythe et al 1994). Handling appears to induce a longterm increase in hippocampal glucocorticoid and 5-HT2 receptors and altered stress responses in adulthood. The serotonergic innervation may have an important role for glucocorticoid receptor expression in the hippocampus (Mitchell et al 1990). Not only does the ontogeny of 5-HT innervation match the development of glucocorticoid binding, but neonatal treatment with the serotonin neurotoxin 5,7-DHT decreases hippocampal glucocorticoid receptors in young rats, and in cultured hippocampal neurons serotonin treatment doubles the number of receptors. Most important, the increase in serotonin turnover with “handling” may be critical for the increase in glucocorticoid receptors. In pups treated with ketanserin at the time of handling, glucocorticoid receptors were not altered (Mitchell et al 1990). Thus, 5-HT release as a consequence of handling (or possibly from subsequent effects on maternal care) may have longterm consequences for hippocampal function, stress responsiveness, and adult behavior. In contrast to the ultrasonic vocalization studies that implicate 5-HT1a receptors, the results with ketanserin suggest that the effects of handling may involve 5-HT2 receptors.

Neuropeptides The opioid peptides were first implicated in the separation response because of the similarity between the behavioral response to withdrawal and the protest response to social separation (Panksepp et al 1978). Indeed, in several species, morphine or mu agonists reduce separation calls (Newman 1988). The question of whether opioids participate physiologically in the separation response has been addressed by administration of opiate antagonist or by selective lesions of mu receptors and the results suggest marked species differences with rat ultrasonic calls not mediated via this receptor whereas calls in non-human primates are increased by naloxone administration (Winslow and Insel 1991a). Presumably these species differences reflect differences in the pattern of receptor expression in the developing brain, but there is, at this time, no clear explanation for these differences in the response to opiate antagonists. Studies using in vivo imaging in primates or humans to localize pathways activated by the separation response still need to be done. Vasopressin and oxytocin are both found early in development, although in the rat oxytocin does not show a fully processed transcript until the postnatal period (Whitnall et al 1985). Receptors for both peptides are found in the developing brain (Shapiro and Insel 1989; Tribollet et al 1989; Tribollet et al 1991). Indeed, there is a transient, but marked “over-production” (relative to the adult) of both oxytocin and vasopressin receptors in limbic brain

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Figure 4. Data represent effects of selected doses of OT and AVP (icv) and their respective antagonists (OTA and V1A) on the ultrasonic vocalizations of 9 –10 day old pups during 2 min separations from mother and littermates. Columns represent means 11 s.e.m. of the rate of calling expressed as % of calling rate in a pre-injection control trial. Asterisks represent significant differences from vehicle control performance determined by Newman-Keuls tests (data from Insel and Winslow 1991; Winslow and Insel 1993).

areas in the first two postnatal weeks (in both rodents and primates). Although these binding sites are particularly enriched in the cingulate cortex (a region implicated in separation distress), we do not know if these putative receptors are functional nor do we know why they disappear by the time of weaning. Exogenous administration of oxytocin or vasopressin reduces the separation response of the rat pup (Figure 4), consistent with the possibility that these binding sites are responsive to their respective peptides and that these peptides have a role in either attachment or the separation response (Insel and Winslow 1991; Winslow and Insel 1993). It might be assumed that oxytocin, which is secreted in high concentration into breast milk (Leake et al 1981), could also quiet the infant by being absorbed through the neonatal gut and transported into brain. This idea seems intuitively appealing, but there are little data to support or refute it. Indeed, administration of a selective antagonist does not appear to increase isolation calls, so the role of either endogenous or maternal oxytocin in the infant remains to be demonstrated (Winslow and Insel 1993). In our most recent studies (Winslow et al 1997), OT knockout mice show a decrease rather than an increase in calling. This paradoxical finding (given that OT administration decreases calling) might suggest that in the absence of OT, pups do not experience social separation as a source of distress. Perhaps the most intriguing evidence implicating oxytocin in the infant’s attachment response comes from studies of olfactory conditioning in rat pups. Nelson and Panksepp have recently reported that oxytocin facilitated a rapid conditioned association to maternal odor cues, but not to non-social stimuli (Nelson and Panksepp 1995). An

oxytocin antagonist actually delayed this form of conditioning. These studies suggest that oxytocin specifically serves a function related to infant attachment to the mother, linking cues in the environment to the memory of mother.

Summary In summary, the vocal response to separation in the rat pup appears to require intact serotonergic pathways. Calls are exquisitely sensitive to both 5HT1a and 5HT1b drugs and lesions that reduce 5HT by 50% appear to decrease isolation calls relatively selectively. Serotonin possibly via the activation of 5HT2 receptors, appears to be part of a cascade of physiological responses to separation with longterm consequences for the stress response and for hippocampal glucocorticoid receptors in the rat. The role of various neuropeptides in the physiological mediation of the rat pup’s vocal response to separation remains to be elucidated and the interaction of 5HT and opiates, OT, or AVP has not yet been studied.

Grooming The Paradigm One of the easiest affiliative behaviors to measure is grooming. Although self-grooming (or autogrooming) is frequently described in neuropharmacologic studies and appears to be increased by 5HT-1c agonists (Bagdy et al 1992), this behavior has little if any relationship to affiliative grooming (or allogrooming), that is, grooming another individual. Allogrooming is observed most easily in non-human primates. In many species, this behavior

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may be the predominant form of socialization (Hinde 1983). Although a human observer might think that a monkey assiduously checking and combing through the fur of another monkey is simply cleaning or delousing a neighbor; allogrooming is, in fact, important for establishing and maintaining alliances and reinforcing the dominance hierarchy irrespective of the presence of parasites. There are clear species differences in the amount of allogrooming and there are, within species, individual differences associated with social status and kinship. Dunbar has suggested that allogrooming is so important for social cohesion among primates that it represents a form of tactile “gossip” that served as an evolutionary precursor to the development of language in hominids (Dunbar 1997). Of course, the measurement of grooming requires that animals be studied in a social environment, ideally in a naturalistic setting. The behavior which appears phenotypically simple is actually complex as there are different “meanings” depending on whether there is a change in the time spent grooming, the time receiving grooming, or the relationship to the groomer. In some species, dominants groom subordinates more, whereas in others, it is the subordinates who do most of the grooming. For the purpose of monitoring affiliation, we assume that an increase in grooming another individual, irrespective of status, means an increased interest in social contact, but this may be an anthropomorphic simplification.

Serotonin Most of what we know about serotonin in non-human primates derives from studies of CSF levels of the metabolite 5-HIAA, an accessible but imperfect marker of CNS serotonin activity (Banki and Molnar 1981). CSF concentrations of 5-HIAA appear to show stable, inter-individual differences which are, in part, heritable (Higley et al 1993). In both human and non-human primates, CSF 5-HIAA has been negatively correlated with aggression or impaired impulse control (less metabolite associated with more impulsive aggression) (Higley et al 1992). More recently, reports in free ranging adolescent male rhesus monkeys have reported that 5-HIAA in CSF is positively correlated with time spent grooming and time spent in close proximity with other members of the social group (Mehlman et al 1995). The correlation between CSF 5-HIAA and time spent grooming was 0.43, but the nature of this relationship (i.e., does grooming increase CSF metabolite concentrations or does the level of metabolite influence dominance status and thereby alter grooming?) is unclear. And the relationship of CSF 5-HIAA (even when sampled from the cisterna magna as in these studies)

to an identified population of serotonin terminals in the CNS remains speculative. Pharmacological studies more directly implicate 5HT in the mediation of grooming behavior. In vervets, levels of 5HT in blood or 5HIAA in CSF have been associated with social status (more metabolite means more likely to be dominant) (Raleigh et al 1983). To understand this relationship better, Raleigh and colleagues removed the dominant male from a social group and treated one of the two remaining males with drugs that non-specifically enhanced serotonergic activity (tryptophan 40 mg/kg/day or fluoxetine 2 mg/kg/day) or reduced serotonergic activity (fenfluramine 2 mg/kg/day or cyproheptadine 60 mg/kg/ day) (Raleigh et al 1991). In a crossover design, they found that in every case (n 5 12), the males treated with the serotonergic enhancing drug became dominant whereas when the same males were treated with the serotonin-reducing drugs, their vehicle treated cagemates became dominant. Thus, in a setting of uncertain social status, serotonergic drugs were able to influence dominance status. What is particularly relevant for the current discussion is that the males did not become dominant via aggression. An analysis of social behavior during the drug treatment phase demonstrated that males treated with tryptophan or fluoxetine achieved dominance by increasing affiliative interactions (including grooming) with females in the group. In primate societies, the dominant male is usually not the biggest but the most politically savvy. Especially in societies where males emigrate (most Old World monkeys), the key to male survival is successful alliances with females. The data from Raleigh et al (1991) are consistent with the hypothesis that increasing serotonergic activity facilitates affiliative gestures in males. Remarkably, a recent report in young adult human volunteers treated with paroxetine suggested increased positive social interaction on problem-solving tasks. The change in affiliative behavior was correlated with paroxetine plasma levels in these subjects (Knutson et al 1997).

Neuropeptides The most compelling work on grooming and neuropeptides also comes from studies in non-human primates. Keverne and his collaborators have studied talapoin monkeys (a highly affiliative species) to investigate the importance of opioid systems for grooming (Keverne et al 1989). When animals were moved from isolation to paired cages, they immediately began reciprocal grooming and this behavior was associated with an increase in b-endorphin in CSF. Interestingly, b-endorphin in the CSF was negatively correlated with social status, so that subordinate males had higher levels. In stable pairs, naloxone (0.5 mg/kg IM) increased grooming invitations nearly 6-fold

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Figure 5. The frequency of affiliative behaviors expressed by subordinate male squirrel monkeys living in a mixed sex triad. Data represent 15 min observations beginning 15 min after intracerebroventricular injection of OT (0.1, 1.0 mg), the OT antagonist OTA (0.5 mg) or a combination of 1.0 mg OT and 0.5 mg OTA (Left Graph). The effects of AVP (0.5, 5.0 mg) are represented in the right graph. Vertical lines at each columns represent 11 s.e.m.; asterisks indicate p , .05 for Dunnetts t comparisons (data from Winslow and Insel 1991c).

and increased the time spent grooming the partner nearly 5-fold. Conversely, morphine (2 mg/kg) at non-sedating doses, decreased grooming by roughly 75% and increased refusals to be groomed. Although there is a report that grooming increases plasma oxytocin concentrations in rats (Stock et al 1988), there has been little direct study of grooming and oxytocin or vasopressin in the CNS. There has been only one study of central administration of oxytocin and vasopressin looking at affiliative behaviors in monkeys (Winslow and Insel 1991c). Squirrel monkeys, a highly social New World species, do not allogroom but they show a number of other “associative behaviors” that differ between dominant and subordinate males. Subordinate males injected icv with oxytocin (1.0 mg) increase their approach and huddle behavior more than 5-fold (Figure 5). Dominant males, with higher baselines of these behaviors, showed an increase in aggression but no change in associative behaviors after this treatment. Conversely, vasopressin (0.5 or 5.0 mg, icv) decreased both aggressive and associative behaviors in both dominant and subordinate males. As dominant squirrel monkeys in this study had plasma testosterone levels that were roughly double the subordinate levels, status related differences in the response to oxytocin may reflect modulation of the brain oxytocin receptors by gonadal steroids.

Summary In summary, allogrooming appears to be a useful index of affiliation, recognizing that its meaning may be somewhat unique to each species and that this behavior is probably of

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greatest importance among primates. Although much of the primate literature on social behavior has heretofore focused on social status, a more strategic view suggests that dominance is achieved by developing associative networks via grooming. Given this complex, interactive view, there is a conspicuous absence of research on the role of grooming in females, with respect to kinship, social status, and serotonin. In males, drugs that enhance serotonin release (and animals with higher 5HIAA) increase grooming and dominance. b-endorphin also has an important relationship to both grooming and dominance, but the relationship of b-endorphin to serotonin remains unclear. This latter point reminds us of a series of recent papers by Ferris and colleagues about another form of grooming, flank-marking, in the golden hamster (Ferris and Delville, 1994; Ferris et al 1997). In this species, flank-marking is a stereotyped behavior that dominant males use to mark their territory. Injections of AVP into the hypothalamus reliably turn on flank marking and increase aggression. An AVP antagonist decreases these behaviors. Ferris and his colleagues have used confocal microscopy to demonstrate serotonin terminals on AVP cells in the target region of the hypothalamus (Ferris et al 1997). With receptor autoradiography they have found 5HT1b receptors in the same region. As fluoxetine (10-20mg/kg) blocked the AVPinduced increase in flank-marking and aggression, they conclude that 5HT via the 1b receptor inhibits fighting by antagonizing the actions of AVP in the hypothalamus.

Pair Bonding The Paradigm The development of adult-adult pair bonds is certainly the least studied form of attachment from a neurobiological perspective. This relative paucity of studies can be attributed to the absence of pair bonds in commonly-used laboratory animals, such as rats and mice. By definition, pair bonds occur in monogamous animals, with approximately 3% of mammals currently considered monogamous (Dewsbury 1988; Kleiman 1977). The percentage of primates that are monogamous is considerably higher (perhaps 15%) (Van Schaik and Dunbar 1990). Studies in non-human primates have provided important insights into both behavioral and endocrine aspects of monogamy (Mendoza and Mason 1986; Snowdon 1990), but research on the neurobiological basis of pair bonding will ultimately require investigations of monogamous non-primate species that can be used for cellular and molecular studies. The prairie vole (Microtus ochrogaster) is a mousesized rodent which lives in burrows across the American midwest. Prairie voles are usually found in multi-generational family groups with a single breeding pair (Getz et al

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1981; Getz and Hofman 1986). They manifest the classic features of monogamy: a breeding pair shares the same nest and territory where they are in frequent contact, males participate in parental care, and intruders of either sex are rejected. Following death of one of the pair, a new mate is accepted only about 20% of the time (the rate is approximately the same whether the male or the female is the survivor) (Getz, et al 1993). Prairie voles also demonstrate a curious pattern of reproductive development: offspring remain sexually suppressed as long as they remain within the natal group. For females, puberty occurs not at a specific age but after exposure to a chemosignal in the urine of an unrelated male (Carter et al 1987). Within 24 hours of exposure to this signal, the female becomes sexually receptive. She mates repeatedly with an unrelated male and, in the process, forms a selective and enduring preference or pair bond (Carter et al 1995). Two aspects of the prairie vole make this species particularly useful for neurobiological investigation. The first is that the highly developed social behaviors described in field studies are also manifest in the lab using either captive or lab-bred animals. In the lab, prairie voles appear highly affiliative, sitting side-by-side more than 50% of the time and attacking adult intruders (Carter et al 1995; Shapiro and Dewsbury, 1990). Both sexes display intense parental care (McGuire and Novak 1984; Oliveras and Novak 1986). Even neonatal prairie voles appear to crave social contact. During a brief social separation, 5 day-old prairie voles emit ultrasonic “distress” calls and secrete corticosterone (Shapiro and Insel 1990). As a second virtue, prairie voles offer the possibility of comparative studies. The closely related montane vole (Microtus montanus) looks remarkably similar to the prairie vole and shares many features of its non-social behaviors, but differs consistently on measures of social behavior (Dewsbury 1988; McGuire 1986). Montane voles are generally found in isolated burrows (in high meadows in the Rockies), show little interest in social contact, and are clearly not monogamous (Jannett 1980). Males show little if any parental care and females frequently abandon their young between 8 and 14 days postpartum (Jannett 1982). In laboratory studies, montane voles spend little time in side-by-side contact, even within the confines of a mouse cage (Shapiro and Dewsbury 1990). As noted above, montane pups at day 5 do not respond to social separation with either isolation calls or corticosterone release, although the pups give both responses to nonsocial stressors (Shapiro and Insel 1990). Prairie and montane voles thus provide an intriguing natural experiment for studying the neural substrates of pair bonding, inviting comparative neuroanatomical, pharmacological, and physiological studies.

Serotonin Thus far, there have not been studies of serotonergic pathways in voles. Although dopamine, working via the D2 receptor appears to be critical for the development of a pair bond (Cascio et al 1998). The role of 5HT remains to be studied.

Neuropeptides Studies of prairie and montane voles have mostly focussed on central pathways for oxytocin and vasopressin. These species differ in the neural distribution of receptors for both peptides as much as they differ in behavior (Insel and Shapiro 1992; Insel et al 1994). These receptors show similar binding characteristics (in terms of kinetics and specificity) in the two species, but the receptors are expressed within entirely different pathways (Figure 6). Recent studies have demonstrated virtually identical cDNAs in both species for the oxytocin and the vasopressin receptor– demonstrating that these species share the same receptor, but differ in its regional expression (Young et al 1996; Young et al 1997). This species difference in regional receptor distribution indicates that different brain areas are responding to these peptides and thus, the central effects of oxytocin and vasopressin should be quite different in prairie and montane voles. For instance, in the prairie vole, oxytocin receptors are found in brain regions associated with reward (nucleus accumbens and prelimbic cortex), suggesting that oxytocin might have reinforcing properties selectively in this species. Conversely, receptors in the lateral septum, found only in the montane vole, might be responsible for the effects of oxytocin or vasopressin on self-grooming, an effect that is observed in the montane vole but not the prairie vole. Of course, such differences in chemical neuroanatomy may be unrelated to the patterns of social organization which first drew our attention to these animals. However, three observations suggest that the differences in receptor distribution may be related to the species differences in social behavior. First, other vole species (pine voles and meadow voles) selected for analogous differences in social organization (i.e., monogamous versus non-monogamous) manifest similar differences in receptor distribution for both oxytocin and vasopressin (Insel and Shapiro 1992; Insel et al 1994). Second, the findings with oxytocin and vasopressin receptors appear relatively specific; the patterns of binding for mu opiate receptors and benzodiazepine receptors (two systems previously implicated in the mediation of social attachment) do not differ across the four vole species (Insel and Shapiro 1992). Finally, after parturition, when the female montane vole becomes briefly parental, the pattern of oxytocin receptor binding

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Figure 6. Autoradiograms generated by binding of 1251-OTA (oxytocin antagonist) and 125I-sarcAVP (vasopressin V1a receptor antagonist) to sagittal sections from adult male prairie and montane voles. Dark areas are regions of specific binding. Note high levels of OT receptor binding in prairie vole prelimbic cortex (PL), anterior olfactory nucleus (AOP), and nucleus accumbens (NAcc), whereas montane vole binding is most intense in lateral septum (LS), accessory olfactory bulb (AOB), and ventromedial nucleus of the hypothalamus (VMN). For V1a receptor binding, prairie vole brain shows high levels in thalamus (Th) and olfactory bulb (OB); whereas montane vole brain shows high levels in lateral septum and superior colliculus (SC). (Reprinted with permission from Insel 1997.)

changes to resemble the pattern observed in the highly parental prairie vole (Insel and Shapiro 1992). Ultimately however, the anatomic evidence remains correlational. While the data are consistent with a role for either oxytocin or vasopressin in the behavioral differences observed in prairie and montane voles, these descriptive experiments fail to address the central question: is either peptide involved in pair bonding? Research with several other species has demonstrated that both peptides are released with mating (Hughes et al 1987; Kendrick et al 1988; Murphy et al 1987) and mating appears important for pair bonding in the prairie vole (Williams et al 1992). But, this does not demonstrate that either peptide is either necessary or sufficient for pair bonding. To address the role of these peptides directly, we have applied two operational measures of pair bonding. The first measure examined a partner preference using a choice test. It was assumed that formation of a pair-bond requires the formation of a preference for the mate over a stranger. Indeed, in tests of this assumption, prairie vole females reliably chose to sit next to their mates while female montane voles were equally likely to sit with a novel male as with the male with whom they had mated. This “partner preference” is enduring and reciprocal. Prairie voles continue to show a preference for their mates even after weeks of separation and this preference can be detected in both males and females (Insel et al 1995; Insel and Hulihan 1995). A second test of bonding was derived from the

observation that male prairie voles become highly aggressive after mating. This aggression is selectively directed at intruders (never the mate) and appears to serve the function of mate guarding (Insel et al 1995). Curiously, the aggression persists for at least a week, even in the absence of the mate, and continues to be expressed towards both adult males and females for several months if the mate remains present (Winslow et al 1993). Montane voles do not show this induction of aggression after mating, further supporting the validity of this measure as an indicator of pair bonding (Winslow et al 1993). If mating facilitates pair bond formation and oxytocin is released with mating, does oxytocin influence the development of the pair bond? As shown in Figure 7, oxytocin (but not vasopressin) given centrally (icv) to females facilitates the development of a partner preference in the absence of mating (Insel and Hulihan 1995; Williams et al 1994). A selective oxytocin antagonist given icv prior to mating blocks formation of the partner preference without interfering with mating (Insel and Hulihan 1995). This effect appears specific: neither CSF nor a vasopressin antagonist given in an identical fashion blocks partner preference formation. Presumably, the oxytocin antagonist prevents the binding of oxytocin to its receptor and thereby blocks the behavioral consequences of mating. These results suggest that oxytocin released with mating is both necessary and sufficient for the formation of a pair-bond in the female prairie vole. Essentially, female

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Figure 7. Mating facilitates partner preference formation in prairie voles, an important step towards pair bonding. Partner preference is assessed during three hours in a three cage apparatus where test animal is placed in a central cage with free access to the partner or a stranger, each of which is tethered in an adjacent cage on opposite sides of the central cage. If the test animal spends more than twice as much time with the partner than the stranger, we score this as a partner preference (shown as dark bar). If the test animal spends twice as much time with the stranger, this is scored as stranger preference (white bar). If the test animal spends roughly equal time with both partner or stranger or less than 10 minutes with either, the score is no preference (hatched bar). In both males and females, mating for 14 hours induces a partner preference whereas unmated animals did not exhibit a preference unless (A) treated with oxytocin if female or (C) treated with vasopressin if male. (B) If females were treated with an oxytocin antagonist prior to mating, mating did not confer a partner preference. (D) Males treated with vasopressin V1a receptor antagonist prior to mating also did not exhibit a partner preference. All drugs were icv, agonists by minipump infusion over 18 hours and antagonists by single point injection (data from Insel and Hulihan 1995; Winslow et al 1993). (Reprinted with permission from Insel 1997.)

prairie voles given the oxytocin antagonist resemble montane voles–they mate normally but show no lasting interest in their mate. What about the male in this process? Males show a partner preference and increased aggression after mating, but it is not oxytocin but vasopressin that is critical. A vasopressin antagonist administered centrally (icv) to male prairie voles prior to mating blocks the development of both the partner preference and the selective aggression (Winslow et al 1993). As with females, the antagonist does not interfere with mating, rather it appears to block the consequences of mating. Moreover, the antagonist is not anti-aggressive, per se. Males treated after mating show no

reduction of attack behavior (Winslow et al 1993). An oxytocin antagonist has no effects on these behaviors, suggesting that the vasopressin effects are specific. Vasopressin may also be sufficient for male pair bonding. When males are not permitted to mate, but are exposed to ovariectomized females, they show neither the partner preference nor the selective aggression. However, when vasopressin is given centrally, in the absence of mating, males show both measures of bonding–the partner preference and the aggression (Winslow et al 1993). Oxytocin given in the same fashion has no effect on these measures (Figure 5). In short, monogamous voles show different patterns of oxytocin and vasopressin receptor distribution in brain and both peptides appear to be important for pair bond formation, a process that occurs in monogamous animals. Recent data on the molecular structure of oxytocin and vasopressin receptors has provided a model for the evolution of monogamy (Insel et al 1997). It is remarkable that the two peptide systems have been adapted for different roles in male and female prairie voles. Apparently, pair bonding in male and female voles activates two different, albeit closely related neural systems.

Summary The comparative vole model has proven ideal for investigating the molecular and cellular aspects of pair bonding. The results support an important role for OT and AVP receptors in a species in which mating is integral to pair bond formation. Recent results from our lab suggest that the interaction of OT and dopamine during mating is critical for the conditioning to a social partner that permits the female prairie vole to form a longterm attachment to her mate. While 5HT has not been studied thus far, the intimate relationship of 5HT terminals and AVP perikarya in the hamster hypothalamus recommend the importance of investigating this interaction in the development of male vole aggression and partner preferences. A working (albeit simplistic) hypothesis at this point is that pair bonding in the female will involve OT and D2 receptors via estrogen dependent mechanisms whereas in the male, AVP and 5HT will interact in an androgen dependent fashion.

Conclusions Studies of affiliation in non-human mammals suggest the importance of serotonin, both in development (the rat pup isolation call) and in adulthood (monkey allogrooming). Neuropeptides, especially opiates, OT, and AVP are candidates for influencing complex social behaviors, possibly

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through an interaction with serotonergic pathways in the hypothalamus as shown for hamster flank marking. What does any of this have to do with humans who don’t normally emit ultrasonic calls, allogroom, or even predictably pair bond after mating? In autism and type II schizophrenia, the absence of affiliation is a prominent feature. Especially in autism, there is a deficit in social interest which can be manifested by a decrease in separation distress, an absence of reciprocal social interaction, and an inability to form social bonds (Bristol et al 1996). Our studies comparing prairie voles and montane voles highlight the risks of extrapolating across species, but if we can understand the basic organization of neural systems for affiliation in rodents and non-human primates (including the neurochemical ways in which species with different forms of social organization differ), then we should know how to approach the affiliative deficits in disorders such as autism and schizophrenia. From what we know currently, serotonin, opiates, OT and AVP are all excellent candidates to begin to look for functional genetic polymorphisms in synthetic and receptor pathways (and, of course, transporters for 5HT), post-mortem changes in regional expression, and even differences in response to challenge in patients. This work was presented at the Neuroscience Discussion Forum “A Decade of Serotonin Research” held at Amelia Island, Florida in November 1997. The conference was sponsored by the Society of Biological Psychiatry through an unrestricted educational grant provided by Eli Lilly and Company.

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