There are also more subtle forms of sensitive periods in social development, in which experience with caregivers early in life shapes later social relationships (35–38). Across taxa, isolation from conspecifics and caregivers results in significant disruptions to social functioning later in life (39–42). Furthermore, research in rats has demonstrated that early experiences of maternal care (e.g., licking and grooming behaviors) can alter both the responsiveness to stressors and maternal behavior in adulthood (37, 43, 44). Song learning in birds and vocal learning in humans, though they occur later in development, are also examples of sensitive periods in development in which the developing organism is dependent on interactions with adult caregivers to learn speciestypical vocal structures (45, 46).

What appears to be common across these different kinds of sensitive periods in development is competitive exclusion—a particular class of sensory input from the environment is particularly influential, to the exclusion of others (47). These early forms of social learning, particularly about the identity, features, and valence of caregivers, provides an important foundation for later learning. Both learning the characteristics of and maintaining the motivation to be proximal to caregivers provide developing organisms with food and protection but also an abundance of opportunities for social learning. Indeed, these early social experiences provide the foundation upon which all future social interactions are built.

In each of these cases, we have a sense of what circuits are involved in the sensitive periods (as well as a number of ways to disrupt those circuits), but critically we do not know the mechanisms of how or why there is a particular kind of sensitivity in the first place. For example, the filial imprinting work in birds has focused on forebrain regions thought to be homologous to mammalian cortex which are involved in visual processing and multimodal association (24–26, 48). But why those particular inputs at that particular time?

#### Established Mechanisms Underlying Sensitive Periods

One theory about how sensitive periods emerge is based on the observation that the sensory systems do not all become functional at the same time, but rather sequentially. In both birds and mammals, the first sensory modality to become functional is the tactile/vestibular system, followed in order by the chemosensory/ olfactory, auditory, and visual systems (49, 50). Based on the pioneering work of Gilbert Gottlieb in avian development, Turkewitz and Kenny proposed that the invariance in the sequential onset of sensory function results in a reduction in the complexity of the sensory experience for the developing organism and a more reliable structure to the prenatal and early postnatal experience (51). Developing organisms are not bombarded with novel stimuli in all sensory modalities upon birth, but instead encounter a drastically reduced sensory experience. Earlier-developing systems (i.e., tactile and olfactory) in fact develop under reduced competition from other sensory modalities. This allows later-developing systems (i.e., visual and auditory) to build on associations formed in earlier-developing systems.

Furthermore, given that many sensory systems begin to develop prior to birth or hatching, the *in utero* and *in ovo* environments provide learning opportunities for the developing embryo. Precocial birds, which are born with all sensory systems functional at birth, benefit from extensive learning that takes place in the egg which they use to support the "emergence, maintenance, and transformation of behavior" (52). For example, in ducks, the preference for the maternal assembly call is dependent upon the prenatal exposure of the embryo either to its own vocalizations or those of its siblings in the days prior to hatching (53). Similarly, bob white quail denied interaction with broodmates after hatching fail to develop preferences for species-specific maternal cues (54). Even altricial rodents, which are less mature at birth, also use olfactory associations formed *in utero* to perform suckling behaviors (55).

In addition, generalized physiological arousal has been identified as a critical component of a young organism's perceptual learning and development. In human infants, for example, there is a strong association between arousal levels and sensitivities to sensory stimulation (56–58). Physiological arousal can be manipulated neurochemically, or by simply making sensory stimuli more salient. For example, only rat pups receiving either tactile stimulation or injected with amphetamine while exposed to an artificial odor preferred to suckle nipples coated in the familiar odor (59). Furthermore, this process can be disrupted by a poorly timed change in arousal state. Injection of norepinephrine into quail embryos in the absence of exposure to appropriate auditory stimulation resulted in disrupted preference for the familiar maternal call (60). This work suggests that normal social development depends on physiological systems that mediate arousal and attention in the appropriate social environments early in life.

#### NEUROENDOCRINE SIGNALS AS A POTENTIAL MECHANISM UNDERLYING SENSITIVE PERIODS

I propose that neuroendocrine mechanisms are also prime candidates for mediating sensitive periods in development. Hormones, which are typically defined as long-distance chemical signals, act directly on the cellular processes of neurons, but they also affect more general physiological systems, such as arousal, gonadal state, and metabolic function. Hormones influence multiple tissues simultaneously and modulate physiological and developmental processes across a wide spatial and temporal distance (61). This enables organisms to simultaneously coordinate many tissues or recruit whole neural circuits for an important task (62). Indeed, hormonal signals can provide a functional link between otherwise unconnected neuronal populations (63).

Most of the developmental effects of hormones have been studied in the context of steroid hormones and sexual differentiation (64–68). The focus of this work has been on how the hormones directly affect cellular function and the connectivity of neural circuits. However, many neuroendocrine signals have the potential to play a role in the organization of the social brain specifically by altering learning processes. Glucocorticoids, sex steroids, and neuropeptides have all been shown to be involved in learning and memory, both directly and indirectly (69). Nevertheless, there remains a gap in our understanding the role that such signals play in influencing the outcome of development in the context of important social experiences. Furthermore, the diversity of social phenotypes both within and between species begs the question as to how the unique features of both the organism's early social experiences, as well as evolved differences in their neuroendocrine function, support the evolution and development of novel social phenotypes. For the purposes of this review, I focus on vasotocinfamily neuropeptides, but many of the general principles of my argument may apply to other neuroendocrine signals, as well.

#### Overview of the Nonapeptides

Over the last several decades, much research effort has been devoted to vasotocin family of neuropeptides (i.e., nonapeptides), which includes [arginine vasotocin (AVT), found in nonmammals and likely the ancestral peptide] and its mammalian homolog AVP; and the OT-like peptides [isotocin (IT), found in fish, mesotocin (MT), found in lung fish and non-eutherian tetrapods, including birds; and OT, found in mammals] (70, 71). The nonapeptides derive from an evolutionarily ancient neuromodulator. In the earliest vertebrates, only one gene was present (AVT), but sometime after *Agnatha* (lampreys and hagfish) a gene duplication event led to the divergence of the vasotocin (AVT/AVP) and OT (IT/MT/OT) lineages (72). Although these two lineages differ in only a single amino acid, the nonapeptides appear to have evolved quite distinct functions.

This is because the nonapeptides coevolved with their receptors, which are classic G-protein coupled receptors. There are typically four receptor subtypes for the nonapeptides within each species: V1a, V1b, V2, and OT (VT4, VT1, VT2, and VT3, respectively, in birds). The amino acid sequences of each receptor subtypes are more similar to each other across species (~90%) than they are to different subtypes within a single species (~45%) (73). When binding to their receptors, nonapeptides can have a multitude of effects on neurons, including changes to gene transcription, recruitment of intracellular calcium, neuroprotective effects, and alterations to long-term potentiation mechanisms (74). The sequencing of the vertebrate nonapeptide receptor genes suggests that the core–ligand receptor interaction sites have remained remarkably conserved, while varying the intracellular components, and thus their downstream effects (75). Receptors for nonapeptides are distributed throughout the brain, but importantly, the distribution of each of the receptor subtypes can vary widely by species, sex, age, and social context (76–78).

The primary sources of nonapeptides are the AVP/OT cell groups of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) nuclei of the hypothalamus, as well as smaller extra-hypothalamic accessory cell groups, including the medial amygdala (MeA), medial bed nucleus of the stria terminalis (BSTm), lateral septum (LS), olfactory bulb (OB), and suprachiasmatic nucleus (SCN) (79, 80). The production of AVT/AVP, particularly in from the extra-hypothalamic cell groups, is often sexually dimorphic (usually male greater than female), organized by sex steroids during development, and sensitive to changes in gonadal state (81–88).

In order to understand the modulatory role of AVT/AVP during development, we need to consider the sources of the peptide, the sites of action in the body, and the functional consequences. There are three primary physiological systems influenced by AVT/AVP. The first can be summarized as AVT/AVP's involvement in vasoconstriction and water balance. AVT/AVP, when released by magnocellular neurons in the PVN and SON of the hypothalamus, is released into the posterior pituitary. From there, AVT/AVP enters general circulation where exerts antidiuretic effects throughout the body.

The second is AVT/AVP's involvement in the stress response. AVT/AVP, when it is released from the anterior pituitary by parvocellular neurons in the PVN, is at the top of the hypothalamic– pituitary–adrenal (HPA) axis. The HPA axis regulates the physiological response to stressors, helping the body mobilize resources in response to challenges in its environment. AVT/ AVP, along with corticotropic releasing factor (CRF) serves as a releasing hormone for adrenocorticotropin-releasing hormone (ACTH) from the anterior pituitary, which is the chemical signal that leads to the release of glucocorticoids from adrenal tissue (89). AVT/AVP is not itself the major releasing hormone for ACTH, but it plays a critical role by potentiating the biological activity of CRF (90). Parvocellular AVT/AVP neurons are highly responsive to stress (91). Acute stress increases the production of both CRF and AVT/AVP in the PVN (92, 93).

Finally, multiple cell groups in the brain contribute to the central pool of nonapeptides with highly diverse functional consequences. Some of the same neurons that project into the pituitary also send projections back into the brain. In addition, the extra-hypothalamic accessory cell groups, including the MeA, BSTm, LS, OB, and SCN, contribute to the central pool of peptides (79, 80). Each of the AVT/AVP cell groups has a different pattern of activity and neural release, which is ultimately a function of the kinds of computation those neurons perform (87). Variation in how the nonapeptides affect the interconnected set of brain nuclei known as the social behavior network and other brain regions is thought to underlie the multiple effects of AVT/AVP across species (94, 95). Indeed, nonapeptides have been implicated in species differences in many diverse social behavioral domains (76, 77, 96–104).

#### Nonapeptides in Development

The vast majority of this comparative work, however, has focused on nonapeptide function in adulthood. We in fact understand remarkably little about how nonapeptides shape social behaviors during development, particularly those behaviors for which plasticity, flexibility, and learning are critical. The effect that nonapeptides have on social learning in each species is influenced by when they act relative to important social experiences and in what brain regions. Across the few vertebrate species in which it has been investigated, the sequence of nonapeptide cell group maturation appears to be conserved. The first AVT/AVP immunostaining is consistently found in the SON followed by the PVN (105–112). In tetrapods, this is followed by production in the extra-hypothalamic cell groups, such as the BSTm and MeA, which exhibit steroid hormone-mediated sexual dimorphism in AVT/AVP staining (81, 113–115).

The most detailed developmental work in the nonapeptide system comes from rodents, particularly the rat. In rats, the neurons of the SON and PVN have formed before birth by 12–14 days postfertilization (dpf), gestation is 21 days in the rat (108). In the rat brain, the first AVP staining is observed between 14–18 dpf, which steadily increases to adult levels by postnatal day 30 (108, 113). Between birth and postnatal day 21, there is a 22- to 30-fold increase in AVP production by the pituitary, suggesting that the neurons that project from the hypothalamus to the pituitary are gradually coming on-line during development. By contrast, the cell groups of the BSTm and MeA show AVP staining only after birth, with the MeA delayed relative to the BSTm. AVP mRNA was only observed in the BSTm on postnatal day 3 and in the MeA on day 5 in male rats and day 14 and day 35, respectively, in female rats (113). The levels of AVP, thus, reach adult levels by postnatal day 35 in the BSTm and day 60 in the MeA in both sexes (113).

A similar developmental trajectory is found in the domestic chicken, despite substantial evolutionary distance and its precocial development. AVT is observed early in development in the chicken embryo SON and PVN, as early as 6 dpf (109–112). AVT is detectable in the BTSm by 12 dpf, which increases until hatching at 17 dpf before dropping precipitously in days after hatching (114). AVT then increases gradually in males until 129 dpf (114). Thus, the onset of function of all the cell groups in the SON, PVN, and BTSm occur while the chicken is still in the egg.

Very limited information on the development of these cell groups exists from other species, but the sequential development of the hypothalamic cell groups appears similar. In humans, AVP is detectable in the SON and PVN at 77 and 91 dpf, respectively (107, 116). Thus, in humans, hypothalamic production of AVP begins before birth. Even in zebra fish, two cell groups in the rostral diencephalon and hypothalamic regions show AVT mRNA expression sequentially starting at 24 h postfertilization (106). The embryonic development of the nonapeptide system has not been explored in songbirds. An early paper that explored the development of the nonapeptide circuitry in canaries found that AVT was expressed in the PVN at 4 weeks, but no staining was observed in the BSTm or LS until later at 8–12 weeks of age (117).

Taken together, these data suggest that the relative timing of the onset of function of the AVT/AVP cell groups is, in fact, remarkably conserved throughout evolution. Furthermore, the sequence of general neurodevelopmental events (from neurogenesis to eye opening) is also very predictable (118). What changes more is the timing of birth or hatching and, thus, early social experiences relative to these ontological changes. Inspired by Workman et al., **Figure 1** depicts the development of the nonapeptide system scaled according to when the brain reaches 20% total brain volume, a milestone that is highly correlated with other neurodevelopmental events (118). This is based on data from three species for which we have some information about the development of the nonapeptide system: rats, humans (SON and PVN only), and chickens. The fact that neurodevelopmental events occur in a highly stereotyped sequence allows us to make predictions about the maturation of the nonapeptide system for cell groups or other species for which we do not have data, as well. **Figure 1** shows the predicted timing of AVT synthesis in the respective cell groups in the zebra finch brain, based on general data on zebra finch neurodevelopment. Data supporting the figure can be found in **Tables 1** and **2**.

A few notes of caution are required. First, it is important to note that these predictions are still quite speculative, given the available data. The Workman et al. model has not been applied to avian systems, so the extent to which we can extrapolate the

Figure 1 | Comparative timeline of arginine vasotocin (AVT)/arginine vasopressin (AVP) cell group development. Conceptual timeline illustrating the production of AVT/AVP across early development in rats, humans, chickens, and zebra finches in three main AVT/AVP cell groups: the supraoptic nucleus (SON, blue), paraventricular nucleus (PVN) of the hypothalamus (PVN, orange), and the medial bed nucleus of the stria terminalis (BSTm, green). The *x*-axis of the timeline is scaled to the neurodevelopmental time point of when the organism is estimated to reach 20% total adult brain volume (indicated by the gray dotted line intersecting each timeline). Days postfertilization (dpf) are indicated by hatchmarks along each timeline. Solid lines indicate solid data (see text for references), whereas dotted lines are predicted results. The data from which this illustration is created include counts of AVT/AVP immunoreactive cells, mRNA expression, and peptide concentrations (see Table 1). Thus, the *y*-axis does not have a scale, as it is not clear how these different data types are comparable across species, cell group, study, etc. The date of hatching or birth for each species is indicated by a red rectangle. Date of eye opening is indicated by an asterisk.

*The type of evidence for each time point is indicated below. If available, we use data from the labeling of the AVT peptide, from either immunohistochemistry (IHC) labeling cell bodies and radioimmunoassay (RIA) from brain and pituitary. In some cases, only data from in situ hybridization (ISH) labeling AVT gene expression is available. Sex differences are indicated, where noted. Cell group abbreviations: supraoptic nucleus (SON) of the hypothalamus, paraventricular nucleus (PVN) of the hypothalamus, medial bed nucleus of the stria terminalis (BSTm), and medial amygdala (MeA).*

Table 2 | Data are presented for 20% brain volume, eye opening, amygdala neurogenesis peak, and the date of birth or hatching.

*Asterisks indicate that the date is predicted based on the model in Ref. (118). DPF, days postfertilization.*

findings to chickens and zebra finches is still unknown. More information about when key neurodevelopmental events occur in the zebra finch brain may also help to determine whether the maturation of the AVT system would be more similar to the altricial but distantly related rat or the precocial but more closely related chicken.

Second, **Figure 1** was created using available data, which is a mix of immunohistochemistry (IHC) labeling of AVT or AVP protein and *in situ* hybridization (ISH) labeling AVT/AVP gene expression (see **Table 1**). It is also based on labeling of these in the cell bodies within the respective brain regions. AVT/AVP is first synthesized as a large protein precursor molecule, which is enzymatically cleaved into the active hormone (80, 128). The active hormone must then be packaged into specialized neurosecretory vesicles and transported to the nerve terminals where it is released. Thus, the presence of either ISH or IHC labeling in the cell bodies is not a definitive indicator that the hormone is being released, particularly during development (105).

Third, we have limited information about where nonapeptides are acting during development. Binding sites have been found in the developing mouse and rat brain in both the amygdala and septum between postnatal days 0 and 8, as well as several brain regions where AVP receptors are not expressed in adulthood, including the hippocampus, dentate gyrus, and caudate nucleus (129, 130). In rats, many brain regions also show significant differences in between juveniles and adult (131). The consequences of these brain-region specific changes in receptor expression across development are almost certainly important, but have proven difficult to explore experimentally.

Finally, sex differences in both AVT/AVP production and receptor expression also likely influence developmental processes. For example, there is a delay in AVT/AVP synthesis in females relative to males in the BSTm and MeA (113, 115). Given these differences, we might predict that males would be more affected by AVT/AVP manipulations or by manipulations earlier in development, as compared to females. Some sex differences in receptor expression have also been found, but we have a poor understanding of the functional consequences of these differences.

Nevertheless, manipulations of nonapeptides in rodents very early in life provide evidence that nonapeptides matter in development (6, 7). For example, vasopressin–deficient Brattleboro rat pups show hyperactivity, reduced huddling, and reduced proximity to other pups in the nest compared to wild-type rats (132). Wild-type rat pups treated with a nine-day exposure to AVP showed increased emotionality, activity levels, and grooming in an open field test as juveniles, as well as smaller overall brain size (133). Acute central administration of AVP in wild-type neonatal rat pups was found to decrease the number of ultrasonic vocalizations and reduced locomotor activity in a maternal isolation test (134). In juvenile male rats, both targeted infusion of AVP into the LS and intracerebroventricular infusion increased preference for investigating novel individuals, whereas a V1aR antagonist increased the preference for investigating familiar individuals (135). In addition, V1aR blockade in the LS increased social play behavior in males and decreased it in females, but only when it tested in a familiar environment (136, 137). Neonatal manipulation of AVT or OT in the socially monogamous prairie vole, leads to sex-specific changes in nonapeptide binding in several brain regions in adults and alterations to social behaviors (138–141). For example, sexually naïve males who were treated with AVP early in life were more aggressive than control males but females were less responsive to AVP treatment (140). It is unclear whether the developmental effects of these nonapeptide manipulations are mediated through physiological effects on the body versus binding within the brain. Nevertheless, these studies provide intriguing evidence that the nonapeptides are involved in the development of social behavior.

#### CONSERVATION AND NOVELTY

Several researchers have proposed that one way to understand the outsize role of neuropeptides such as AVT/AVP in the evolution of behavioral diversity is through studying the differential expression of the peptides and their receptors (142, 143). The evolution of neuropeptide signaling systems may be highly constrained within the nervous system because mutations altering the proteins themselves would have deleterious effects on the receptor–ligand interactions. However, tweaks to where and in what quantities the peptides and their receptors are expressed via changes to gene regulation provide opportunities to modify the activity of neural circuits (142). Indeed, a number of examples have been identified. For example, variation in the production of AVT has been associated with species differences in social behaviors in birds (144). The number of IT-producing cells in the POA has been associated with cooperative breeding in cichlid fish (103). And the expression of V1aR is associated with variation in mating behaviors in voles (76, 145, 146). However, perhaps an important mechanism underlying this behavioral diversity across species is not just where but also *when* nonapeptides are acting during development.

#### Sensorimotor Processing Hypothesis

Much of the research for the role of nonapeptides in social behavior has focused on brain regions with more generalized roles in social processing. However, the "compartmentalization" of AVT/AVP function can extend to include the modulation of species-specific behavioral circuits throughout the central nervous system (142, 147). In their sensorimotor hypothesis, Rose and Moore posit that AVT/AVP can act on sensory pathways to modulate the responsiveness of neurons to particular kinds of sensory stimuli as well as act on motor pathways to modulate behavioral output (147). In this case, the nonapeptide signal is directly modulating the specific circuits that are necessary for the production of the behavior. This theory emerged from comparative work which suggests that AVT modulates the activity of neurons in each step of a sensorimotor processing circuit which controls a complex courtship behavior in male newts (*Taricha granulosa*) (147).  AVT enhances this behavior by modulating sensory processing in the visual and olfactory domains as well as motor output at the level of the spinal cord (149–151).

A parallel story may also be true in the case of complex learned behaviors, such as bird song. Interestingly, there is limited evidence that anterior forebrain song learning pathway is sensitive to AVT, at least in adults AVT (88, 152–154). However, each step in the circuit controlling the expression of vocal behavior in birds appears to be partially modulated by AVT (152, 154, 155). Several auditory structures in the forebrain, including the caudomedial mesopallium and the caudomedial nidopallium, highly express V1aR in zebra finches (154). The robust nucleus of the arcopallium (RA, homologous to laryngeal motor cortex) exhibits limited receptor expression, but two nuclei involved in the motor pathway of song production contain AVT receptors. There is AVT immunoreactivity and binding in the intercollicular nucleus (ICo, a region implicated in vocal control) in several songbird species (152, 153, 156, 157). In addition, the key motor nucleus, nXIIts, which innervates the syrinx and is considered part of the song system, contains high levels of mRNA for all three subtypes of AVT receptor (154). However, a sensorimotor account of the role played by these regions during song learning remains to be tested.

#### Social Gating Hypothesis

Evolutionary novelty in behavior may also arise when new structures or circuits are modulated in new ways starting early in development. Syal and Finlay claim that what is necessary for the evolution of novel behaviors is changes to how the sensory and motor circuits are attached to the socio-motivational circuitry during early social interactions with caregivers, family, and conspecifics (158). In fact, they view the reciprocally connected network of brain nuclei known as the social behavior network (which includes the major nonapeptide cell groups), as the conserved neural structure that assembles the relevant sensory dimensions of a representation of other individuals (i.e., caregivers, mating partners, rivals) and attaches that representation to motivations and actions appropriate to their social context (158). The social behavior network is highly connected to the mesolimbic reward system via the BSTm, MeA, and LS, which all contain nonapeptide cell groups. Other structures commonly associated with reward and motivation, such as the ventral tegmental area, ventral pallidum, and nucleus accumbens, also densely express receptors for nonapeptides (159).

In this context, the modulatory signal produced by the nonapeptide cell groups, by acting on receptors throughout the brain, can be used to bias attention toward certain kinds of sensory stimuli or to reward the performance of certain behaviors. Consequently, it is easy to imagine how even tiny tweaks to the system, such as gene mutations that change the regulation of a receptor gene or slightly alter its downstream functions, might have large effects on whole neural circuits. Thus, the nonapeptide system may provide a mechanism whereby evolution generates novel social behavior using an otherwise highly conserved brain.

The effect of changes to the nonapeptide system would, thus, be expected to be even more consequential in development, particularly when coupled with salient social experiences. If indeed nonapeptides are gating social learning, then the nonapeptides may function by biasing a young organisms' attention toward the behaviors exhibited by their family or other socially relevant conspecifics. For example, a primary reason why the development of the nonapeptide system may underlie important social development is because of its central role in olfactory processing. Early social experiences are often highly olfactory and thermotactile in nature. For example, suckling behavior in rat pups is dependent upon odor processing in the accessory olfactory system and MeA, which allows them to learn the odor of their mother's amniotic fluid and of the saliva of their broodmates to guide nipple attachment (160). Even zebra finches, which do not have an accessory olfactory system and which are thought to be more responsive to auditory and visual stimuli, show olfactory preferences for their natal nest (161). Thus, early olfactory experiences provide some of the first forms of social learning about conspecifics at the same time that the relevant behavioral circuits begin responding to nonapeptides.

Increased attention to relevant social stimuli would provide opportunities for social learning, which could also be reinforced by socio-motivational circuits. Early sensitivity to social stimuli would support future social learning, leading to accumulating effects. On the other hand, genetic mutations that reduce social approach or attention during development might reduce the probability that predictable aspects of the social environment are learned at all. Of course, these kinds of effects depend critically on the kinds of input that an organism receives from its environment during these social interactions. It is possible to think of the organizational effects of nonapeptide circuitry independent of its social environment, but more likely, the kinds of social experiences an organism has—and their sensitivity to those social experience—coevolved with each other. Neuropeptide systems during development may have, thus, evolved to allow organisms to plastically respond to their environment as they mature. However, by allowing for variable outcomes in adulthood, these evolvable systems also provide the raw material for evolution to act.

#### DEVELOPMENTAL EFFECTS OF AVT ON AFFILIATION AND SONG LEARNING IN THE ZEBRA FINCH

Recent experimental evidence from manipulating the nonapeptide system early in life in zebra finch provides support for the idea nonapeptides play an organizational role in on a broad suite of social behaviors (162–164). Intracranial injections of either AVT or [Manning Compound (MC), a V1aR antagonist] in hatchlings (days 2–8 post-hatching) altered social interest in the parents and conspecifics after fledging, suggesting that the nonapeptides are serving to gate a number of social approach behaviors in juvenile zebra finches (162). In addition, early life nonapeptide treatment also altered affiliative behaviors and courtship song in adult male, but not female, zebra finches. Both AVT and Control males showed an increased affiliative interest in females as they reached reproductive maturity (162). However, AVT-treated males showed less sexually motivated courting of females compared to Controls and instead formed highly affiliative pair bonds with their female partner (163). By contrast, MC males did not show the normal increase in affiliative interest in females as they reached maturity and showed only modest levels of both courtship and affiliation in their interactions with females (162, 163). Furthermore, nonapeptide treatment also altered neural activity and the expression of V1aR in the BSTm and MeA (163). Taken together, these findings suggest that AVT-injected males may have had more experience attending to social cues or a stronger association between affiliative interactions and reward compared to both MC and Control males, resulting in different approaches to reproduction.

This change in the affiliative interest in parents and conspecifics also had functional consequences for social learning. Male zebra finches injected with MC as hatchlings both showed decreased interest in their parents during development and ultimately sang a song that was a worse acoustic match to their father's song in adulthood compared to Controls (162, 164). By contrast, AVT males showed increased affiliative interest in their parents and family and more effectively copy their father's song (162, 164). Interestingly, affiliation with parents at 30 days post-hatch was correlated with song quality in adulthood. These data suggest that the nonapeptides may bias the motivation of developing zebra finches to attend to the behaviors of the father during development, which ultimately allows them to more accurately learn courtship song from their father. This is ultimately consistent with Syal and Finlay's hypothesis that the nonapeptides gate complex vocal learning in song birds by altering social motivation, supporting their suggestion that the nonapeptides may play an equally critical role in language learning in humans (158).

Thus, social phenotypes may evolve via relatively simple alterations to the actions of a single nonapeptide during development. In zebra finches, AVT altered early social behaviors, potentially affecting the opportunities for social learning. However, it also affected the organization of the neural substrate underlying these social behaviors. It will likely prove impossible to disentangle the direct effects of nonapeptides on the brain during development from their indirect effects resulting from how they alter the trajectory of learning from early social experiences. Indeed, this conceptual challenge is at the heart of the nature "versus" nurture debate (4).

### CONCLUSION

Nevertheless, these results provide support for the idea that the actions of nonapeptides in development may play an important role in the evolution of novel social behavior. The field of evolutionary developmental biology (evo-devo) has long been concerned with how evolution shapes developmental processes to generate phenotypic novelty. However, the insights from evo-devo have rarely expanded into the social domain (165–167). Neuropeptides and hormonal systems are well-situated to play that role, given that they alter the activities of whole neural circuits. However, we are just scratching the surface in our understanding of the diversity of mechanisms which may facilitate the evolution and development of social behaviors.

Indeed, the nonapeptides are almost certainly not the only chemicals that play a role in the evolution of diverse social phenotypes. We now know of more than 100 different peptides and other signaling molecules, each of which is expressed in only a small population of neurons, and all of which signal to neurons throughout the brain via specific receptors. The endless forms of neural systems and behavior appear to be result of evolutionary changes to compartmentalization of neuropeptide signaling systems (142). However, the complex nature of diverse signaling systems suggests that they can only be fully understood by integrating research at all levels of analysis—investigating both their molecular and developmental mechanisms, as well as their adaptive significance in the life of an organism.

#### AUTHOR CONTRIBUTIONS

NB wrote the article as part of her dissertation work at Cornell University.

### ACKNOWLEDGMENTS

I am grateful for the feedback of E. Adkins-Regan and M. H. Goldstein on this manuscript, as well as their support and insight throughout my graduate research.

### REFERENCES

### FUNDING

This research was supported by an NSF Doctoral Dissertation Improvement Grant (NSF, IOS – 1310908); NSF, IOS – 1146891; and NIH Training Grant 5T32HD055177-05.

(*Taricha granulosa*) revealed by immunohistochemical and in situ hybridization techniques. *J Comp Neurol* (1997) 385:43–70. doi:10.1002/(SICI)1096-9861(19970818)385:1<43:AID-CNE3>3.0. CO;2-C

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Baran. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Triarchic Psychopathy Dimensions in Chimpanzees (Pan troglodytes): Investigating Associations with Genetic Variation in the Vasopressin Receptor 1A Gene

Robert D. Latzman<sup>1</sup> \*, Steven J. Schapiro2, 3 and William D. Hopkins 4, 5

<sup>1</sup> Department of Psychology, Georgia State University, Atlanta, GA, United States, <sup>2</sup> Michael E. Keeling Center for Comparative Medicine and Research, University of Texas MD Anderson Cancer Center, Bastrop, TX, United States, <sup>3</sup> Department of Experimental Medicine, University of Copenhagen, Copenhagen, Denmark, <sup>4</sup> Neuroscience Institute, Georgia State University, Atlanta, GA, United States, <sup>5</sup> Division of Developmental and Cognitive Neurosciences, Yerkes National Primate Research Center, Atlanta, GA, United States

#### Edited by:

Heather K. Caldwell, Kent State University, United States

#### Reviewed by:

Gábor B. Makara, Institute of Experimental Medicine, Hungarian Academy of Sciences, Hungary Jacques Epelbaum, Institut National de la Santé et de la Recherche Médicale, France

> \*Correspondence: Robert D. Latzman<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience

Received: 28 May 2017 Accepted: 30 June 2017 Published: 17 July 2017

#### Citation:

Latzman RD, Schapiro SJ and Hopkins WD (2017) Triarchic Psychopathy Dimensions in Chimpanzees (Pan troglodytes): Investigating Associations with Genetic Variation in the Vasopressin Receptor 1A Gene. Front. Neurosci. 11:407. doi: 10.3389/fnins.2017.00407 Vasopressin is a neuropeptide known to be associated with the development and evolution of complex socio-emotional behaviors including those relevant to psychopathic personality. In both humans and chimpanzees, recent research suggests a strong genetic contribution to individual variation in psychopathic traits. To date, however, little is known concerning specific genes that might explain the observed heritability of psychopathy. In a relatively large sample of captive chimpanzees (N = 164), the current study thus sought to investigate gene-environment associations between triarchic psychopathy dimensions (i.e., disinhibition, meanness, and boldness) and (1) early social rearing experiences and (2) polymorphisms in the promoter region of the V1A receptor gene (AVPR1A). Among chimpanzees raised by their biological conspecific mothers, AVPR1A was found to uniquely explain variability in disinhibition and in sex-specific ways for boldness and a total psychopathy score; however, in contrast, no significant associations were found between AVPR1A and any of the triarchic psychopathy dimensions in chimpanzees raised the first 3 years of life in a human nursery. Thus, when considered in its entirety, results suggest an important contributory influence of V1A receptor genotype variation in the explanation of the development of psychopathy under some but not all early rearing conditions. Results of the current study provide additional support for the assertion that psychopathic tendencies are rooted in basic, evolutionarily-meaningful dispositions, and provide support for a primate-translational operationalization of key neurobehavioral constructs relevant both to psychopathy and to broader forms of psychopathology.

Keywords: vasopressin, AVPR1A, psychopathy, chimpanzees, nonhuman primate models

### INTRODUCTION

Psychopathic personality (psychopathy) is a condition that involves severe disturbance in behavioral control, social relations, and emotional experiences concealed by an outward appearance of normalcy. Although historically studied predominantly in adult forensic samples, it has become clear that psychopathy is a multi-faceted condition that includes tendencies grounded in basic

biobehavioral dispositions that vary continuously within the human population (Patrick et al., 2009; Lilienfeld et al., 2015). That is, individuals vary on psychopathic traits in degree, rather than kind. Viewed in this way, understanding of psychopathy can be advanced through study of psychopathy-related trait dimensions in a range of populations, including both clinical and non-clinical samples (Lilienfeld, 1994; Hall and Benning, 2006; Salekin, 2006). Consistent with this conceptualization, recent work has sought to more accurately capture the dimensions of the construct, through the explication of its component dispositional trait dimensions (e.g., Patrick et al., 2009; Marcus et al., 2011; Poythress and Hall, 2011). Developed for this purpose, the triarchic model (Patrick et al., 2009) characterizes psychopathy as a configuration of three dimensional traits explicitly linked to underlying biological systems: boldness, meanness, and disinhibition.

Within this framework, investigations of these biobehavioral dimensions have been extended to our closest living relatives, chimpanzees (Latzman et al., 2016a), providing a basis for comparative research on the evolutionary and neurobiological foundations of psychopathy. Similar to in humans (Farrington, 2006; Larsson et al., 2006; Tuvblad et al., 2016), recent quantitative genetics work in chimpanzees suggests that variability in psychopathy dimensions is heritable (Latzman et al., 2017). To date, however, little is known concerning specific genes that might explain the heritability of psychopathy. In a relatively large sample of chimpanzees, the current study thus aimed to examine one particularly promising gene, AVPR1A, a gene that underlies arginine-vasopressin (AVP), a neuropeptide known to associate with a range of psychopathy-relevant social behaviors.

The now influential triarchic model of psychopathy (Patrick et al., 2009; Patrick and Drislane, 2015) characterizes the symptomatic components of psychopathy in terms of three biobehavioral trait constructs (i.e., traits with clear referents in biology and behavior): disinhibition, meanness, and boldness. Disinhibition reflects an externalizing liability and phenotypic propensity toward impulse control problems. These problems include a lack of planfulness and foresight, difficulties regulating affect and urges and delaying immediate gratification, and deficient behavioral restraint. Meanness corresponds to the callous aggression subdimension of the externalizing spectrum of psychopathology and includes deficient empathy, disdain for and lack of close relationships, exploitativeness, rebelliousness, excitement seeking, and empowerment through cruelty (Krueger et al., 2007). Lastly, boldness encompasses low levels of fear/avoidance (Kramer et al., 2012), expressed as a capacity to remain calm in situations involving threat, an ability to recover quickly from stressful events, high self-assurance and social efficacy, and an easiness with unfamiliarity and danger (Lilienfeld et al., 2012, in press).

As noted above, Latzman et al. (2016a) developed a chimpanzee operationalization of psychopathic personality organized around the triarchic model. Specifically, drawing on caretaker-rated items from an existing primate personality instrument, Latzman et al. used a consensus rating approach to formulate scale measures of the three triarchic model constructs for use with chimpanzees. These Chimpanzee Triarchic (CHMP-Tri) scales were then validated both in terms of their associations with performance on behavioral tasks and their translational relevance to humans. As noted by Latzman et al. (2016a), it is important to note that this model was not developed to derive some ways of characterizing some chimpanzees as "psychopaths" in a clinical way nor was it to imply that chimpanzees can be psychopaths. Rather, the goal was to evaluate the triarchic model from a comparative and evolutionary standpoint.

Results from this work indicate that the triarchic model of psychopathy can be operationalized effectively in chimpanzees, an animal species uniquely well-suited for neurobiological investigations of individual variation in broad, transdiagnostic biobehavioral traits (Latzman et al., 2016b). Such an approach is particularly opportune given the National Institute of Mental Health's (NIMH) research domain criteria (RDoC; Insel et al., 2010; Kozak and Cuthbert, 2016) initiative, encouraging investigators to consider psychopathology in terms of neurobehavioral dispositions. Indeed, as described previously (i.e., Latzman et al., 2016a) the dimensional constructs of the triarchic model can be viewed as trait-dispositional counterparts to RDoC constructs (Yancey et al., 2016).

It has long been theorized that psychopathy has heritable biological foundations (e.g., Karpman, 1946; Lykken, 1995), and an accumulating empirical literature supports the idea that genetic influences contribute to variance in psychopathic personality tendencies (Waldman and Rhee, 2006). Indeed, a replicable human literature has reported appreciable heritabilities for psychopathic tendencies (e.g., Blonigen et al., 2005, 2006; Viding et al., 2005; Brook et al., 2010; Bezdjian et al., 2011; Tuvblad et al., 2016). Taken together, the available research literature with humans clearly indicates an important contribution of genes to psychopathic tendencies.

Using the CHMP-Tri model, the finding of significant heritabilities of psychopathic tendencies has recently been confirmed in chimpanzees (i.e., Latzman et al., 2017). Consistent with findings in humans, results indicate significant genetic contributions to individual variability in psychopathic tendencies. Further, within the population of apes included in this study, some were raised by their biological mothers, whereas others were raised by humans for the first 3 years of life in a nursery. As described in more detail below, this quasi-experimental manipulation allowed for the explicit consideration of early social rearing experiences on estimates of heritability. When examined separately by early rearing background, consistent with previous findings for general personality dimensions (Latzman et al., 2015), the heritability of psychopathy dimensions varied by early social learning experiences: Whereas all three triarchic dimensions showed significant heritability among mother-reared participants, heritability was not evident for any dimension in the nurseryreared subsample (Latzman et al., 2017). All told, the existing literature, for both human and chimpanzee samples, provides clear evidence of a genetic contribution to psychopathy. To date, however, little is known concerning the specific genes associated with psychopathy.

Genes that underlie arginine-vasopressin (AVP), a phylogenetically conserved neuropeptide, constitute a particularly promising candidate, given the role of AVP in a range of complex social behaviors in both humans and nonhuman animals (Donaldson and Young, 2008). For example, within a sample of patients meeting diagnostic criteria for various personality disorders, cerebrospinal fluid AVP levels were found to correlate with a history of aggression (Coccaro et al., 1998). In addition to associations with direct measures of AVP, converging findings suggest an association between AVP receptor polymorphisms and a range of psychopathy-relevant social behaviors. For example, AVPR1A, the vasopressin V1A receptor gene, has been shown to be related to several social behaviors including pair bonding, territoriality, and aggression among voles, particularly males (e.g., Young and Wang, 2004; Hammock and Young, 2005, 2006). With regard to primates specifically, studies with humans have suggested an association between a similar repetitive element in the AVPR1A promoter and relevant social behaviors including altruism (Wassink et al., 2004) and pair bonding relationships (Walum et al., 2008). Further, with regard to personality traits in humans, AVPR1A promoter polymorphisms have been found to be associated with variability in a number of psychopathy-relevant traits (Patrick and Drislane, 2015) including increased novelty seeking, decreased harm avoidance (Walum et al., 2008) and increased reward dependence (Bachner-Melman et al., 2005).

Many of the association studies reviewed above have focused on the RS3 polymorphic repetitive element. In humans, the RS3 repeat region is housed within a larger, ∼350 bp tandem duplicated region. The first of these duplicated regions, DupA, spans −3730 to −4074 bp relative to the transcription start site and contains a GT20−<sup>26</sup> microsatellite, known as STR1. The second duplicated region, DupB, spans −3382 to −3729 bp and contains the complex microsatellite, RS3 ((CT)6−14(GT)8−24). In chimpanzees, approximately 65% of the AVPR1A alleles have a complete deletion of the DupB region, resulting in a 357 bp difference between the DupB<sup>+</sup> and DupB<sup>−</sup> alleles (Donaldson et al., 2008). As a result of the complete deletion of RS3 in some individuals, chimpanzees are a uniquely valuable animal species for assessing the potential effect of the RS3 polymorphic repetitive element on individual variability in neurobehavioral dispositional dimensions such as those aspects described in the triarchic model.

In chimpanzees, a small but generally consistent literature suggests an association between AVPR1A and personality. For example, Hopkins et al. (2012) found that traits of Dominance and Conscientiousness were associated with polymorphic variation in AVPR1A, particularly among males. Latzman et al. (2014) have reported similar findings with AVPR1A associated with factor-analytically derived Disinhibition and Dominance constructs. Similar to Hopkins et al., these associations were found to vary by participant sex. Staes et al. (2015) also reported converging results. Specifically, Staes et al. found sexspecific AVPR1A associations with behavioral observations of sociability. Further, findings of associations between AVPR1A and chimpanzee personality have also been reported by Wilson et al. (2017) who found AVPR1A to be associated with Conscientiousness and Extraversion, although not in sex-specific ways. All told, AVPR1A appears to be a promising candidate gene for research investigating the genetic basis of interpersonal dispositional traits, such as those described by the triarchic model of psychopathy.

In addition to unique AVPR1A polymorphisms, as described in more detail below, chimpanzees in the current study were raised in different rearing environments early in life. In both human and nonhuman animals, the genetic contribution to particular traits likely depends on distinct factors in the environment, resulting in the relevance of genes in some environments but not in others (Charmantier and Garant, 2005; Rutter et al., 2006). That is, the genetic contributions to various outcomes may differ depending on the environment. Across human (i.e., Moffitt et al., 2006), monkey (i.e., Suomi, 2011), and chimpanzee (i.e., Latzman et al., 2015, 2017) samples, research has suggested an interactive contribution of early adversity and genetic variation to a broad range of outcomes. Research in nonhuman animals (i.e., Charmantier and Garant, 2005), including chimpanzees (Latzman et al., 2015, 2017), has revealed similar variability in heritability estimates as a function of differences in early adversity. Indeed, as described above, recent CHMP-Tri findings suggest that the heritability of psychopathy dimensions varies by early social rearing experiences (Latzman et al., 2017). Given these existing lines of evidence, the role of early social rearing experiences is important to consider.

The overarching aims of the current study were to investigate the effects of DupB genotype on triarchic psychopathy dimensions and whether these effects appear sex-specific. Although no studies to date have investigated AVPR1A RS3 polymorphisms and psychopathy specifically, molecular genetic studies on the association between other specific candidate genes and psychopathy-related behaviors and traits suggest an important moderating role for early adversity in moderating associations (e.g., Capitanio et al., 2006; Kim-Cohen et al., 2006; Buckholtz and Meyer-Lindenberg, 2008; Karere et al., 2009). Similar findings have been shown among chimpanzees with regard to heritability estimates of psychopathy traits (Latzman et al., 2017). Specifically, whereas all psychopathy dimensions were found to be heritable among mother-reared apes, none of the heritability estimates were significant among nursery-reared apes. We thus decided to examine associations separately by early rearing experience.

Given previous findings of significant associations between AVPR1A and Conscientiousness, Dominance and Disinhibition (e.g., Hopkins et al., 2012; Latzman et al., 2014), we expected genotypic variability to relate as well to the CHMP-Tri dimensions, in distinct ways. Specifically, given previous chimpanzee findings with regard to Disinhibition (Latzman et al., 2014) and Conscientiousness (Hopkins et al., 2012), and clear links between triarchic disinhibition and each of these two constructs, we expected significant associations to emerge between AVPR1A genotype and CHMP-Tri Disinhibition, particularly for males. Specifically, we predicted that DupB+/<sup>−</sup> males would show lower scores on this triarchic trait dimension. Further, given previous findings of lower levels of Dominance (Latzman et al., 2014), along with higher levels of anxiety-related behaviors (i.e., scratching, Latzman et al., 2016b; Mahovetz et al., 2016) for DupB+/<sup>−</sup> males, we predicted that DupB+/<sup>−</sup> males would score lower on CHMP-Tri Boldness, a triarchic disposititon associated with low threat-sensitivity. We did not advance any a priori hypotheses regarding CHMP-Tri Meanness. Whereas, previous human findings of an association between AVPR1A genotype and Dictator game performance (Knafo et al., 2008) are suggestive of an association for meanness, prior work with chimpanzees has reported no association between AVPR1A variability and scores on Agreeableness, the personality trait most strongly associated with triarchic meanness (Patrick and Drislane, 2015). Finally, given previous findings of a genetic foundation for a higher-order psychopathic personality dimension in both humans (e.g., Larsson et al., 2006) and chimpanzees (i.e., Latzman et al., 2017), we investigated the association between DupB genotype and a total CHMP-Tri score. Importantly, given previous findings of significant genetic contributions to CHMP-Tri scales among mother- but not nursery-reared chimpanzees, we expected to find significant AVPR1A effects only in the mother-reared sample.

#### MATERIALS AND METHODS

#### Participants

Chimpanzees were members of two genetically distinct colonies of apes housed at the Yerkes National Primate Research Center (YNPRC) in Atlanta, Georgia and at the National Center for Chimpanzee Care (NCCC) at The University of Texas MD Anderson Cancer Center in Bastrop, Texas. Participants for the current study included 82 adult and sub-adult chimpanzees at YNPRC, including 57 females and 25 males ranging in age from 9 to 53 years (Mage = 22.15, SD = 8.96) and 96 adult and subadult chimpanzees at NCCC, including 46 females and 50 males ranging in age from 8 to 41 years (Mage = 22.88, SD = 6.12). After removing 14 chimpanzees for whom AVPR1A data were not available (e.g., blood sample were not available, DNA yield was not sufficient) participants from both colonies were combined for analyses resulting in a final sample of 164 chimpanzees.

Early rearing experiences varied among individuals, with 119 being mother-reared and 59 human nursery-reared. Motherreared chimpanzees remained under the care of their mothers for at least 2.5 years of life and were raised in "nuclear" family groups of chimpanzees, with group sizes ranging from 4 to 20 individuals. Nursery-reared chimpanzees were separated from their mothers within the first 30 days of life, due to unresponsive care, injury, or illness. These chimpanzees were placed in incubators, fed standard human infant formula, and cared for by humans until they could care adequately for themselves, at which time they were placed with other infants of the same age until they were 3 years old (Bard et al., 1992; Bard, 1994). At 3 years of age, the nursery-reared chimpanzees were integrated into larger social groups of adult and sub-adult chimpanzees.

It should be noted that all of the nursery-reared chimpanzees were raised in this manner to protect the infants' well-being. That is, the chimpanzees in this study were not nurseryreared by design, with the goal of subsequently determining the effects of early life experiences on development. The data for these subjects are therefore ex post facto and opportunistic; indeed, we capitalized on the fact that some of the chimpanzees received different rearing experiences in order to evaluate whether this might have long-term consequences on personality development. Importantly, as described previously (Bogart et al., 2014; Latzman et al., 2017), based on the composition of the rearing groups, potential rearing differences are not conflated with familial environment. That is, group membership reflects early experiences, rather than familial aggregation of group placement decisions. As reported previously by Latzman et al. (2017) with regard to relatedness within each rearing group, 52 different sires and 79 different dams contributed to the mother-reared group, and 34 different sires and 42 different dams contributed to the nursery-reared group. Further, as described in detail previously, the genetic diversity within each group was comparable suggesting that group membership reflects early experiences rather than familial aggregation of group placement decisions (see Latzman et al., 2017). The full pedigree structure for this sample has been described previously (see Hopkins et al., 2014b).

All aspects of the research complied with the American Psychological Association's Guidelines for Ethical Conduct in the Care and Use of Nonhuman Animals in Research (American Psychological Association, 2012), followed the Institute of Medicine (US) and National Research Council (US) Committee on the Use of Chimpanzees in Biomedical and Behavioral Research (2011) guidelines for research with chimpanzees, and was done with the approval of the Institutional Animal Care and Use Committees of the universities at which the research was conducted. All chimpanzees are housed in social groups ranging from 2 to 16 individuals in indoor-outdoor compounds, with free access to both portions of their enclosures 24 h a day. During the winter, the indoor facilities are heated, while air conditioning or fans and misters are provided in the hotter summer months. Lighting in the outdoor facility follows the typical seasonal cyclic change in sunrise and sunset. Standard tungsten lighting is provided in the indoor facility and the lights are on a 12 h on-off cycle. The chimpanzees are fed two to five times per day with a diet that consists of fruits, vegetables, and commercially produced primate chow. In addition, they receive a number of foraging and enrichment opportunities each day. Environmental enrichment, such as simulated tool use tasks or non-nutritive substrates, is provided to the chimpanzees on a daily basis. At no time are apes food- or water-deprived.

#### Assessment of Triarchic Psychopathy Dimensions

Chimpanzee Triarchic (CHMP-Tri) scales previously developed through a consensus-based approach (Latzman et al., 2016a) were used in the current study. Consistent with the triarchic model of psychopathy in humans, the three CHMP-Tri scales assess Boldness (6-items), Meanness (5-items), and Disinhibition (7 items). As described by Latzman et al. (2016a), chimpanzees were rated by colony-staff members; typically two to three independent raters, who had worked with the animals for an extended period of time and reported having "enough experience for an accurate rating" (Freeman et al., 2013, p. 1044), rated each chimpanzee. Items for each scale were rated using a 7-point Likert-type format, with response options ranging from 1 (least descriptive of the chimpanzee) to 7 (most descriptive of the chimpanzee). Internal consistencies (Cronbach's alpha) for the three scales have been shown to be acceptable, especially considering their brevity: 0.82 for Boldness, 0.77 for Disinhibition, and 0.67 for Meanness (Latzman et al., 2016a).

#### DNA Extraction, Genotyping and Analysis

As described previously (e.g., Donaldson et al., 2008), DNA samples were isolated from buccal swabs or blood samples using Puregene DNA purification system (Gentra, Minneapolis, MN, USA). Following extraction, stock DNA was separated into three aliquots: one for onsite storage at −80◦C, one for offsite storage, and a working stock for genotyping. Samples were tracked via a secure Filemaker Pro 8 database that linked sample codes for each aliquot, demographics for each subject, DNA quantification and purity analysis results, and genotype data.

Each individual was genotyped for the AVPR1A DupA/B region using the primers and conditions reported in previous studies (e.g., Hopkins et al., 2012; Latzman et al., 2014). Briefly, we used forward primer 5′ -GCATGGTAGCCTCTCTTTAAT and a reverse primer of 5′ - CATACACATGGAAAGCACCTAA with an annealing temperature of 57◦C for 30 cycles: 95◦C, 5 min; 30 × (95◦C, 30 s; 57◦C, 30 s; 72◦C, 3 min; 72◦C, 10 min; 4◦C, hold). Polymerase chain reaction (PCR) amplification was undertaken using the Epicentre Failsafe kit using premixH (Illumina Inc., Madison, WI, USA) according to the manufacturer's directions. Genotyping was performed in a volume of 20 µl containing 20 ng target genomic DNA. PCR products were resolved on a 2% agarose gel (SeaKem Agarose LE, Lonza, Basel, Switzerland) at 100 V for 45 min with a 100 bp DNA ladder (New England Biolabs, Ipswich, MA, USA) in tris-borate-EDTA (TBE). The DupB-containing allele resulted in a band of ∼900 bp, while the DupB minus allele was ∼570 bp long, and genotypes were visually assigned (Donaldson et al., 2008). All genotypes were run in duplicate with gel analysis and were checked before the data set was finalized. Forty-three males and 64 females were homozygous for the short allele (DupB−/−) and 26 males and 31 females had the long allele (DupB+/−), yielding overall genotype frequencies of 65.2 and 34.8%. These frequencies are consistent with those previously identified in wild-caught chimpanzees (Donaldson et al., 2008). Further, as reported in previous studies, the AVPR1A genotype distribution in these two colonies of apes does not deviate from Hardy-Weinberg equilibrium (Hopkins et al., 2014a).

#### Data Analysis

To examine associations between CHMP-Tri dimensions and AVPR1A DupB genotype, we used multivariate analysis of variance (MANOVA). Specifically, we used CHMP-Tri scores as the dependent variables and sex as a fixed factor, and, given the age range of the sample, we included age as a covariate. The potential moderating role of sex was examined for a number of reasons. In addition to vasopressin systems in the brain being (1) found to be sexually dimorphic and (2) thought to regulate social behaviors in sex-specific ways (De Vries et al., 1981), investigations in chimpanzees of associations between AVPR1A and general dispositional traits have resulted in sexuallydimorphic results (e.g., Hopkins et al., 2012; Latzman et al., 2014, 2016b). Given prior work with chimpanzees demonstrating important moderating impacts of early rearing experience on the heritability of personality broadly (i.e., Latzman et al., 2015), and psychopathy more specifically (i.e., Latzman et al., 2017), analyses were conducted separately for mother- and nurseryreared chimpanzees.

To confirm that associations between AVPR1A DupB genotype and CHMP-Tri scores were a direct reflection of AVPR1A rather than a result of all shared genes, a series of post-hoc analyses were performed in the Sequential Oligogenic Linkage Analysis Routines (SOLAR; Almasy and Blangero, 1998) program. Specifically, as described in detail previously (see Fears et al., 2009, 2011), and consistent with recent quantitative genetic work on CHMP-Tri scales (Latzman et al., 2017), SOLAR uses a variance components approach that relies on maximum likelihood estimation to compute a polygenic variance term for a dependent measure of interest (i.e., CHMP-Tri scores) when considering the entire pedigree. To determine the contribution of AVPR1A DupB genotype explicitly, we included AVPR1A and AVPR1A<sup>∗</sup> sex as covariates in the polygenic models for each CHMP-Tri score found associated with AVPR1A in our MANOVA analyses. The contribution of genotype and genotype<sup>∗</sup> sex to the explanation of the CHMP-Tri scores independent of genetic relatedness was evaluated by testing the statistical significance of their associations within the full model.

#### RESULTS

#### AVPR1A Variation and Chmp-Tri Psychopathy Dimensions

Within the mother-reared sample, a significant main effect was found for AVPR1A[F(3, 104) = 8.78, p < 0.001, η<sup>p</sup> <sup>2</sup> = 0.10] in the prediction of CHMP-Tri scores. Subsequent univariate F-tests revealed a significant main effect association for DupB genotype with CHMP-Tri Disinhibition [F(1, 106) = 6.66, p = 0.01, η<sup>p</sup> <sup>2</sup> = 0.06]. Mean standardized CHMP-Tri Disinhibition scores in DupB−/<sup>−</sup> and DupB+/<sup>−</sup> are shown in **Figure 1**. Specifically, across sexes, Dup+/<sup>−</sup> apes scored significantly higher in disinhibitory tendencies than DupB−/<sup>−</sup> apes. By contrast, no direct effects between the DupB genotype and either CHMP-Tri Boldness [F(1, 106) = 2.75, p > 0.10, η<sup>p</sup> <sup>2</sup> = 0.03] or Meanness emerged [F(1, 106) = 1.28, p > 0.25, η<sup>p</sup> <sup>2</sup> = 0.01].

However, a significant two-way interaction between AVPR1A and sex [F(3, 104) = 4.87, p < 0.01, η<sup>p</sup> <sup>2</sup> = 0.12] was found in predicting CHMP-Tri scores as a whole.  Mean standardized CHMP-Tri Boldness scores in DupB−/<sup>−</sup> and DupB+/<sup>−</sup> males and females are shown in **Figure 2**. Whereas, female Dup−/<sup>+</sup> apes evidenced higher scores on this trait dimensions, male Dup−/+ apes showed lower scores on CHMP-Tri Boldness. No significant effects for the AVPR1A<sup>∗</sup> sex interaction term were found in predicting CHMP-Tri Meanness [F(1, 106) = 2.71, p > 0.10, η<sup>p</sup> <sup>2</sup> = 0.03] or Disinhibition [F(1, 106) = 0.51, p > 0.45, ηp <sup>2</sup> = 0.01].

Within the nursery-reared sample, no significant main effects [F(3, 46) = 0.06, p > 0.95, η<sup>p</sup> <sup>2</sup> = 0.004] or interactions [F(3, 46) = 0.16, p > 0.90, η<sup>p</sup> <sup>2</sup> = 0.01] were found. No predictive effects were found for any individual CHMP-Tri scale (all Fs < 0.20, ps > 0.65).

#### AVPR1A Variation and Total CHMP-Tri Psychopathy

Finally, the association between DupB genotype and total CHMP-Tri psychopathy was investigated. Within the motherreared sample, whereas no significant main effect for AVPR1A emerged [F(1, 106) = 0.83, p > 0.35, η<sup>p</sup> <sup>2</sup> = 0.01], a significant twoway interaction between AVPR1A genotype and sex [F(1, 106) = 7.13, p < 0.01, η<sup>p</sup> <sup>2</sup> = 0.06] was found in the prediction of total CHMP-Tri scores. Mean standardized CHMP-Tri total scores in DupB−/<sup>−</sup> and DupB+/<sup>−</sup> are shown in **Figure 3**. Whereas, female DupB−/<sup>+</sup> apes evidenced lower scores, male Dup−/<sup>+</sup> apes showed higher scores on CHMP-Tri total score; the opposite was true for DupB−/<sup>−</sup> apes. Within the nursery-reared sample, no

FIGURE 3 | Mean CHMP-Tri total scores (± SE) for mother-reared males and females with DupB+/<sup>−</sup> and DupB+/<sup>−</sup> genotypes. <sup>n</sup> <sup>=</sup> 29 DupB−/<sup>−</sup> males and 48 DupB−/<sup>−</sup> females. <sup>n</sup> <sup>=</sup> 16 DupB+/<sup>−</sup> males and 18 DupB+/<sup>−</sup> females.

significant main effects [F(1, 53) = 0.03, p > 0.85, η<sup>p</sup> <sup>2</sup> = 0.001] or interactions [F(1, 53) = 0.03, p > 0.85, η<sup>p</sup> <sup>2</sup> = 0.001] were found.

### Contribution of AVPR1A and AVPR1A∗Sex in Polygenic Models

As described above, to confirm the robustness of our MANOVA findings within the mother-reared group, tests of AVPR1A and AVPR1A<sup>∗</sup> sex as potential covariates within full polygenic models were next run. Consistent with MANOVA findings, these post-hoc analyses suggested a significant specific effect for AVPR1A DupB genotype for CHMP-Tri Disinhibition explaining 8.56% of the proportion of variance (p = 0.01). Further, also consistent with MANOVA findings described above, AVPR1A<sup>∗</sup> sex evidenced a significant effect for both CHMP-Tri Boldness explaining 8.32% of the variance (p = 0.0009) and CHMP-Tri Total score explaining 5.86% of the variance (p = 0.005).

#### DISCUSSION

The current study represents the first investigation to date of the association between variation in AVPR1A, a gene that underlies AVP, and psychopathy tendencies as a function of differences in early rearing experiences. Consistent with expectations, AVPR1A DupB genotype was found to explain variability in psychopathy dimensions uniquely and in sex-specific ways. Results underscore the translational value of a nonhuman primate model for investigating psychopathy dimensions and provide strong support for the notion of triarchic psychopathy dimensions as biologically-based and evolutionarily derived (Patrick et al., 2009; Latzman et al., 2016a).

#### AVPR1A and Triarchic Psychopathy

Consistent with previous findings in both humans and chimpanzees with regard to psychopathy-related behaviors and traits, results suggest a potentially important etiological pathway from AVP to triarchic psychopathy, at least for mother-reared apes. Specifically, in line with previous findings of higher scores on the personality traits of Disinhibition/low Conscientiousness (Hopkins et al., 2012; Latzman et al., 2014), traits linked to triarchic disinhibition (Patrick and Drislane, 2015), DupB+/<sup>−</sup> apes evidenced lower scores on CHMP-Tri Disinhibition in the current sample. In contrast to previous findings of this association being male-specific, however, this association did not vary by sex in the current study. Contrary to both expectations and previous findings for related traits and behaviors, DupB+/<sup>−</sup> was found to be associated with higher CHMP-Tri Boldness scores for males. This is surprising for a number of reasons, including previous findings of higher levels of anxiety-related behaviors (i.e., scratching, Latzman et al., 2016b; Mahovetz et al., 2016) for DupB+/<sup>−</sup> males and the contention that triarchic boldness reflects the phenotypic expression of low threat-sensitivity. It will thus be important for future research to more explicitly investigate this finding. Finally, total CHMP-Tri psychopathy was found to associate with AVPR1A in sex-specific ways. Importantly, in a series of post-hoc analyses, the robustness of findings and their specificity to AVPR1A rather than all shared genes, were confirmed through a series of polygenic models in which genetic relatedness among apes were considered. All told, although not entirely consistent with expectations, the current findings in the mother-reared sample suggest an important role for AVP on variation in psychopathy dimensions.

In direct contrast to findings among mother-reared chimpanzees, and consistent with recent biometric results (Latzman et al., 2017), none of the psychopathy dimensions were found to associate with the AVPR1A RS3 polymorphism in the nursery-reared sample, providing evidence of different etiologies as a function of rearing. These findings are not only consistent with biometric results in chimpanzees, but also with accumulating evidence in the human (e.g. Moffitt et al., 2006; Rutter et al., 2006) and nonhuman animal literatures (e.g., Charmantier and Garant, 2005; Karere et al., 2009) for variations in the effects of genes as a function of environmental context. Importantly, whereas in humans it is quite difficult to disentangle etiological influences due to confounding of environmental and genetic influences, findings for our chimpanzee sample are less likely to reflect this confound. Indeed, as described previously (i.e., Bogart et al., 2014; Latzman et al., 2017), although offspring in each of the two early rearing groups were not entirely heterogeneous, the degree of genetic diversity was comparable between them.

#### Triarchic Model, AVP, and the NIMH Research Domain Criteria (RDoC)

A notable feature of the dispositional constructs of the triarchic model is that they are framed explicitly in neurobiological terms (Patrick et al., 2009; Patrick and Drislane, 2015). As mentioned at the outset, an analysis of psychopathy in terms of neurobehavioral dispositions is apt, given the NIMH's RDoC initiative (Insel et al., 2010; Kozak and Cuthbert, 2016), which endeavors to explicate the neurobiological bases of mental illness and reframe conceptions of psychopathology around constructs with specific brain referents. The RDoC research framework specifies biobehavioral constructs, grouped within major domains of functioning, as explanatory referents for understanding clinical problems—and encourages investigation of these constructs using measures from multiple assessment domains ("units of analysis"). Clear counterparts to the triarchic model dimensions exist within the RDoC framework. Indeed, Disinhibition fits within the RDoC construct of "response inhibition" within the Cognitive Systems domain; boldness fits within the construct of "acute threat" in the Negative Valence Systems domain; and meanness fits within the construct of "affiliation and attachment" in the Social Systems domain. The dimensional constructs of the triarchic model can thus be viewed as trait-dispositional counterparts to these RDoC constructs (Yancey et al., 2016).

The direct relevance of the current research to the RDoC initiative is further bolstered by the consideration of relations between these three biobehavioral phenotypes and AVPR1A. Indeed, the vasopressin system is explicitly referred to as a suggested unit of analysis across a variety of RDoC domains and constructs, including within the Negative Valence System, Social Processes, and Arousal and Regulatory Systems Domains. For example, vasopressin is thought to relate to the Acute Threat ("Fear") construct within the Negative Valence System, a construct with direct links to boldness in the triarchic model.

Although the current study focused specifically on psychopathy subdimensions described within the triarchic model and associations with the AVPR1A RS3 polymorphism, taken together with a growing body of research (i.e., Latzman et al., 2016b), results from the current study provide clear support for primate-translational operationalizations of specific constructs within the RDoC framework. Despite recent decisions by the National Institutes of Health (National Institutes of Health, 2011) to scale back research of some types involving captive chimpanzees, work undertaken for the current study fits clearly within the ethical framework of scientifically justifiable research with chimpanzees as outlined by the Institute of Medicine (Institute of Medicine (US) and National Research Council (US) Committee on the Use of Chimpanzees in Biomedical and Behavioral Research, 2011). In conjunction with work being conducted on other RDoC-relevant lines (e.g., Hopkins et al., 2014a; Latzman et al., 2016b), the current work highlights the importance of including a chimpanzee comparative-translational component in the NIMH RDoC research program. Along with findings from human studies, work of this kind can provide exceptionally valuable insights into core biobehavioral processes relevant to psychological illness and health (Latzman and Hopkins, 2016).

#### Limitations

The current study is not without limitations. First, the sample size, particularly in the case of the nursery-reared subgroup, was relatively modest. Additional research is thus needed to replicate the current findings and establish more stable estimates for contributions of AVP to psychopathy. Nonetheless, it is important to note that although potential concerns regarding sufficient power to detect effects within the nursery-reared sample are appropriate, effect sizes for these associations approached zero (e.g., η<sup>p</sup> <sup>2</sup> ≤ 0.01). Additionally, while widelyused in both the human and nonhuman primate literatures, our use of scores on the CHMP-Tri scales, derived from caretaker ratings of a set of adjective descriptors with accompanying narrative definitions, is only one of a number of potential approaches to assessing the dimensions described within the triarchic model. Indeed, multi-domain operationalizations of triarchic dimensions are possible through the use of composite psychoneurometric indices of the various dimensions (e.g., Patrick et al., 2013; Yancey et al., 2016). It will be important for future research to replicate the current findings considering various domains of measurement.

Additionally, there have been a number of replicability concerns raised with regard to candidate gene studies (e.g., Munafo, 2009). Although AVPR1A has repeatedly been found to underlie variation in AVP, the current study considered a single polymorphism and did not directly assess circulating levels of AVP. Further, we are not able to determine whether the differences that emerged are due directly to gene expression caused by the presence or absence of the DupB region. Nonetheless, as described earlier, across nonhuman animal studies, AVPR1A has emerged as a reliable correlate of a variety of social behaviors and traits. One important strength of animal studies is that, as compared to humans, nonhuman animal participants are raised in homogeneous, controlled environments; indeed, as described above, chimpanzee participants in the current study were raised in a common, controlled environment. Nonetheless, it will clearly be important for future studies to replicate our findings and also include more direct measures of AVP to confirm that our findings are a result of AVPR1A expression or some other potential pathway. Further, it will be important for future studies to examine additional related neuropeptides known to be associated with social behavior, such as oxytocin and associated genes.

Finally, it is important to note that chimpanzees encounter a variety of potentially impactful early experiences, whether raised by their biological mothers or in human-managed nursery settings. Given this, as noted previously (i.e., Latzman et al., 2015), our classification of participants into subgroups based on the ostensibly topographical manner in which they were raised likely obscures important variability within each group. Notably, however, our approach of grouping chimpanzee participants in this manner likely resulted in a more conservative indication of the role of early social rearing experiences, potentially enhancing confidence in conclusions advanced from current findings. Relatedly, the nursery-rearing experience of apes is not directly parallel to experiences of early adversity in humans. As described previously (e.g., Latzman et al., 2017), nurseryreared apes are removed from their mothers as a result of caregiving needs (e.g., inadequate maternal care, injury, illness)

#### REFERENCES

Almasy, L., and Blangero, J. (1998). Multipoint quantitative-trait linkage analysis in general pedigrees. Am. J. Hum. Genet. 62, 1198–1211. doi: 10.1086/301844

American Psychological Association (2012). Guidelines for Ethical Conduct in the Care and Use of Nonhuman Animals in Research. Available online and subsequently placed in an adequate (i.e., less adverse) condition. Nonetheless, whereas this is not the best parallel to experiences of physical adversity, this experience is likely more similar to human experiences of social adversity. Indeed, maternal deprivation at an early age is associated with a number of dysfunctional behaviors in humans (Gunnar and Quevedo, 2007). Thus, although likely not completely analogous to the early adversity encountered by many humans, it is clear that early maternal separation results in a number of behavioral sequelae indicative of adversity.

#### CONCLUSION

Using a powerful and unparalleled animal model, the current study points to contributions of AVP influences to psychopathic tendencies, with an important role for a specific environmental factor—early rearing experience—in affecting this contribution. Taken together, results suggest an important contributory influence of neuropeptide variation in the explanation of the development of psychopathy. Results of the current study further provide additional compelling evidence that psychopathic tendencies are rooted in basic, evolutionarilymeaningful dispositions (Fowles and Dindo, 2006; Patrick et al., 2009; Skeem et al., 2011; Patrick and Drislane, 2015; Latzman et al., 2016a, 2017), and provide support for a primate-translational operationalization of key neurobehavioral constructs relevant both to psychopathy and to broader forms of psychopathology. As such, the current work highlights the value of a chimpanzee comparative-translational component to the NIMH RDoC research framework (Latzman and Hopkins, 2016).

#### AUTHOR CONTRIBUTIONS

RL and WH conceived of the study. WH and SS oversaw data collection. RL performed all analyses and drafted the paper. WH and SS provided critical revisions.

#### ACKNOWLEDGMENTS

This research was supported in part by National Institutes of Health Grant MH92923 and NS-42867 and by a Georgia State University Brains and Behavior seed grant. The chimpanzees at the National Center for Chimpanzee Care are supported by NIH Cooperative Agreement U42 OD-011197. Additional support was provided by the National Center for Research Resources P51RR165 to Yerkes National Primate Research Center. Thank you to Christopher Patrick for his helpful comments on an earlier version of this paper.

at: http://www.apa.org/science/leadership/care/guidelines.aspx. (Accessed May 12, 2017).

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constructs: development and initial validation. Clin. Psychol. Sci. 4, 50–66. doi: 10.1177/2167702615568989

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Latzman, Schapiro and Hopkins. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Arginine Vasopressin Effects on Subjective Judgments and Neural Responses to Same and Other-Sex Faces in Men and Women

*James K. Rilling1,2,3,4\*, Ting Li1 , Xiangchuan Chen2 , Pritam Gautam1 , Ebrahim Haroon2 and Richmond R. Thompson5*

*1Anthropology, Emory University, Atlanta, GA, United States, 2Psychiatry and Behavioral Sciences, Emory University, Atlanta, GA, United States, 3Center for Translational and Social Neuroscience, Emory University, Atlanta, GA, United States, 4Center for Behavioral Neuroscience, Atlanta, GA, United States, 5Psychology, Bowdoin College, Brunswick, ME, United States*

Arginine vasopressin (AVP) influences social and emotional behaviors across a wide range of species. In humans, intranasal AVP has been previously shown to alter physiological responses to and subjective judgments of same-sex faces in both men and women. The present study attempted to elucidate the neural mechanism for these effects by randomizing 40 healthy men and 40 healthy women to treatment with either 40 IU intranasal AVP or a saline placebo approximately 30 min before imaging their brain function with fMRI as they viewed same and other-sex faces. All subjects were also scanned a second time several days later with no treatment to evaluate the persistence of AVP effects over time. AVP acutely increased positive ratings of same-sex faces in women, with some evidence that these effects persisted until the second scan. While AVP had no acute effects on same-sex ratings in men, AVP increased positive ratings of same-sex faces several days later. On the other hand, AVP had no effect on other-sex face judgments in either sex. AVP modulation of brain function was focused on the nucleus accumbens (NAc) and the lateral septum, two reward processing areas involved in the formation of social bonds. AVP provoked acute increases in right NAc and bilateral lateral septum responses to female faces among men, with left lateral septum responses persisting over time while right NAc responses reversed over time. Finally, AVP modulated hypothalamic activation to faces in both men and women. The present study therefore indicates that intranasal AVP affects subjective ratings and neural responses to same and other-sex faces in men and women, with some effects persisting and others emerging over time. Future studies should investigate whether AVP effects are modulated by individual variables such as genotype, personality, or attachment style as previously reported for other nonapeptides.

Keywords: vasopressin, fMRI, face processing, sex differences, nucleus accumbens

#### INTRODUCTION

Arginine vasopressin (AVP) is a nine amino acid peptide that is synthesized in the hypothalamus and released into the general circulation where it acts as a hormone to regulate blood pressure and water retention in the body (1, 2). In addition, AVP-producing neurons within and outside the hypothalamus release AVP into the brain where it can act *via* vasopressin receptors (and possibly related oxytocin receptors) to influence social and emotional behaviors.

Arginine vasopressin and arginine vasotocin (AVT, its non-mammalian homolog) effects on brain functions related to social behavior have been explored in numerous non-human animals. These

#### *Edited by:*

*Aras Petrulis, Georgia State University, United States*

#### *Reviewed by:*

*Caroline F. Zink, Lieber Institute for Brain Development, United States Elliott Albers, Georgia State University, United States*

> *\*Correspondence: James K. Rilling<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology*

*Received: 26 May 2017 Accepted: 31 July 2017 Published: 21 August 2017*

#### *Citation:*

*Rilling JK, Li T, Chen X, Gautam P, Haroon E and Thompson RR (2017) Arginine Vasopressin Effects on Subjective Judgments and Neural Responses to Same and Other-Sex Faces in Men and Women. Front. Endocrinol. 8:200. doi: 10.3389/fendo.2017.00200*

**164**

studies have identified both species-specific neuromodulatory circuits that have evolved in relation to unique life histories, as well as circuits that are highly conserved across species. Within these circuits, target areas have been identified in which AVT/AVP can stimulate courtship [hindbrain; rough-skin newt; (3)], promote affiliation related to pair bonding [septum and ventral pallidum, prairie voles; (4, 5)], and increase gregariousness [septum, zebra finches; (6)]. Areas in which the peptides influence aggression and aggressive communication [anterior hypothalamus, hamsters and prairie voles (7–9); amygdala, rats (10); septum, finches and sparrows (11, 12); preoptic area, plainfin midshipmen (13)], as well as social withdrawal [amygdala, rats (14); hindbrain, goldfish (15)] have also been identified. From this body of work, it has become clear that these peptides can influence a variety of social responses, promoting affiliative interactions in some species or contexts and aggressive or antisocial responses in others, *via* actions in different brain circuits [further discussed in Ref. (16)].

Arginine vasopressin effects on human social cognition and behavior have been studied using intranasal AVP administration, which is believed to cross the blood–brain barrier (17). Most studies have been done in men, where intranasal AVP has been found to facilitate cooperation (18, 19), enhance recognition of sexual cues (20), and enhance encoding of happy and angry faces (21). In addition, among both men and women, intranasal AVP increases empathic concern in those who received high levels of paternal warmth (22), as well as anxiety and skin conductance responses to angry faces (23). However, AVP can also have different effects in men and women for some social responses. In men, AVP induced agonistic facial motor patterns and decreased perception of friendliness to faces of unfamiliar men. On the other hand, in women, AVP induced affiliative facial motor patterns and increased perceptions of friendliness in faces of unfamiliar women (23). Together, these studies suggest that, in humans, AVP has sex- and perhaps context-dependent influences on a variety of social responses, as it also does in other vertebrates.

Although we know less about where within the human brain AVP acts to influence particular social responses than we do in other animals, fMRI studies have identified some regions in which AVP alters patterns of activity in parallel with its effects on social behavior and/or emotional processes. In men, intranasal AVP increases the amygdala response to emotional scenes (24), consistent with increased self-reported anxiety (23). To the contrary, it decreased both amygdala and anterior insula activation in response to negative social interactions between men in the iterated Prisoner's Dilemma (PD) Game (25). On the other hand, intranasal AVP augmented bilateral insula activation to positive social interactions in men the PD game while having the opposite effect in women (26). Thus, AVP may increase the salience of positive social interactions, while decreasing the salience of negative social interactions among men in some contexts. AVP has also been shown to decrease activation within the temporoparietal junction, a key node of the theory of mind network, when viewing unfamiliar but not familiar faces (27). Finally, AVP has been shown to modulate putative emotion regulation circuitry in the human brain. For example, AVP increased connectivity of the right amygdala with the medial prefrontal cortex (mPFC) during the processing of socially threatening scenes, which was interpreted as reflecting a reduced suppressive effect of mPFC on amygdala activity (24). In another study, it induced a relative increase in activation within the subgenual ACC, another key emotion regulation area (28), in response to emotional faces.

To further explore the neural mechanisms underlying AVP's effects on responses to social stimuli, the present study investigates whether intranasal AVP modulates the BOLD fMRI response to viewing unfamiliar same and other-sex faces in both men and women. Given known effects of AVP on learning (21, 29, 30) and long-lasting, organizational effects on social responsiveness that it and related nonapeptides can have during development (31–34), we also investigated whether any observed effects persist beyond the period of AVP exposure. Although our previous work focused on a lower dose (20 IU), preliminary data from a parallel study suggested that a higher dose (40 IU) more effectively stimulates positive assessments potentially related to affiliative responses to faces in women and may also promote positive responses in men, whereas lower doses promote negative responses (under review, this issue). Our strongest prediction was, therefore, that 40 IU AVP would increase positive responses toward the faces of other women in women. Although lower doses promote negative responses toward same-sex faces in men (23), these preliminary data also led us to predict that this higher dose (40 IU) would promote positive responses in men. We also predicted that if AVP has effects in humans that are, as in other species such as prairie voles and finches, dependent on social contexts, including the sex of the individual/stimulus with which subjects interact (35, 36), then AVP may produce different effects toward sameand other-sex stimuli, In particular, if AVP promotes affiliative responses related to pair bonding in human males (37), then we predicted that any positive effects in males would be selective for female faces. Further, we predicted that positive responses would be concurrent with increased activation in the ventral striatum, in which nonapeptides, including AVP, have been shown to promote affiliative responses in prairie voles, particularly in relation to pair bonding (see above), and in which activity is generally associated with positive social responses in humans (38, 39). In contrast, we predicted any antisocial effects might be concurrent with increased activation in regions in which AVP/AVT act to enhance aggression/antisocial behaviors in other animals, most notably the amygdala and hypothalamus (see references in second paragraph). Predictions about the lateral septum were more difficult; AVT can promote aggression *via* actions there, but also gregariousness, and AVP in the lateral septum promotes affiliative processes related to pair bonding. Further, based on previous findings in humans (28), we predicted that AVP would increase the subgenual ACC response to viewing faces.

#### MATERIALS AND METHODS

#### Subjects

Participants were 40 healthy men and 40 healthy women between the ages of 21 and 30 (mean = 23.89 ± SD = 2.19). All participants were heterosexual and not in a committed relationship. Participants were randomized to either 40 IU intranasal AVP (20 men and 20 women) on scan 1 followed by no treatment on scan 2, or to placebo (PL) (20 men and 20 women) on scan 1 followed by no treatment at scan 2. Scans were collected within 2 weeks of each other. The mean interval between scans was 4.3 days (SD = 2.25 days). Randomization was performed by the Emory Investigational Drug Service (IDS) using Research Randomizer,1 which randomizes each subject by using the method of randomly permuted blocks.

All potential subjects completed a full medical history questionnaire. Subjects with a history of seizures or other neurological disorders, alcoholism, or any other substance abuse, hypertension, cardiovascular disease, diabetes, and other endocrine diseases or malignancy were excluded from the study. Subjects who reported a history of asthma or migraine headaches were excluded if their symptoms were persistent, disabling, and required one or more medication adjustments within the past month. Subjects with a history of head trauma, psychiatric illness, or use of medications with known psychoactive effects over the past year were generally excluded. However, a *post hoc*, secondary review of screening forms revealed inclusion of one subject who reported mild head trauma and another who indicated seizure due to fever at the age of 2. Subjects with claustrophobia were excluded at the discretion of the Principal Investigator. Subjects were allowed to continue on their current medications if the agents in question were not reported to alter brain activity in regions of interest. Some of these medications included birth control and antihistamines for allergy.

All subjects gave written informed consent, and the study was approved by the Emory University Institutional Review Board and the U.S. Food and Drug Administration. Preparation of study medication and details of randomization were maintained by the Emory IDS and all study personnel including the PI were blind to group assignment. Administration of 40 IU vasopressin was generally safe. None of the subjects developed any major side effects of study medication, including anaphylaxis. One subject experienced a transient increase in blood pressure; however, this subject was in the PL group.

### Preparation and Administration of AVP and Placebo

#### Intranasal AVP

Lyophilized AVP purchased from Polypeptide Group (Hillerod, Denmark) was diluted in sterile saline at concentration of 40 U/0.5 ml. The solution was immediately sterilized *via* a 0.22 μm filter before being transferred to sterile conical tube and stored at −80°C until use. On the day of the study, the drug was transferred to a nasal spray bottle after thawing from which the subjects self administered. Both prior to and after freezing, three AVP samples were tested for sterility and potency by Eagle Analytics. Samples measured 103, 112, and 90.2% of 40 IU, respectively.

#### Intranasal Placebo

The PL group self-administered 0.5 ml of PL spray comprised of sterile saline, pH adjusted and filtered in a similar manner as above, but not containing the neuropeptide, prepared ahead of time and stored at −80°C until use.

#### Administration of AVP or PL

Both experimenters and subjects were blind to the treatment subjects received. All solutions were administered intranasally. The AVP group self-administered 40 IU of AVP (Polypeptide Group, Limhamn, Sweden). This required five nasal puffs to administer 0.5 ml of solution. The PL group self-administered five nasal puffs of PL. Subjects were instructed to place the nasal applicator in one nostril and depress the lever until they felt a mist of spray in the nostril, to then breathe in deeply through the nose, and afterward to place the applicator in the other nostril and repeat the process.

#### Monitoring Vital Signs

To monitor for unintended side-effects of AVP administration, subjects' ear temperature, heart rate, and blood pressure were measured prior to drug administration and again approximately 20 min later.

Following intranasal administration of AVP, CSF concentrations begin rising within 10 min, continue to increase for up to 80 min, and remain above those of PL-treated subjects at 100–120 min after administration (17). Thompson et al. (23) tested subjects at 15 and 50 min after intranasal vasopressin administration. Accordingly, our goal was for subjects to be fully immersed in the task at 50 min post-drug administration. We, therefore, aimed to start both the task and fMRI scan at 30 min after drug administration. In actuality, this time period averaged 31.27 min (SD = 3.82) across subjects.

#### Task

Nine head-shot photographs were taken from each of three Caucasian male and three Caucasian female models, displaying neutral expressions. All photos were similar, but unique in terms of dress and lighting.

For scan 1, subjects viewed pictures of two male models, two female models, and one object (a coffee mug). Three pictures of each model and object were displayed per run and there were a total of three runs. We repeated presentations of the same models to increase familiarity with the stimuli during the test to, at least in part, mimic the repeated contact with particular individuals that would typically occur during a social interaction.

Thus, subjects viewed a total of 45 pictures. A single trial involved an 8 s presentation of the stimulus, a variable fixation interval (2, 4, or 6 s) in which subjects viewed a cross in the center of the screen, a 4 s interval during which subjects were asked to rate the stimulus on approachability on a scale from −3 (threatening and unapproachable) to 3 (friendly and approachable), a 0.5 s fixation interval, another 4 s interval during which subjects rated the stimulus on attractiveness on a scale from −3 (unattractive) to 3 (attractive), and finally another variable fixation interval (2, 4, or 6 s). Pictures were presented in pseudorandom order. Total task duration was approximately 21 min.

Scan 2 stimuli were the same as for scan 1, except that we included nine pictures from one additional male model and nine pictures from one additional female model to assess whether any persisting effects of AVP treatment were specific to faces that were seen previously.

<sup>1</sup>http://www.randomizer.org.

E-prime software (Psychology Software Tools, Pittsburgh) was used for stimulus presentation. Stimuli were projected onto a screen that subjects could view through a mirror mounted on the head coil in the MRI scanner. Subject responses were recorded using a response box.

### Neuroimaging Data Acquisition

#### Anatomical Image Acquisition

Subjects lay motionless in a supine position in the scanner with padded head restraint to minimize head movement during scanning. Each scanning session began with a 15 s scout, followed by a 5 min T1-weighted MPRAGE scan (TR = 2,600 ms, TE = 3.02 ms, matrix = 256 × 256, FOV = 256 mm, slice thickness = 1.00 mm, gap = 0 mm).

#### fMRI Image Acquisition

Functional scans used an EPI sequence with the following parameters: TR = 2,000 ms, TE = 28 ms, matrix = 64 × 64, FOV = 224 mm, slice thickness = 2.5 mm, 34 axial slices. TE was minimally decreased from the typical value (32 ms) in order to reduce magnetic susceptibility artifact in the orbitofrontal region. The duration of each EPI scan was about 7 min (15 pictures × 8 s per picture, plus 8 s for fixation, and 8 s to rate each picture on two different adjectives).

#### Analysis of Subjective Ratings

Two sample *t*-tests were used to test for between-subject effects of AVP treatment (vs. PL) on approachability and attractiveness ratings of same and other-sex faces at scan 1 to test for acute effects of the drug, and again at scan 2 to test for more prolonged effects, whether acute effects were present or not.

#### Analysis of Neuroimaging Data

The analysis was conducted with the Oxford Center for Functional Magnetic Resonance Imaging of the Brain's software library (FSL).2

The preprocessing pipeline of the fMRI data involves (1) motion correction using the MCFLIRT (40), (2) non-brain tissue removal using the BET (41), (3) slice timing correction, (4) high-pass temporal filtering with a cut-off of 200 s, (5) spatially smoothing with a Gaussian kernel of full-width at half maximum (FWHM) of 4 mm, and (6) normalizing to MNI space *via* corresponding extracted T1 brain using Boundary-Based-Registration (42).

For each subject, the preprocessed fMRI data were analyzed using a general linear model (GLM). Regressors were specified for male faces, female faces, and objects seen at both scans, and also for the novel male and female faces seen at scan 2. Each task regressor was convolved with a standardized model of the hemodynamic response function. Contrasts of beta values for male faces vs. objects and female faces vs. objects were generated for use in group analyses. The individual-level GLM was implemented using FMRIB's Improved Linear Model (FILM).

Given widespread evidence for sex differences in the AVP system (43, 44), analyses were conducted separately for males and females. For group analyses, a two sample *t*-test was used to compare the contrast (face-object) for same and other-sex faces between the AVP and PL groups at scan 1. Another two-sample *t*-test was used to compare the same contrasts between the AVP and PL groups at scan 2 (i.e., carryover effects). Whole brain exploratory analyses were thresholded using clusters determined by *z* > 3.1 (voxel-wise 1-tailed *p* < 0.001), and a family wise error (FWE)-corrected cluster significance threshold of *p* < 0.05 was applied to the suprathreshold clusters. Region of interest (ROI) analyses were also conducted within bilateral nucleus accumbens (NAc), amygdala, lateral septum, and hypothalamus. NAc and amygdala were defined using the Harvard-Oxford Subcortical Structural Atlas implemented in FSL3 with 50% probability as a threshold. The lateral septum and hypothalamus were manually defined based on the coordinates and anatomy of these ROIs and surrounding brain structures, referring to the "Atlas of the Human Brain" (45). All ROIs were defined in the MNI space (Figure S1 in Supplementary Material). Results of ROI analyses were corrected for multiple comparisons at the voxel level (*p* < 0.05) using Gaussian Random Field Theory. In addition, we imposed a minimum spatial extent threshold of three voxels. Results for each contrast were also Bonferroni corrected for the number of ROIs investigated (8), such that significance required *p* < 0.006.

Neuroimaging data from one female subject in the placebo group was unusable due to technical problems. Subjects were compensated with a total of \$50 on each of the two visits.

### RESULTS

#### Participant Demographics in the AVP and PL Groups

There was no difference in age between participants randomized to AVP vs. PL for either men [AVP group: mean = 23.05, SD = 1.99; PL group: mean = 23.50, SD = 2.46; *t*(38) = −0.64, *p* = 0.53] or women [AVP group: mean = 23.40, SD = 1.98; PL group: mean = 23.45, SD = 2.31; *t*(38) = −0.07, *p* = 0.94]. The racial distribution across groups was follows: female AVP = 5 Caucasian, 7 African American, 7 Asian, 1 mixed race; female PL = 8 Caucasian, 5 African American, 6 Asian, 1 Hispanic; Male AVP = 5 Caucasian, 3 African American, 11 Asian, 1 Hispanic; Male PL = 10 Caucasian, 3 African American, 5 Asian, 1 Mixed, 1 not available/other.

### Attractiveness and Approachability Ratings

#### Female Participants

Arginine vasopressin treatment increased female participant's attractiveness ratings of female faces compared with PL treatment at scan 1 [*t*(38) = 2.51, *p* = 0.017]. There was a trend for this effect to persist until scan 2 when no treatment was given, although this result was only marginal [*t*(38) = 1.83, *p* = 0.075]. In the PL group, the number of days between scan 1 and scan 2 was not correlated with scan 2 attractiveness ratings

<sup>2</sup>http://www.fmrib.ox.ac.uk/fsl/.

<sup>3</sup>https://fsl.fmrib.ox.ac.uk/fsl/fslwiki.

of familiar female faces (*r* = −0.03, *p* = −0.91). However, in the AVP group, there was a significant positive correlation (*r* = 0.55, *p* = 0.01) such that female faces were rated as more attractive with increasing scan interval. Thus, AVP effects appear to become more pronounced with longer scan interval (Figure S2 in Supplementary Material). To determine if these marginal carryover effects generalized to the novel faces seen at scan 2, we also compared attractiveness ratings for novel female faces between the AVP and PL groups and found no significant difference [*t*(38) = 1.46, *p* = 0.15]. However, the effect of AVP did not significantly differ for familiar and novel faces [*F*(1, 38) = 0.05, *p* = 0.83] (**Figure 1**). Approachability ratings for female faces did not differ between the AVP and PL group on either scan 1 or scan 2. Nor was there any significant effect of AVP treatment on either attractiveness or approachability ratings of male faces, at either scan 1 or scan 2. Finally, there was no significant effect of AVP treatment on either attractiveness or approachability ratings of objects, at either scan 1 or scan 2 (Table S1 in Supplementary Material).

#### Male Participants

Although AVP did not increase attractiveness ratings of male faces at scan 1, it did significantly increased attractiveness ratings of male faces at scan 2 [*t*(38) = 2.28, *p* = 0.03]. In the PL group, the number of days between scan 1 and scan 2 was negatively correlated with scan 2 attractiveness ratings of familiar male faces (*r* = −0.51, *p* = 0.02). That is, men rated male faces they had seen previously as less attractive as the scan interval increased. On the other hand, in the AVP group, there was no correlation between scan interval and scan 2 attractiveness ratings of familiar male faces (*r* = −0.15, *p* = 0.54). AVP effects appear to become more pronounced with longer scan intervals (Figure S1 in Supplementary Material). To determine if these marginal carryover effects generalized to the novel faces seen at scan 2, we also compared attractiveness ratings for novel male faces between the AVP and PL groups and found no significant difference [*t*(38) = 0.90, *p* = 0.38]. However, the effect of AVP did not significantly differ for familiar and novel faces [*F*(1, 38) = 1.14, *p* = 0.29] (**Figure 2**). AVP had no significant effect on approachability ratings of male faces at either scan 1 or scan 2. There was also no effect of AVP treatment on either attractiveness or approachability ratings of female faces at either scan 1 or scan 2. Finally, there was no significant effect of AVP treatment on either attractiveness or approachability ratings of objects, at either scan 1 or scan 2 (Table S1 in Supplementary Material).

#### Neuroimaging Data Whole Brain Analyses

In whole brain analyses, there was no effect of AVP treatment vs. PL treatment on the BOLD response to either same or other-sex faces in either men or women, at either scan 1 or scan 2.

#### ROI Analyses

#### *Female Participants*

For women viewing female faces, there was no effect of AVP treatment at scan 1. On scan 2, AVP increased the response to female faces in the left hypothalamus (4 voxels, peak activation at MNI coordinate = −2, −4, −16; peak *z* = 2.68, *p* = 0.004) (**Figure 3**).

For women viewing male faces, AVP treatment decreased the left hypothalamus response at scan 1 (6 voxels, peak activation at MNI coordinate = −4, −4, −8; peak *z* = 2.65, *p* = 0.004) (**Figure 4**). There was no effect of AVP treatment on scan 2.

#### *Male Participants*

For men viewing female faces, AVP treatment on scan 1 increased the right NAc response compared with PL (3 voxels,

FIGURE 1 | Subjective ratings of same-sex faces, other-sex faces, and objects in women, as a function of arginine vasopressin (AVP) vs. PL treatment and first vs. second scan. For attractiveness ratings of female faces, data for novel faces at scan 2 are plotted to the right of familiar faces. Error bars = ± 1 SE. \**p* < 0.05 for AVP vs. PL at scan 1.

FIGURE 2 | Subjective ratings of same-sex faces, other-sex faces, and objects in men, as a function of arginine vasopressin (AVP) vs. PL treatment and first vs. second scan. Error bars = ± 1 SE. \**p* < 0.05 for AVP vs. PL at scan 2.

peak activation at MNI coordinate = 6, 14, −8; peak *z* = 2.94, *p* = 0.002) (**Figure 5A**). However, the opposite effect was observed at scan 2, when AVP decreased both the right (23 voxels, peak activation at MNI coordinate = 8, 10, −6; peak *z* = −2.74, *p* = 0.003) (**Figure 5A**) and left NAc response to female faces (4 voxels, peak activation at MNI coordinate = −6, 2, −8; peak *z* = −2.88, *p* = 0.002). In addition to these effects within NAc, AVP treatment at scan 1 also increased the bilateral lateral septum response compared with PL (left septum = 6 voxels, peak activation at MNI coordinate = −2, −2, 12; peak *z* = 2.67, *p* = 0.004) (right septum = 3 voxels, peak activation at MNI coordinate = 4, −2, 14; peak *z* = 2.58, *p* = 0.005) (**Figure 5B**). AVP also increased the left, but not right, lateral septum response to female faces at scan 2 (3 voxels, peak activation at MNI coordinate = −2, 2, 10; peak *z* = 2.59, *p* = 0.005) (**Figure 5B**). While AVP had no effect on activation in the hypothalamus at scan 1, AVP decreased the left hypothalamus response to female faces at scan 2 (3 voxels, peak activation at MNI coordinate = −4, 2, −8; peak *z* = 2.78, *p* = 0.003) (**Figure 5C**). Finally, AVP had no effect on activation in the amygdala at either scan 1 or scan 2. While we could not accurately define a ventral pallidum ROI given its very small size and the lack of anatomical landmarks to guide its definition in the anatomical MRI, inspection of the uncorrected (*p* < 0.05) whole brain results showed a lack of AVP-related activation in the vicinity of the ventral pallidum.

For men viewing male faces, there was no effect of AVP treatment at either scan 1 or scan2.

#### DISCUSSION

Here, we show that administration of 40 IU intranasal AVP approximately 30 min before viewing faces influences both neural responses to and subjective ratings of those faces. In women, AVP treatment on the first scan day increased attractiveness ratings of female faces, and there was a trend for this effect to persist until scan 2 when no treatment was administered. AVP also increased

the left hypothalamus response to female faces, but only on scan 2. Additionally, AVP decreased the left hypothalamus response to male faces on scan 1 only. In men, despite no effect on subjective ratings at scan 1, AVP increased attractiveness ratings of male faces at scan 2. AVP also increased the right NAc and bilateral lateral septum responses to female faces on scan 1. These effects persisted to scan 2 for the left lateral septum, but reversed for right NAc. Finally, AVP decreased activation to female faces within the left hypothalamus, but only for scan 2. Although these patterns are complex, three important points emerge. First, intranasal AVP can promote positive responses to same-sex faces in women and men, though on different time scales. Second, there is not always congruence between behavioral effects and influences in brain areas with functions presumably related to the behavioral responses; AVP increased attractiveness ratings in women on scan 1, but not activation in NAc, in which activity is correlated with positive assessments (46). Conversely, in men, AVP selectively increased activation in response to female faces in several areas, but did not affect behavioral responses. These patterns suggest either that some behavioral outputs may depend more on complex network or emergent processes than we currently capture, that areas in which activity was influenced are unrelated to the behaviors measured, and/or that our statistical methods are too conservative to capture such associations. Third, a single dose of intranasal AVP has the potential to induce long-lasting effects on behavioral and neural responses.

In a previous study, 20 IU intranasal AVP increased approachability ratings of women viewing female faces with neutral expressions (23). While we did not strictly replicate this effect, AVP-treated women did rate female faces as more attractive compared with PL-treated women. We believe attractiveness ratings in women are a generalized assessment of same-sex individuals and not a specific measure of sexual or romantic interest because heterosexual women in this and in our parallel studies rated the faces of other women as more attractive than the faces of men. Together, the two studies indicate that AVP selectively promotes positive assessments of the faces of other women in women. We did not measure the menstrual cycle phase of women in this study, and so cannot say whether AVP effects in women are moderated by cyclic hormones. In contrast to the effects of lower doses, which decreased social assessment of other men in our previous study, the current findings indicate that higher doses can, over time (see further discussion below), increase positive assessments of other men. Thus, different doses may produce divergent behavioral responses in men, a possibility also supported by dose differences found in our parallel study (under review, this issue). That AVP selectively enhanced assessments of same-sex faces in men and women is consistent with AVP/AVT's effects in other species being dependent on the sex of stimulus (35, 36).

Arginine vasopressin's ability to acutely increase assessments of other women in women on scan 1 or of other men in men on scan 2 was not associated with detectable alterations of brain activity, suggesting that those behavioral effects depend on modulation in areas outside the ROIs we examined, or on complex alterations in patterned activity that we did not measure. We had expected that AVP treatments that increase positive social assessments would also increase the ventral striatum response, which has been associated with increased positive ratings of other individuals (46). However, this was not the case. While these results are unexpected, NAc need not be tracking positive ratings in relation to the rewarding aspect of the faces. Indeed, if increased ratings of other female faces are part of a "tend and befriend" stress responses strategy, particularly in women (47), then AVP's behavioral influences may not be directly related to reward and/or positive affect, but rather to stress/anxiety reduction. On the other hand, there was a parallel effect on behavioral and neural responses on scan 2 in women; AVP delivered prior to scan 1 marginally increased attractiveness ratings on scan 2 and increased responses to female faces in the lateral hypothalamus. AVP enhances aggression *via* actions in the hypothalamus in male hamsters but decreases aggression in female hamsters (48), though it is not yet known if AVP's ability to inhibit aggression in females depends on activation or inhibition of hypothalamic activity. We did not measure responses directly associated with aggression, though increased social assessments of other women might be associated with decreased aggressive responses toward them. However, it is unclear why associations between increased hypothalamic activation on scan 2 would be related to increased attractiveness ratings in the absence of similar linkages on scan 1, or of linkages between the decreased activation in hypothalamic and behavioral responses to male faces on scan 1, unless AVP's acute and lasting influences on behavioral and brain responses are associated with different mechanisms (see further discussion below). It is, therefore, also possible that influences on hypothalamic responses are completely unrelated to social assessments. Perhaps most important is simply that AVP did influence hypothalamic responses to faces, as AVP influences on hypothalamic responses to social stimuli, which mediate a variety of social behaviors in other vertebrates, have not previously been reported in humans.

In previously unpaired male prairie voles, AVP elevations in the ventral striatum that are concurrent with social contact with a novel female facilitate affiliative responses toward that individual. AVP appears to link the reward from mating with the identity of a particular female, resulting in a preference to associate with that female over others (49). Despite the independent evolution of pair bonding in prairie voles and humans and associated differences in life histories associated with pairing, males in both species are capable of forming such bonds in reproductive contexts, and AVP has been indirectly implicated in that process in human males (37). Although the design of this experiment was not identical to those used to test AVP's role in pair bonding in prairie voles, in part due to the limitations associated with manipulating human subjects, we did presumably elevate AVP in single men while they were exposed to novel female faces. We recognized that the induction of social preferences related to pair bond formation in prairie voles requires continuous AVP administration during sustained social contact with a novel female (4, 36), so we did not expect our more limited manipulation to induce selective social attachments to the briefly presented faces. Nonetheless, we predicted that intranasal AVP might, particularly in light of the sustained elevations of the peptide in the brain that follow intranasal delivery (17) (discussed further below), enhance ratings indicative of increased attractiveness toward or tendencies to interact/affiliate with novel females while decreasing similar assessments of other males in relation to potential mate competition/guarding functions. We also predicted elevations of AVP during those limited "interactions" might increase activity in areas of the brain in which AVP promotes affiliative responses related to pair bonding with females and aggression toward males in voles if convergent peptide mechanisms associated with the promotion of emotional attachments in reproductive contexts evolved in the two species. These predictions were only partially supported. Although AVP did specifically augment bilateral responses in the lateral septum and ventral striatum to female faces, the responses in the ventral striatum were in the NAc, not the ventral pallidum, where AVP promotes affiliative responses relating to pairing in male prairie voles, and it did not augment ratings of female faces in this or our parallel study, in which two doses of AVP were administered, and in which men were only exposed to a single female face, but had more exposure to that face. Nor did the dose used in this study decrease responses to other males, but rather enhanced them over time. It is possible that our rating responses do not reflect responses related to affiliative and aggressive processes associated with pair-bonding/mate-guarding, or that our tests did not stimulate sufficient, concurrent dopamine release, which is also involved in stimulating partner preferences in male prairie voles (49). Indeed, the female faces were not even smiling and thus unlikely to have represented a potential romantic interest/ partner. Of course, we also must acknowledge the possibility that AVP does not, as it does in male prairie voles, facilitate affiliative processes related to pair bonding in human males, and if it does, that it does so through different neural mechanisms, i.e., through activations in the lateral septum and NAc, rather than the ventral pallidum, and perhaps also through long-term downregulation of lateral hypothalamic responses to familiar females, which were lower only on scan 2. Indeed, comparative studies have suggested that common AVT/AVP mechanisms do not underlie pair bond formation in reproductive contexts in species in which such tendencies have independently evolved (50–52).

The reversal of AVP effects on male NAc activation to female faces at scan 2 was unexpected. However, this result may be consistent with another fMRI study showing that intranasal AVP effects differ for familiar and unfamiliar faces (27), as the same female faces were used on scan 1 and scan 2 in our study. **Figure 5B** shows an increase in NAc activation from scan 1 to scan 2 in the PL group. This might reflect increased reward from the female face over time as familiarity is established. It is possible that AVP accelerates this process so that NAc activity is already augmented at scan 1 in men who received AVP. The subsequent decrease in NAc activation from scan 1 to scan 2 could reflect a social habituation effect of AVP as has been reported previously for the closely related nonapeptide OT (53).

Arginine vasopressin modulation of lateral septum and NAc activation was specific to men, and AVP had opposing effects on hypothalamic activation in men and women viewing female faces. These results are consistent with accumulating evidence for sex differences in AVP effects in both humans and other animals (43, 44). In rats, for example, treatment with a V1aR antagonist in the lateral septum significantly increased social play in males while decreasing social play in females (54). In hamsters, hypothalamic AVP injection stimulated aggression in males, while inhibiting aggression in females (48). In humans, intranasal AVP augmented bilateral insula activation to positive social interactions in men, while having the opposite effect in women (26). Finally, with a face viewing paradigm similar to that used here, we previously showed that lower doses of AVP induced agonistic facial motor patterns and decreased perception of friendliness to same-sex faces in men, while inducing affiliative facial motor patterns and increased perceptions of friendliness to same-sex faces in women (23).  Describing sex differences in human V1aR is an important task for future research.

No effects were observed in either whole brain analyses or in ROI analyses focused on the amygdala. The amygdala plays a critical role in threat detection (55–57), and AVP has been shown to modulate amygdala response in various contexts in both humans and non-humans (25, 58–60). Given that 20 IU intranasal AVP was previously shown to stimulate agonistic facial motor patterns and to increase skin conductance responses to same-sex faces in men (23), we expected AVP to increase amygdala activation to same-sex faces in men; however, this was not observed. The current study used 40 IU intranasal AVP. It is possible that the hypothesized effects on amygdala activation in face processing contexts would emerge at the 20 IU dose, which our parallel study found yields a more negative response than the higher dose.