Physiology & Behavior 141 (2015) 154–163

Contents lists available at ScienceDirect

Physiology & Behavior journal homepage: www.elsevier.com/locate/phb

Oxytocin mechanisms of stress response and aggression in a territorial finch James L. Goodson, Sara E. Schrock, Marcy A. Kingsbury ⁎ Department of Biology, Indiana University, Bloomington, IN 47405 USA

H I G H L I G H T S • Oxytocin receptor antagonism reduces aggression in a territorial finch. • Hypothalamic oxytocin cell groups respond to stressors (subjugation and pursuit). • Oxytocin inhibition of the HPA axis may be permissive for aggression.

a r t i c l e

i n f o

Article history: Received 16 July 2014 Received in revised form 12 January 2015 Accepted 13 January 2015 Available online 14 January 2015 Keywords: Oxytocin Mesotocin Aggression Corticosterone Stress Coping Defense Songbird Violet-eared waxbill

a b s t r a c t All jawed vertebrates produce a form of oxytocin (OT), and in birds, mammals and fish, OT is strongly associated with affiliation. However, remarkably few data are available on the roles of OT and OT receptors (OTRs) in aggression. Because OT and OTRs exert anxiolytic effects in mammals (although context-specific) and modulate stress coping, we hypothesized that OTR activation is at least permissive for territorial aggression. Indeed, we find that peripheral injections of an OTR antagonist significantly reduce male–male and female–female aggression in a highly territorial finch. This finding suggests the hypothesis that aggression is accompanied by an increase in transcriptional (Fos) activity of OT neurons, but contrary to this hypothesis, we find that dominant male residents do not elevate OT-Fos colocalization following an aggressive encounter and that OT-Fos colocalization in the preoptic area and hypothalamus correlates negatively with aggression. Furthermore, OT-Fos colocalization increases dramatically in males that were aggressively subjugated or pursued by a human hand, likely reflecting OT modulation of stress response. Because OT inhibits the hypothalamo–pituitary–adrenal axis, the antagonist effects may reflect the fact that aggressive birds and mammals tend to be hyporesponsive to stress. If this is correct, then 1) the observed effects of OTR antagonism may reflect alterations in corticosterone feedback to the brain rather than centrally mediated OTR effects, and 2) the negative correlation between OT-Fos colocalization and aggression may reflect the fact that more aggressive, stress hyporesponsive males require less inhibition of the hypothalamo–pituitary–adrenal axis than do less aggressive males, despite the requirement of that inhibition for the normal display of aggression. © 2015 Elsevier Inc. All rights reserved.

1. Introduction The modulation of social cognition, affiliation and anxiety by the neuropeptides oxytocin (OT)1 and vasopressin (VP) has become a

⁎ Corresponding author. E-mail address: [email protected] (M.A. Kingsbury). 1 Vertebrates express a variety of OT forms, and although the canonical form in mammals is Leu8-OT, some mammals also express Pro8-OT or Ile8-OT (mesotocin) [1–3]. This latter form is expressed in birds and other non-mammalian tetrapods. Similarly, multiple forms of VP have been identified, including the canonical Arg8-VP form found in most (but not all) mammals, and Ile3-VP (vasotocin), which is found in non-mammalian vertebrates [1,3]. Thus for clarity, we will simply refer to all OT forms as “OT” and all VP forms as “VP” (following other recent precedents [3,4]). We will also refer to homologous oxytocic receptors (of which one appears to be present in all vertebrates) as OTRs, following the nomenclature of Ocampo Daza et al. [5], and also Yamaguchi et al. [6].

http://dx.doi.org/10.1016/j.physbeh.2015.01.016 0031-9384/© 2015 Elsevier Inc. All rights reserved.

major field of inquiry, with more than 100 relevant papers being published annually [7]. This literature supports the general view that OT reduces anxiety and promotes many aspects of affiliation in mammals [8–10]. OT also facilitates social approach in goldfish (Carassius auratus) [11], and similarly, we have found that flocking behavior in zebra finches (Taeniopygia guttata) is promoted by both central OTR activation [12] and OT production in the paraventricular nucleus of the hypothalamus (PVN; a female-specific effect) [4], suggesting an evolutionarily deep history of OT effects on sociality. OT immunoreactivity fluctuates in relation to seasonal flocking in sparrows [13], consistent with these findings in finches. However, OT and OT receptors (OTRs) also influence many other aspects of physiology and behavior, including appetite, immune function, glucocorticoid secretion and thermoregulation [14–17]. Of particular relevance here, OT promotes maternal aggression in selected rat lines ([18]; but see [19]), although it is not clear that OT effects

J.L. Goodson et al. / Physiology & Behavior 141 (2015) 154–163

in this context will be observed in other contexts of aggression. In fact, exogenous OT is capable of reducing aggression in male rats, despite the fact that OTR antagonism is largely without effects [20,21]. However, it must be considered that endogenous OT may modulate behavior via heterologous binding to V1a receptors (V1aRs), which are known to mediate at least some of OT's effects (e.g., [22,23]). In contrast to the voluminous data on the affiliation effects of OT, relatively little is known about the role(s) that endogenous OT plays in same-sex offensive aggression. OT effects on intrasexual aggression have been primarily examined within a developmental framework (e.g., using neonatal OT injections in prairie voles, Microtus ochrogaster, and Mandarin voles, Lasiopodomys mandarinus [24,25] and intranasal OT administrations in pigs [26]) or using knockout mice that lack OT or the OTR [22,27]. The data obtained using these methods has provided consistent evidence that OT facilitates aggression, contrary to the popular conception of OT as a “prosocial peptide” [28–30]. Regardless, such manipulations do not directly address the influence of OT in adult animals, since these developmental manipulations likely act neonatally to organize brain and behavior [24,25]. Remarkably, only one study has clearly demonstrated that endogenous OT modulates resident-intruder aggression in adults. This experiment shows that OT infusions into the preoptic area-anterior hypothalamus (POA-AH) decrease resident-intruder aggression in female Syrian hamsters (Mesocricetus auratus), and more importantly, that OTR antagonism facilitates aggression [31]. However, broad generalization from this finding to males, and to other brain areas and species is difficult, because 1) AH infusions of VP exert opposing effects on aggression in male and female hamsters ([32]; and thus OT may likewise exert sexspecific effects, particularly given that OT-VP receptors tend to be promiscuous; e.g., [33–35]); 2) OT effects on maternal aggression are mediated in areas of the brain outside of the POA-AH, including the amygdala [18,19]; and 3) the neural distribution of OTRs is species-specific [12,33, 36–39]. Importantly, because OT is important for anxiolysis, fear reduction and stress coping [40–42], and because aggressive birds, fish, and some rodents tend to be hyporesponsive to stress (defined primarily in terms of glucocorticoid secretion [43–47]), we hypothesized that OTR activation is at a minimum permissive for territorial aggression, even if it does not actively promote it (but note that stress reactivity likely interacts with coping style to modulate aggression, rather than having unitary effects; [48,49]). One caveat is that endogenous OT does not exert anxiolytic effects across all contexts, but rather in specific contexts that are otherwise associated with OT release, such as lactation and parturition [50]. In fact, recent antisense experiments in zebra finches (conducted after the completion of the present experiments) demonstrate that OT neurons of the paraventricular hypothalamus (PVN) do not modulate anxiety when subjects are tested in a nonsocial context, and actually promote passive coping behavior [4] (note that although these more recent data could not inform our hypotheses for the experiments presented here, they are nonetheless important to consider in relation to the results, and will be revisited in the Discussion section). A second consideration is that OT inhibits basal and stress-induced activity of the hypothalamo–pituitary–adrenal (HPA) axis in a manner that appears to be uncoupled from the central modulation of anxiety [51], at least in rodents. The species under investigation here, the violet-eared waxbill (Estrildidae: Uraeginthus granatina) is exceptionally territorial [52,53], and thus we hypothesized that aggressive interactions in this species will drive endogenous release of OT, which could thus modulate both the HPA axis and central anxiety processes (presuming socially- and/ or stress-mediated OT release; see [50] for a consideration of contextspecific effects on anxiety), thereby facilitating aggression. Other known effects of OT may also serve to facilitate aggressive response, such as enhanced sensory processing of social stimuli, and modulation of autonomic function (reviews: [7,54]).

155

Despite the potential for the independent modulation of central and peripheral processes [55], it is also possible that the central and peripheral effects of OT (e.g., sensory and autonomic effects) are coupled to some extent. This is because the parvocellular OT population of the PVN that projects to the anterior pituitary also projects centrally, including the autonomic brainstem; and magnocellular OT populations that project to the posterior pituitary also release peptide centrally from axons, soma and dendrites (reviews: [7,54,56]). Thus, in order to examine the combined central and peripheral effect of oxytocic signaling on aggression, we here quantified residentintruder aggression following peripheral injections of an OTR antagonist or vehicle in male and female violet-eared waxbills. In a second experiment we then quantified the Fos responses of OT neurons to handling (control), pursuit by a human hand, aggressive subjugation, or aggressive domination. OT-Fos colocalization was quantified for the two largest hypothalamic populations, which lie in the PVN and supraoptic nucleus (SON), as well as within the smaller OT populations of the medial preoptic nucleus (POM; magnocellular) and AH (embedded in the hypothalamo-pituitary tract; parvocellular). 2. Materials and methods 2.1. Subjects Captive-bred violet-eared waxbills (n = 9 females and 24 males) were individually housed in cages 61 cm W × 36 cm D × 43 cm H on a 14L:10D photoperiod and provided finch mix and water ad libitum. Experiments were conducted under non-breeding conditions and subjects were separated from opposite-sex partners for a minimum of two weeks prior to testing. Violet-eared waxbills are generally encountered as singletons or as pairs in the wild (unless dependent young are present) ([53]; J.L. Goodson, pers. obs.), and thus the period of isolation is not expected to produce behavioral abnormalities. Violet-eared waxbills are aggressive year-round [52]. Experiments were performed in compliance with federal and institutional guidelines and were approved by the Institutional Animal Care and Use Committee of Indiana University. 2.2. OTR antagonist experiment 2.2.1. Behavioral screenings Same-sex pairs of violet-eared waxbills were screened for aggression in short resident-intruder assays (most less than 1 min; none more than 2 min; terminated as soon as an obvious dominance relationship appeared) in order to select subordinate (intruder) and dominant (resident) subjects. Due to the limited number of birds available, most birds were used as both stimuli and subjects, with at least 1 week between tests. This was possible because most birds could dominate an intruder in their home cage, but were subordinated as intruders. Only subjects who dominated same-sex intruders in their own home cage were used as subjects. 2.2.2. Injections Thirty minutes prior to testing, subjects (9 females and 10 males) were injected into the inguinal leg fold with either 0.05 cm 3 saline vehicle or vehicle containing 5 μg OTR antagonist (desGly-NH 2 ,d(CH 2 ) 5 [Tyr(Me) 2 ,Thr 4 ] ornithine vasotocin) in a counterbalanced, repeated-measures design with 2 days allowed between tests. This dose has been used in previous studies and effects have been replicated using a much lower dose centrally [12]. Importantly, effects of systemic administration of the OTR antagonist do not produce general behavioral alterations. For instance, subjects do not decrease approach to conspecifics [12], and despite potent effects on nest-building behavior in female zebra finches, the behavior of males is not altered [57], suggesting that effects are highly specific. Another important consideration is that an iodinated form of this same

156

J.L. Goodson et al. / Physiology & Behavior 141 (2015) 154–163

antagonist yields binding pattern that is closely matched to OTR (VT3) mRNA expression in zebra finches and sparrows, but does not match mRNA expression patterns for other OT-VP receptor types [33,38]. 2.2.3. Resident-intruder aggression A same-sex intruder was introduced into the subject's home cage and observations were conducted for 7 min or until 40 displacements were observed, in which case behaviors were prorated. A variety of observations suggest that prorating in this species provides an accurate estimate of behavior [58,59]. We recorded displacements, threats, pecks, and agonistic calls. 2.3. Fos experiment 2.3.1. Behavior The Fos experiment was conducted using males only. Pairs of male violet-eared waxbills were screened for aggression in short residentintruder assays as described above and at least one month was allowed between screenings and testing. Based on behavior in these screenings, we were able to create treatment groups that did not differ in their levels of aggression, based on their behavior as residents during the screenings. Of the 20 males used for the Fos experiment, 10 had been used for the OTR antagonist study described above (including all subjects in the Dominant condition) and the remaining 10 had been tested in an identical paradigm, but following peripheral injection of a dopamine antagonist. Prior to testing and brain collection, we acclimated subjects to a sound isolation booth (3 h per day for 2 days), and on the third day left the subjects in the booth overnight. Tests were conducted the following morning. Subjects were assigned to one of four conditions (n = 5 each): Control, Subordinate, Dominant or Defense. Dominant and Subordinate subjects were exposed to a resident-intruder encounter as described above, with a fixed test duration of 7 min (i.e., no test termination or prorating). Because Subordinate subjects were handled twice (to introduce them to the resident's cage and to remove them), other subjects were likewise handled twice. Subjects in the Defense condition were pursued by a human hand 40 times over a period of 7 min. Birds were perfused 90 min after the start of testing. 2.4. Immunofluorescent labeling and quantification Subjects were intracardially perfused with 0.1 M phosphatebuffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde. Brains were postfixed overnight, sunk for two days in 30% sucrose, and sectioned on a cryostat into three series of 40 μm sections. We followed standard lab protocols for the immunofluorescent labeling of OT and Fos using free-floating sections [4,60]. These antibodies were labeled using secondaries conjugated to Alexa Fluor 488 (green) and 594 (red), respectively (Invitrogen, Carlsbad, CA). We used a rabbit anti-Fos antibody (Santa Cruz Biotechnology, Santa Cruz, CA; used at 1:1000) that has been extensively used and validated in birds (e.g., [61]), and a guinea pig anti-OT antibody (Bachem, Torrance, CA; used at 1:1000) from a lot that we have previously shown does not cross-react with arginine vasotocin (Ile3-vasopressin) in fish ([62]; please note that this is a polyclonal antibody and that we find that newer lots raised from a different guinea pig do indeed cross-react with Ile3-VP [vasotocin]). In addition, we find that preabsorption of this antibody with 10 μM Ile8-OT (mesotocin) eliminates labeling, whereas identical preabsorptions with vasotocin do not. Photomicrographs of sections containing OT populations in the POM, SON, PVN and AH were shot on a Zeiss AxioImager microscope outfitted with a z-drive and optical dissector (Apotome; Carl Zeiss Inc., Göttingen, Germany). Cell counts were conducted using Adobe Photoshop CS5 (Adobe Systems, Seattle, WA) and Image J (National Institutes of Health, Bethesda, MD) as previously described [63,64].

2.5. Statistical analyses Behavioral effects of the OTR antagonist were analyzed using repeated-measures ANOVA with Sex as a between-subjects factor and Treatment as the repeated measure. OT-Fos double-labeling was analyzed using one-way ANOVA with the single factor of Condition (Control, Subordinate, Dominant, Defense). Other data were analyzed using linear regressions, as described below. Violet-eared waxbills are among the most difficult finches to breed in captivity [53] and thus our n's for these experiments are relatively modest. However, the extraordinary robustness of aggressive behavior in this species (e.g., [59, 65]) allows for excellent behavioral quantification, which should yield substantial reductions in measurement error. 3. Results 3.1. OTR antagonism decreases aggression in male and female waxbills As shown in Fig. 1A, displacements of the intruder were significantly reduced by OTR antagonist injections (main effect of Treatment: F(1,17) = 5.38, p = 0.03, repeated-measures ANOVA) as was the total number of aggressive behaviors (Fig. 1B; main effect of Treatment: F(1,17) = 4.68, p = 0.04). Other than displacements, only threats were exhibited with sufficient frequency for separate analysis, but antagonist treatments did not significantly alter this behavior (main effect of Treatment: F(1,17) = 1.23, p N 0.10). Males were significantly more aggressive than females (for total aggression, main effect of Sex: F(1,17) = 5.16, p = 0.04), although females were still very aggressive, averaging approximately 32 displacements and 60 aggressive behaviors following injections of vehicle (Fig. 1). No Sex*Treatment interactions were observed, and the latency to displace the intruder was not affected by OTR antagonism (all p N 0.10). 3.2. OT neurons are Fos-activated by stressors Representative co-labeling of OT and Fos is shown in Fig. 2. As shown in Fig. 3A, OT-Fos colocalization in the POM (i.e., the percent of POM OT-immunoreactive, −ir, neurons that are Fos-ir+) is substantially greater in Defense and Subordinate subjects than in Control and Dominant subjects. A similar pattern is observed in the SON (Fig. 3B), although this does not reach significance. In the PVN, only Defense subjects exhibit significant elevations of OT-Fos colocalization (Fig. 3C). Finally, OT-Fos colocalization in the AH is significantly elevated above Control levels in Defense and Subordinate subjects, as in the POM, but Dominant subjects do not differ significantly from any other group (Fig. 3D). 3.3. OT neuron activation correlates with aggression Based on the behavioral effects of OTR antagonist injections, we predicted that Dominant subjects would show the greatest OT-Fos colocalization. As just described, this is not the case. Rather, only Defense and Subordinate subjects exhibit significant increases in colocalization above control levels. However, the Control “baseline” level of colocalization is very high, with comparable levels in Dominant subjects (~15–35%; Fig. 3), and this colocalization may reflect constitutive neuromodulation, phasic response to handling (all subjects were handled twice), and/or phasic response to the testing environment. All of these factors apply equally to the Control and Dominant subjects, and thus we hypothesized that aggression is positively correlated with the ability to cope, either constitutively or in response to phasic stressors. This led to the prediction that OT neuromodulation would correlate positively with aggression in Dominant subjects. In considering such phenotypic variation in OT neuromodulation, we considered that 50% OT-Fos colocalization would presumably yield less neuromodulation in subjects that had relatively few OT neurons

J.L. Goodson et al. / Physiology & Behavior 141 (2015) 154–163

157

Fig. 1. Aggressive displacements (A) and total numbers of aggressive behaviors (B) exhibited by male and female violet-eared waxbills in 7-min resident-intruder tests. Subjects were tested in a within-subjects design following injections of saline vehicle or an OTR antagonist (OTA). Data are shown as means ± S.E.M. *p b 0.05, main effect of Treatment, Treatment*Sex repeated-measures ANOVA; n = 9 females and 10 males.

as opposed to relatively many OT neurons. We therefore present our analyses here based upon total numbers of OT-ir neurons expressing Fos. This yields multiple significant results (described below), which comparable analyses based on the percent of OT-ir neurons that express Fos do not (all p N 0.10). Contrary to our predictions, numbers of OT-Fos co-labeled neurons actually correlate negatively with aggression. As with the group data shown in Fig. 3, all four OT cell groups tend to follow the same pattern, although the effect is significant only for the PVN (Fig. 4A). However, because all cell groups tend to follow the same pattern, we conducted an additional analysis with all cell groups combined, and again find that OT-Fos co-labeling correlates negatively with aggression (Fig. 4B).

level of stress entailed in the two experiments was likely very different. In the antagonist study, animals were handled once for a peripheral injection that was given 30 min prior to resident-intruder testing. In contrast, subjects in the Fos study were acclimated and housed in small sound booths prior to testing, and were handled both before and after testing. An examination of displacements exhibited by dominant animals during resident-intruder testing across both experiments does suggest that the Fos testing environment was likely more stressful, in that dominant birds exhibited significantly less displacements when tested in the sound booths, relative to testing in the home environment (Fig. 5). 4. Discussion

3.4. Aggression by dominant animals varies between test environment 4.1. Paradoxical effects of OTR antagonism and OT neuronal response Based on the paradoxical results from the antagonist and Fos studies, and the observation that the activation of a stress response likely explains the results of the Fos study, we further examined whether the

The present experiments provide novel evidence that endogenous activation of OTRs promotes or is permissive for resident-intruder

Fig. 2. Representative OT-Fos double-labeling in the PVN (A), SON (B), AH (C) and POM (D) of subjects in the Defense group. Arrows highlight double-labeled neurons. The box in panel A corresponds to the location of the inset. Scale bars = 50 mm for panels A–D; 10 mm for the panel A inset. Abbreviations: AH, anterior hypothalamus; ot, optic tract; POM, medial preoptic nucleus; PVN, paraventricular nucleus; SON, supraoptic nucleus; vaf, ventral amygdalofugal pathway (occipitomesencephalic tract).

158

J.L. Goodson et al. / Physiology & Behavior 141 (2015) 154–163

Fig. 4. Total aggression exhibited by dominant male violet-eared waxbills correlates negatively with the number of OT-Fos double labeled cells in the PVN (A) and across all hypothalamic cell groups (B; log-transformed for normality).

Fig. 3. OT-Fos colocalization in male violet-eared waxbills following handled control conditions (Control), 40 pursuits by a human hand (Defense), or a resident-intruder encounter in which the subject was dominant (Dominant) or subordinate (Subordinate). Data are shown as means ± S.E.M. for the POM (A), SON (B), PVN (C) and AH (D). Different letters above the error bars denote significant pairwise comparisons following significant ANOVA (p b 0.05); n = 5 per condition. Abbreviations: AH, anterior hypothalamus; POM, medial preoptic nucleus; PVN, paraventricular nucleus; SON, supraoptic nucleus.

aggression. We first show that antagonism of OTRs reduces aggression, yielding the prediction that aggression is positively associated with the activation of OT neurons. However, the results for OT-Fos colocalization are inconsistent with that prediction. Significant elevations in colocalization are observed only for Subordinate and Defense subjects, indicating that OT neurons are strongly activated by stressors. Further inconsistent with our expectations, we find that the number of cells that co-label for OT and Fos correlates negatively with aggression in Dominant subjects, despite the fact that Dominant subjects did not exhibit an increase in OT-Fos colocalization above the level of Control subjects. This finding may reflect a baseline (constitutive) modulation of aggression-related processes by OT neurons. Alternatively, the test conditions for the Fos experiment (isolation in the sound booths, being handled twice and observed) may have induced a phasic, stress-related increase in OT-Fos colocalization in all subjects, including Dominant subjects and Controls. Indeed, as described below, the Fos testing environment was likely much more stressful than the subjects' home environment, where the antagonist experiment was conducted. Thus, it is likely that OT neurons

J.L. Goodson et al. / Physiology & Behavior 141 (2015) 154–163

Fig. 5. Evidence that levels of stress varied between the antagonist (OTA) experiment and the Fos experiment (as assessed in a group of males that participated in both the OTA experiment and the Dominant condition of the Fos experiment). Subjects in the antagonist experiment had been housed for many months in the same room and were handled only once for injection 30 min prior to testing. For the Fos experiment, subjects were acclimated for 3 h per day for 2 days in a sound attenuated booth, and then remained in the booth overnight prior to the Fos experiment. Subjects were handled immediately before introduction of the intruder and immediately afterwards. As shown here, displacements were significantly suppressed in the Fos experiment relative to the OTA experiment (based on control saline data). Data are shown as means ± S.E.M. *p b 0.05, paired t-test.

in all subjects exhibited some amount of stress-induced Fos response. If this is correct, and if more aggressive violet-eared waxbills are hyporesponsive to stress (as in other species [43–47]), then stress-induced activation of OT neurons should correlate negatively with aggression, which is what we found. And importantly, this relationship can be expected even if OT inhibition of the HPA axis is permissive for aggression. Thus, this interpretation of results suggests that endogenous activation of OTRs is necessary for normal levels of aggression, even if the most aggressive animals exhibit less stress-induced activation of OT neurons. As expounded upon below, we believe that this interpretation provides the most parsimonious explanation of our results. However, we must consider that the avian OTR binds both the avian OT and VP forms (mesotocin and vasotocin) with high affinity [33], and thus it is possible that the behavioral effects of OTR antagonism reflect endogenous actions of VP and not OT neurons. Thus, we first address this possibility before focusing specifically on OT. 4.2. Are OTR-mediated effects driven by endogenous OT or VP? The present experiments were designed to examine the relationships between the behavioral effects of OTR antagonism and OT neuronal activation. However, because avian OTRs bind VP with a high affinity ([33]; also see [38]), we must also consider the possibility that VP is mediating some of the observed effects. If this were the case, the decreased aggression observed here following OTR antagonism would suggest that VP (via binding to OTRs) increases aggression. However, virtually all lines of evidence suggest that VP circuits inhibit territorial aggression in songbirds. For instance, in both violet-eared waxbills and territorial sparrows, intraseptal VP infusions decrease residentintruder aggression [58,66]. Conversely, central administrations of a V1aR antagonist increase aggression in male zebra finches after they have pair bonded and are defending nests [67]. In addition, Fos activation of PVN VP neurons is negatively related to aggression in sparrows and mice [68,69], and VP neurons in the medial bed nucleus of the stria terminalis (BSTm) show no response to socially aversive and/or aggressive interactions in finches, chickens, or mice [68–70]. In fact, knockdown of VP production in the BSTm reduces affiliation and profoundly increases aggression in male zebra finches [71]. Consistent with this observation, the density of VP-ir elements in the BSTm correlates negatively with aggression in territorial sparrows, and differentiates species that are more or less aggressive [13]. Relative to

159

aggressive (short attack latency) mice, less aggressive mice also exhibit more VP neurons in the BSTm and greater VP fiber densities in the lateral septum [72]. However, endogenous VP does act within the AH to promote aggression in male Syrian hamsters (M. auratus) that have been isolated and trained as fighters ([73]; for discussion see [74]), and also in male prairie voles following mating [75]. Both of these effects are associated with experience-dependent up-regulation of V1aRs, and notably, no such effects are observed in male hamsters that have been socially housed [76]. Furthermore, endogenous VP inhibits aggression in the AH of female hamsters, and VP-Fos colocalization in the AH and other VP cell groups correlates positively with bites received by subordinate male mice [69]. In fact, no VP cell group in dominant male mice exhibits a positive Fos response to aggressive interactions [69]. To a great extent, the negative relationship between aggression and the Fos activation of PVN VP neurons in sparrows and mice [68,69] is consistent with the idea that more aggressive males show lower socially induced activation of the HPA than do less aggressive males, at least in some species of birds and rodents (review: [49]). This is because VP released into the anterior pituitary (presumably derived from the PVN) is a potent secretagogue for adrenocorticotropic hormone (ACTH), and in fact, VP may be a more potent ACTH secretagogue in birds than is corticotropin-releasing hormone [77].

4.3. OT modulation of behavioral and hormonal stress responses: a resolution to the paradox? Although we have focused primarily on the relationship of OT to aggression, the most obvious effect obtained here is the robust activation of OT neurons following exposure to stressors such as social subjugation or pursuit by a potential human “predator.” Consistent with this observation, OT reduces HPA responses to a wide variety of social and physical stressors in rodents [51,78], and comparable findings are obtained in primates, including humans [40,79–81]. In general, OT dampens autonomic responses to stress, as well (rodents: [82,83]), and modulates behavioral aspects of stress coping. The behavioral effects on stress coping are complex. For instance, intraventricular infusions of OT or OT agonist decrease immobility in the mouse tail suspension test [84] and forced swim test [85,86], with some evidence that these effects are mediated via the OTR [85]; however site-specific OT manipulations in the central amygdala of rats produce an opposite pattern of results in the forced swim assay [87], suggesting that there are site-specific, species-specific, or other complex OT effects on stress coping that are not yet fully understood. PVN OT neurons likewise promote passive stress coping in zebra finches [4]. Interestingly, whereas OT effects on stress coping and physiological stress responses are observed in a diversity of nonsocial paradigms, such as forced swimming and restraint [4,87], effects on anxiety-like behaviors tend to be restricted to social and reproductive contexts [50]. Moreover, it is important to note that most anxiety research involves behavioral tests of animals in a non-social context, which may represent a confound to conclusions drawn about the role of OT in anxiety, particularly when this research is compared to research in humans or non-human primates focusing on social anxiety [40,79–81]. Our hypotheses for the present experiments were focused largely on the central, behavioral functions of OT. Thus, we hypothesized that OT would be at least permissive for aggression, based on findings that socially induced OT release in mammals is anxiolytic [88] and that OT promotes active stress coping, at least in some paradigms [89]. However, whereas the behavioral effects of OTR antagonism are consistent with those hypotheses (i.e., antagonism reduces aggression), the results of the OT-Fos experiment are not. Specifically, if the antagonist results reflect OT effects on anxiolysis and/or stress coping, then OT-Fos coexpression should correlate positively with aggression, which is not the case.

160

J.L. Goodson et al. / Physiology & Behavior 141 (2015) 154–163

The most parsimonious resolution to this apparent paradox is complex. First, if OT exerts tonic inhibitory effects on the HPA axis in waxbills, as shown in rats [51], then peripheral OTR antagonism should increase basal HPA activity. Given that corticosterone (CORT) inhibits territorial behavior in songbirds [90,91], OTR antagonism could thereby reduce aggression via increases in CORT release. Notably, in contrast to the Fos experiment, the antagonist experiment was conducted in the home cage environment and entailed only a single handling 30 min prior to testing. Hence, findings from the antagonist experiment may reflect tonic inhibitory OT effects on the HPA axis, not stress-induced actions of OT. This idea is consistent with our antagonist data, but we would also expect OT-Fos co-labeling to correlate positively with aggression (based on the OT inhibition of CORT secretion), which is not the case (Fig. 4). However, the level of stress entailed in the two experiments was likely very different, given that subjects were housed in small sound booths for the Fos experiment and were handled both before and after testing. Indeed, the behavioral data do suggest that the Fos testing environment was stressful, given that the dominant birds were significantly less aggressive (by ~80%) when tested in the sound booths, relative to testing in the home environment (Fig. 5). Whether the acclimation, housing and handling conditions in the Fos experiment may have represented an acute, chronic or acute short-term stressor is currently unclear. It is also worth noting that VEWs may be more territorial in their home cage (which was also the cage they occupied with their mate) used for the antagonist experiment in contrast to the testing cage used for the Fos experiment that the animals had occupied the night before testing. If the testing conditions for the Fos experiment induced a stress response, and hence induced OT-Fos co-labeling across all subjects, we would expect that the more stress-responsive birds would exhibit greater OT-Fos co-labeling. Thus, it may be that aggressive male waxbills are hyporesponsive to stress, in terms of CORT secretion, as shown in other species [43–47]. This does not mean that OT neurons (particularly parvocellular PVN neurons, which project to the anterior pituitary) are not still exerting inhibitory effects on the HPA axis. Rather, we hypothesize that the more aggressive birds simply require less of a “brake” on that axis in order to exhibit aggression, since they are less stress responsive. In this scenario, the stress-induced OT-Fos response is effectively masking the underlying positive association between OT and aggression, which we hypothesize is mediated via OT's inhibition of the HPA. An alternative explanation for the negative correlation between OT-Fos co-labeling and aggression in dominate animals is that more aggressive animals may acclimate faster to testing conditions rather than being hyporesponsive to stress. However, we would still hypothesize that the more aggressive birds require less of a “brake” on the HPA axis to display aggression due to less OT-Fos co-labeling as compared to less aggressive birds. Both interpretations yield the testable hypothesis that baseline CORT will correlate negatively with aggression. Because we no longer maintain a lab population of violet-eared waxbills, and that they are not commercially available, we cannot address this hypothesis directly (note that our long-term population of waxbills was originally derived from African birds that we collected in the wild). Nonetheless, our data do suggest that the Fos testing environment was stressful, as addressed above. An important implication of this hypothesis is that it might explain the inconsistent results obtained following central OTR antagonist infusions in male rats, in which no effects on aggression are observed [20,21], and those obtained here following peripheral antagonist infusions in male and female violet-eared waxbills, in which aggression is reduced. In contrast to peripheral injections, central antagonist infusions likely have little or no access to the anterior pituitary, and thus the seemingly inconsistent results may reflect differences in the sites of potential action. We must also consider that OT cell groups of the SON, MPO and AH may each play a unique role in stress response that is somewhat

uncoupled from the functions of the PVN and other OT neuronal populations. The present data suggest that this is unlikely, given that the patterns of neuronal activation for each cell group are generally similar, as are their relationships to behavior. This is not to say that each cell group does not modulate different aspects of the stress response (autonomic, hormonal and behavioral), but rather that those activities appear to be coordinated. In fact, recent experiments in zebra finches demonstrate that complex dimensions of behavioral phenotype (social competence/dominance, gregariousness, and anxiety) are all significantly predicted by functional interactions across multiple OT-VP cell groups [60]. Nonetheless, if OT's relationship to aggression is primarily mediated via effects on CORT, we should expect that the OT neurons of the PVN (which project to the anterior pituitary) will show the strongest coupling to behavior. As shown in Fig. 4, this is indeed the case. 4.4. Evolving perspectives on a “prosocial” peptide A wide variety of work shows that OT promotes affiliation behaviors across the vertebrate taxa, including maternal care, trust, and pair bonding [7,9,54]. This has led to a popular view of OT as a “cuddle hormone” and it is increasingly common for behavioral scientists to present OT as a molecule that selectively promotes prosocial behavior [29,92–94]. The tendency to view OT in this light is somewhat understandable, because the vast majority of relevant literature does show that OT promotes affiliation. Problematically, however, other possible functions have not been examined in detail, despite the very broad effects that OT exerts on autonomic, sensory, and anxiety processes [7,95]. This lack of experimental breadth is particularly of concern with regard to offensive aggression. Only a single study has examined the effects of site-specific OTR antagonism on offensive aggression in adult animals. That study demonstrates that OT acts within the POA-AH to reduce resident-intruder aggression in female Syrian hamsters [31], although endogenous OT acts within the central amygdala and paraventricular hypothalamus to facilitate maternal aggression in rats [18]. Whether these different effects in hamsters and rats relate to the differences in social context and/or specific brain region remains to be determined. Although two additional studies have shown that intraventricular infusions of exogenous OT reduce male-male aggression in rats, no facilitation of aggression is observed following OTR antagonism [20, 21]. In light of these results and those presented in Fig. 5, particular attention should be paid to the species, sex, phenotype and experimental context in which the behavior is being measured. For instance, in another study examining aggression in male VEWs, a V1aR antagonist reduced mate competition aggression but had no effect on residentintruder aggression or male/male aggression in a novel cage, highlighting the importance of experimental context when measuring aggressive behavior [59]. Furthermore, the V1aR antagonist actually increased aggression in subordinate, less aggressive males within the resident intruder paradigm (with no effect in dominant males), demonstrating phenotypic-specific effects, in addition to context-specific effects [59]. We now show that an OTR antagonist significantly reduces aggression in both male and female violet-eared waxbills, indicating that endogenous activation of the avian OTR (VT3) is at least permissive for aggression. The magnitude of the effect is particularly noteworthy in females, in which displacements are reduced by an average of 71%. Importantly, an iodinated form of the OTR antagonist used here exhibits a pattern of binding that is closely matched to the distribution of OTR (VT3) mRNA [33,38]. Hence, we are confident that the pharmacological results reflect endogenous effects of OTR activation. Furthermore, previous experiments using peripheral and central delivery of this OTR antagonist have shown that the different routes of administration yield similar results [12], suggesting that the antagonist does gain entrance to the brain, although we propose that the present findings reflect actions at the level of the pituitary (see previous section). Importantly, although our findings our novel with respect to aggression, they are certainly not the first to show that OT can promote

J.L. Goodson et al. / Physiology & Behavior 141 (2015) 154–163

negative behavior and perceptions, as is increasingly well documented in humans. For instance, intranasal OT administration reduces trust and cooperation in borderline personality patients [96], and promotes parochial altruism, ethnocentrism, and out-group derogation in healthy men [97,98]. Recent work also shows that, in contrast to acute OT administrations [99], chronic intranasal OT administrations actually impair pair bonding in male prairie voles (as defined by a reduction in preference for the familiar partner [100]; but see [101]) and promote aggression in pigs, as well as dysregulate the HPA axis [26]. Furthermore, although not an antisocial effect, intranasal OT administrations reduce activity in the nucleus accumbens of men viewing pictures of their own children relative to viewing pictures of other children, suggesting that OT does not increase the “social reward” associated with father-offspring bonds [102]. The effects of a single OT administration during development on the display of adult social behaviors are equally complex. Female prairie voles that received a low-dose of OT took longer to approach their pups but developed a partner preference like controls in contrast to females that received a high OT dose which retrieved their pups more often but failed to develop a partner preference [103]. Finally, after a single OT administration at birth, female prairie voles (but not males) displayed increased aggression and less social behavior compared to control females or females injected with an OTA antagonist at birth [24]. Thus, the present dataset is but one piece of a larger body of evidence that undermines the simplistic notion that OT is a “prosocial neuropeptide” [29,94]. Whether the present findings are predictive for other taxa remains to be determined, but this seems likely, given the extensive similarities in OT anatomy and function across vertebrate classes. For instance, OTRs in both birds and mammals promote maternal care [9,104], facilitate various aspects of same-sex affiliation (e.g., flocking in gregarious finches, [7]), and are necessary for pair bonding in prairie voles and zebra finches [54,105]. 5. Conclusions Although affiliation-related effects of oxytocic signaling have been addressed in hundreds of studies, only three prior experiments have examined the relationships between oxytocic signaling and same-sex aggression using OT manipulations in adult animals [20,21,31]. Of these, only one has demonstrated a function for endogenous OTR-mediated signaling, reflecting site-specific actions in the POA-AH. Using peripheral injections of an OTR antagonist that is known to bind to selectively to the avian OTR, we have here demonstrated that endogenous activation of OTRs is required for the normal display of both male and female aggression in the highly territorial violet-eared waxbill. Importantly, this experiment was conducted in the home cage environment with minimal handling. We further demonstrate that multiple OT cell groups exhibit significant Fos responses to the stressors of subjugation and pursuit by a human hand. However, evidence suggests that all subjects were stressed by the experimental environment (which included isolation in a sound booth and multiple handlings), and we find that OT-Fos co-labeling actually correlates negatively with aggression. This is observed despite the fact that Dominant animals do not differ from Controls in OT-Fos co-labeling. After considering and discarding various alternative hypotheses, we propose that this negative relationship reflects a greater stress response in the less aggressive animals, and hence greater OT neuronal activation and HPA response. Considering that the more aggressive animals are likely hyporesponsive to stress, as shown in other species [43–47], we hypothesize that the more aggressive animals require less OT-mediated inhibition of the HPA axis, although HPA inhibition is nonetheless required for the normal display of aggression. The present results suggest the importance of considering peripherally-mediated effects of OTR antagonism, and yield multiple testable hypotheses regarding the interrelationships between behavioral phenotype, OT signaling, CORT and aggression that should be addressed in future experiments.

161

Acknowledgments Funding for this work was provided by Indiana University. This paper is dedicated in loving memory to Jim Goodson (November 17, 1965–August, 14, 2014), who advocated for a comprehensive understanding of nonapeptide signaling via the examination of site-specific effects in the brain, neuronal modulation throughout the social behavior network, and the consideration of sex, species, phenotype and behavioral context when measuring social behaviors.

References [1] R. Acher, J. Chauvet, The neurohypophysial endocrine regulatory cascade: precursors, mediators, receptors, and effectors, Front. Neuroendocrinol. 16 (1995) 237–289. [2] A.G. Lee, D.R. Cool, W.C. Grunwald Jr., D.E. Neal, C.L. Buckmaster, M.Y. Cheng, et al., A novel form of oxytocin in New World monkeys, Biol. Lett. 7 (2011) 584–587. [3] A.M. Kelly, J.L. Goodson, Social functions of individual vasopressin-oxytocin cell groups in vertebrates: what do we really know? Front. Neuroendocrinol. 35 (2014) 512–529. [4] A.M. Kelly, J.L. Goodson, Hypothalamic oxytocin and vasopressin neurons exert sex-specific effects on pair bonding, gregariousness and aggression in finches, Proc. Natl. Acad. Sci. U. S. A. 111 (2014) 6069–6074. [5] D. Ocampo Daza, M. Lewicka, D. Larhammar, The oxytocin/vasopressin receptor family has at least five members in the gnathostome lineage, including two distinct V2 subtypes, Gen. Comp. Endocrinol. 175 (2012) 135–143. [6] Y. Yamaguchi, H. Kaiya, N. Konno, E. Iwata, M. Miyazato, M. Uchiyama, et al., The fifth neurohypophysial hormone receptor is structurally related to the V2-type receptor but functionally similar to V1-type receptors, Gen. Comp. Endocrinol. 178 (2012) 519–528. [7] J.L. Goodson, R.R. Thompson, Nonapeptide mechanisms of social cognition, behavior and species-specific social systems, Curr. Opin. Neurobiol. 20 (2010) 784–794. [8] C.S. Carter, A.J. Grippo, H. Pournajafi-Nazarloo, M.G. Ruscio, S.W. Porges, Oxytocin, vasopressin and sociality, Prog. Brain Res. 170 (2008) 331–336. [9] I.D. Neumann, The advantage of social living: brain neuropeptides mediate the beneficial consequences of sex and motherhood, Front. Neuroendocrinol. 30 (2009) 483–496. [10] A.H. Veenema, I.D. Neumann, Central vasopressin and oxytocin release: regulation of complex social behaviours, Prog. Brain Res. 170 (2008) 261–276. [11] R.R. Thompson, J.C. Walton, Peptide effects on social behavior: effects of vasotocin and isotocin on social approach behavior in male goldfish (Carassius auratus), Behav. Neurosci. 118 (2004) 620–626. [12] J.L. Goodson, S.E. Schrock, J.D. Klatt, D. Kabelik, M.A. Kingsbury, Mesotocin and nonapeptide receptors promote estrildid flocking behavior, Science 325 (2009) 862–866. [13] J.L. Goodson, L.C. Wilson, S.E. Schrock, To flock or fight: neurochemical signatures of divergent life histories in sparrows, Proc. Natl. Acad. Sci. U. S. A. 109 (Suppl. 1) (2012) 10685–10692. [14] J.P. Herman, J. Flak, R. Jankord, Chronic stress plasticity in the hypothalamic paraventricular nucleus, Prog. Brain Res. 170 (2008) 353–364. [15] B. Robinzon, T.I. Koike, H.L. Neldon, S.L. Kinzler, I.R. Hendry, M.E. el Halawani, Physiological effects of arginine vasotocin and mesotocin in cockerels, Br. Poult. Sci. 29 (1988) 639–652. [16] G. Leng, T. Onaka, C. Caquineau, N. Sabatier, V.A. Tobin, Y. Takayanagi, Oxytocin and appetite, Prog. Brain Res. 170 (2008) 137–151. [17] D. Atasoy, J.N. Betley, H.H. Su, S.M. Sternson, Deconstruction of a neural circuit for hunger, Nature 488 (2012) 172–177. [18] O.J. Bosch, S.L. Meddle, D.I. Beiderbeck, A.J. Douglas, I.D. Neumann, Brain oxytocin correlates with maternal aggression: link to anxiety, J. Neurosci. 25 (2005) 6807–6815. [19] D.A. Lubin, J.C. Elliott, M.C. Black, J.M. Johns, An oxytocin antagonist infused into the central nucleus of the amygdala increases maternal aggressive behavior, Behav. Neurosci. 117 (2003) 195–201. [20] F. Calcagnoli, S.F. de Boer, M. Althaus, J.A. den Boer, J.M. Koolhaas, Antiaggressive activity of central oxytocin in male rats, Psychopharmacology (Berlin) 229 (2013) 639–651. [21] F. Calcagnoli, N. Meyer, S.F. de Boer, M. Althaus, J.M. Koolhaas, Chronic enhancement of brain oxytocin levels causes enduring anti-aggressive and pro-social explorative behavioral effects in male rats, Horm. Behav. 65 (2014) 427–433. [22] M. Sala, D. Braida, D. Lentini, M. Busnelli, E. Bulgheroni, V. Capurro, et al., Pharmacologic rescue of impaired cognitive flexibility, social deficits, increased aggression, and seizure susceptibility in oxytocin receptor null mice: a neurobehavioral model of autism, Biol. Psychiatry 69 (2011) 875–882. [23] A. Schorscher-Petcu, S. Sotocinal, S. Ciura, A. Dupre, J. Ritchie, R.E. Sorge, et al., Oxytocin-induced analgesia and scratching are mediated by the vasopressin-1A receptor in the mouse, J. Neurosci. 30 (2010) 8274–8284. [24] K.L. Bales, C.S. Carter, Sex differences and developmental effects of oxytocin on aggression and social behavior in prairie voles (Microtus ochrogaster), Horm. Behav. 44 (2003) 178–184. [25] R. Jia, F.D. Tai, S.C. An, H. Broders, X.L. Ding, Q. Kong, et al., Effects of neonatal oxytocin treatment on aggression and neural activities in mandarin voles, Physiol. Behav. 95 (2008) 56–62.

162

J.L. Goodson et al. / Physiology & Behavior 141 (2015) 154–163

[26] J.L. Rault, C.S. Carter, J.P. Garner, J.N. Marchant-Forde, B.T. Richert, D.C. Lay Jr., Repeated intranasal oxytocin administration in early life dysregulates the HPA axis and alters social behavior, Physiol. Behav. 112–113 (2013) 40–48. [27] A.C. DeVries, W.S. Young III, R.J. Nelson, Reduced aggressive behaviour in mice with targeted disruption of the oxytocin gene, J. Neuroendocrinol. 9 (1997) 363–368. [28] K. Macdonald, T.M. Macdonald, The peptide that binds: a systematic review of oxytocin and its prosocial effects in humans, Harv. Rev. Psychiatry 18 (2010) 1–21. [29] A. Meyer-Lindenberg, Impact of prosocial neuropeptides on human brain function, Prog. Brain Res. 170 (2008) 463–470. [30] N. Striepens, K.M. Kendrick, W. Maier, R. Hurlemann, Prosocial effects of oxytocin and clinical evidence for its therapeutic potential, Front. Neuroendocrinol. 32 (2011) 426–450. [31] A.C. Harmon, K.L. Huhman, T.O. Moore, H.E. Albers, Oxytocin inhibits aggression in female Syrian hamsters, J. Neuroendocrinol. 14 (2002) 963–969. [32] S.J. Gutzler, M. Karom, W.D. Erwin, H.E. Albers, Arginine-vasopressin and the regulation of aggression in female Syrian hamsters (Mesocricetus auratus), Eur. J. Neurosci. 31 (2010) 1655–1663. [33] C.H. Leung, C.T. Goode, L.J. Young, D.L. Maney, Neural distribution of nonapeptide binding sites in two species of songbird, J. Comp. Neurol. 513 (2009) 197–208. [34] Y. Liu, J.T. Curtis, Z. Wang, Vasopressin in the lateral septum regulates pair bond formation in male prairie voles (Microtus ochrogaster), Behav. Neurosci. 115 (2001) 910–919. [35] B.T. Searcy, C.S. Bradford, R.R. Thompson, T.M. Filtz, F.L. Moore, Identification and characterization of mesotocin and V1a-like vasotocin receptors in a urodele amphibian, Taricha granulosa, Gen. Comp. Endocrinol. 170 (2011) 131–143. [36] T.R. Insel, R. Gelhard, L.E. Shapiro, The comparative distribution of forebrain receptors for neurohypophyseal peptides in monogamous and polygamous mice, Neuroscience 43 (1991) 623–630. [37] T.R. Insel, L.E. Shapiro, Oxytocin receptor distribution reflects social organization in monogamous and polygamous voles, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 5981–5985. [38] C.H. Leung, D.F. Abebe, S.E. Earp, C.T. Goode, A.V. Grozhik, P. Mididoddi, et al., Neural distribution of vasotocin receptor mRNA in two species of songbird, Endocrinology 152 (2011) 4865–4881. [39] A.K. Beery, E.A. Lacey, D.D. Francis, Oxytocin and vasopressin receptor distributions in a solitary and a social species of tuco-tuco (Ctenomys haigi and Ctenomys sociabilis), J. Comp. Neurol. 507 (2008) 1847–1859. [40] M. Quirin, J. Kuhl, R. Dusing, Oxytocin buffers cortisol responses to stress in individuals with impaired emotion regulation abilities, Psychoneuroendocrinology 36 (2011) 898–904. [41] H.S. Knobloch, A. Charlet, L.C. Hoffmann, M. Eliava, S. Khrulev, A.H. Cetin, et al., Evoked axonal oxytocin release in the central amygdala attenuates fear response, Neuron 73 (2012) 553–566. [42] L.D. Kubzansky, W.B. Mendes, A.A. Appleton, J. Block, G.K. Adler, A heartfelt response: oxytocin effects on response to social stress in men and women, Biol. Psychol. 90 (2012) 1–9. [43] K. Ebner, C.T. Wotjak, R. Landgraf, M. Engelmann, Neuroendocrine and behavioral response to social confrontation: residents versus intruders, active versus passive coping styles, Horm. Behav. 47 (2005) 14–21. [44] C. Carere, T.G. Groothuis, E. Mostl, S. Daan, J.M. Koolhaas, Fecal corticosteroids in a territorial bird selected for different personalities: daily rhythm and the response to social stress, Horm. Behav. 43 (2003) 540–548. [45] S. Kralj-Fiser, I.B. Scheiber, A. Blejec, E. Moestl, K. Kotrschal, Individualities in a flock of free-roaming greylag geese: behavioral and physiological consistency over time and across situations, Horm. Behav. 51 (2007) 239–248. [46] A.H. Veenema, J.M. Koolhaas, E.R. de Kloet, Basal and stress-induced differences in HPA axis, 5-HT responsiveness, and hippocampal cell proliferation in two mouse lines, Ann. N. Y. Acad. Sci. 1018 (2004) 255–265. [47] O. Overli, C. Sorensen, K.G.T. Pulman, T.G. Pottinger, W. Korzan, C.H. Summers, et al., Evolutionary background for stress-coping styles: relationships between physiological, behavioral, and cognitive traits in non-mammalian vertebrates, Neurosci. Biobehav. Rev. 31 (2007) 396–412. [48] J.M. Koolhaas, S.F. de Boer, B. Buwalda, K. van Reenen, Individual variation in coping with stress: a multidimensional approach of ultimate and proximate mechanisms, Brain Behav. Evol. 70 (2007) 218–226. [49] J.M. Koolhaas, S.F. de Boer, C.M. Coppens, B. Buwalda, Neuroendocrinology of coping styles: towards understanding the biology of individual variation, Front. Neuroendocrinol. 31 (2010) 307–321. [50] I.D. Neumann, L. Torner, A. Wigger, Brain oxytocin: differential inhibition of neuroendocrine stress responses and anxiety-related behaviour in virgin, pregnant and lactating rats, Neuroscience 95 (2000) 567–575. [51] I.D. Neumann, A. Wigger, L. Torner, F. Holsboer, R. Landgraf, Brain oxytocin inhibits basal and stress-induced activity of the hypothalamo–pituitary–adrenal axis in male and female rats: partial action within the paraventricular nucleus, J. Neuroendocrinol. 12 (2000) 235–243. [52] J.L. Goodson, M.A. Kingsbury, Nonapeptides and the evolution of social group sizes in birds, Front. Neuroanat. 5 (2011) 13. [53] D. Goodwin, Estrildid Finches of the World, Cornell University Press, Ithaca, NY, 1982. [54] H.E. Ross, L.J. Young, Oxytocin and the neural mechanisms regulating social cognition and affiliative behavior, Front. Neuroendocrinol. 30 (2009) 534–547. [55] M. Engelmann, C.T. Wotjak, K. Ebner, R. Landgraf, Behavioural impact of intraseptally released vasopressin and oxytocin in rats, Exp. Physiol. 85 (2000) (Spec No:125S-30S). [56] M. Ludwig, G. Leng, Dendritic peptide release and peptide-dependent behaviours, Nat. Rev. Neurosci. 7 (2006) 126–136.

[57] J.D. Klatt, J.L. Goodson, Sex-specific activity and function of hypothalamic nonapeptide neurons during nest-building in zebra finches, Horm. Behav. 64 (2013) 818–824. [58] J.L. Goodson, Vasotocin and vasoactive intestinal polypeptide modulate aggression in a territorial songbird, the violet-eared waxbill (Estrildidae: Uraeginthus granatina), Gen. Comp. Endocrinol. 111 (1998) 233–244. [59] J.L. Goodson, D. Kabelik, S.E. Schrock, Dynamic neuromodulation of aggression by vasotocin: influence of social context and social phenotype in territorial songbirds, Biol. Lett. 5 (2009) 554–556. [60] A.M. Kelly, J.L. Goodson, Personality is tightly coupled to vasopressin–oxytocin neuron activity in a gregarious finch, Front. Behav. Neurosci. 8 (2014) 55. [61] S.J. Alger, S.N. Maasch, L.V. Riters, Lesions to the medial preoptic nucleus affect immediate early gene immunolabeling in brain regions involved in song control and social behavior in male European starlings, Eur. J. Neurosci. 29 (2009) 970–982. [62] J.L. Goodson, A.K. Evans, A.H. Bass, Putative isotocin distributions in sonic fish: relation to vasotocin and vocal-acoustic circuitry, J. Comp. Neurol. 462 (2003) 1–14. [63] J.L. Goodson, D. Kabelik, A.M. Kelly, J. Rinaldi, J.D. Klatt, Midbrain dopamine neurons reflect affiliation phenotypes in finches and are tightly coupled to courtship, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 8737–8742. [64] J.L. Goodson, Y. Wang, Valence-sensitive neurons exhibit divergent functional profiles in gregarious and asocial species, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 17013–17017. [65] J.L. Goodson, A.K. Kelly, M.A. Kingsbury, R.R. Thompson, An aggression-specific cell type in the hypothalamus of finches, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 13847–13852. [66] J.L. Goodson, Territorial aggression and dawn song are modulated by septal vasotocin and vasoactive intestinal polypeptide in male field sparrows (Spizella pusilla), Horm. Behav. 34 (1998) 67–77. [67] D. Kabelik, J.D. Klatt, M.A. Kingsbury, J.L. Goodson, Endogenous vasotocin exerts context-dependent behavioral effects in a semi-naturalistic colony environment, Horm. Behav. 56 (2009) 101–107. [68] J.L. Goodson, D. Kabelik, Dynamic limbic networks and social diversity in vertebrates: from neural context to neuromodulatory patterning, Front. Neuroendocrinol. 30 (2009) 429–441. [69] J.M. Ho, J.H. Murray, G.E. Demas, J.L. Goodson, Vasopressin cell groups exhibit strongly divergent responses to copulation and male–male interactions in mice, Horm. Behav. 58 (2010) 368–377. [70] J. Xie, W.J. Kuenzel, P.J. Sharp, A. Jurkevich, Appetitive and consummatory sexual and agonistic behaviour elicits FOS expression in aromatase and vasotocin neurones within the preoptic area and bed nucleus of the stria terminalis of male domestic chickens, J. Neuroendocrinol. 23 (2011) 232–243. [71] A.M. Kelly, J.L. Goodson, Functional significance of a phylogenetically widespread sexual dimorphism in vasotocin/vasopressin production, Horm. Behav. 64 (2013) 840–846. [72] J.C. Compaan, R.M. Buijs, C.W. Pool, A.J.H. Deruiter, J.M. Koolhaas, Differential lateral septal vasopressin innervation in aggressive and nonaggressive male mice, Brain Res. Bull. 30 (1993) 1–6. [73] C.F. Ferris, R.H. Melloni, G. Koppel, K.W. Perry, R.W. Fuller, Y. Delville, Vasopressin/ serotonin interactions in the anterior hypothalamus control aggressive behavior in golden hamsters, J. Neurosci. 17 (1997) 4331–4340. [74] H.E. Albers, The regulation of social recognition, social communication and aggression: vasopressin in the social behavior neural network, Horm. Behav. 61 (2012) 283–292. [75] K.L. Gobrogge, Y. Liu, L.J. Young, Z. Wang, Anterior hypothalamic vasopressin regulates pair-bonding and drug-induced aggression in a monogamous rodent, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 19144–19149. [76] H.E. Albers, A. Dean, M.C. Karom, D. Smith, K.L. Huhman, Role of V1a vasopressin receptors in the control of aggression in Syrian hamsters, Brain Res. 1073–1074 (2006) 425–430. [77] W.J. Kuenzel, A. Jurkevich, Molecular neuroendocrine events during stress in poultry, Poult. Sci. 89 (2010) 832–840. [78] R.J. Windle, N. Shanks, S.L. Lightman, C.D. Ingram, Central oxytocin administration reduces stress-induced corticosterone release and anxiety behavior in rats, Endocrinology 138 (1997) 2829–2834. [79] S.S. Hall, A.A. Lightbody, B.E. McCarthy, K.J. Parker, A.L. Reiss, Effects of intranasal oxytocin on social anxiety in males with fragile X syndrome, Psychoneuroendocrinology 37 (2012) 509–518. [80] M. Heinrichs, T. Baumgartner, C. Kirschbaum, U. Ehlert, Social support and oxytocin interact to suppress cortisol and subjective responses to psychosocial stress, Biol. Psychiatry 54 (2003) 1389–1398. [81] K.J. Parker, C.L. Buckmaster, A.F. Schatzberg, D.M. Lyons, Intranasal oxytocin administration attenuates the ACTH stress response in monkeys, Psychoneuroendocrinology 30 (2005) 924–929. [82] A.J. Grippo, D.M. Trahanas, R.R. Zimmerman II, S.W. Porges, C.S. Carter, Oxytocin protects against negative behavioral and autonomic consequences of long-term social isolation, Psychoneuroendocrinology 34 (2009) 1542–1553. [83] A.J. Grippo, H. Pournajafi-Nazarloo, L. Sanzenbacher, D.M. Trahanas, N. McNeal, D.A. Clarke, et al., Peripheral oxytocin administration buffers autonomic but not behavioral responses to environmental stressors in isolated prairie voles, Stress 15 (2012) 149–161. [84] R.H. Ring, L.E. Schechter, S.K. Leonard, J.M. Dwyer, B.J. Platt, R. Graf, et al., Receptor and behavioral pharmacology of WAY-267464, a non-peptide oxytocin receptor agonist, Neuropharmacology 58 (2010) 69–77.

J.L. Goodson et al. / Physiology & Behavior 141 (2015) 154–163 [85] S. Chaviaras, P. Mak, D. Ralph, L. Krishnan, J.H. Broadbear, Assessing the antidepressant-like effects of carbetocin, an oxytocin agonist, using a modification of the forced swimming test, Psychopharmacology (Berlin) 210 (2010) 35–43. [86] E. Loyens, D. De Bundel, H. Demaegdt, S.Y. Chai, P. Vanderheyden, Y. Michotte, et al., Antidepressant-like effects of oxytocin in mice are dependent on the presence of insulin-regulated aminopeptidase, Int. J. Neuropsychopharmacol. (2012) 1–11. [87] K. Ebner, O.J. Bosch, S.A. Kromer, N. Singewald, I.D. Neumann, Release of oxytocin in the rat central amygdala modulates stress-coping behavior and the release of excitatory amino acids, Neuropsychopharmacology 30 (2005) 223–230. [88] A.S. Smith, Z. Wang, Salubrious effects of oxytocin on social stress-induced deficits, Horm. Behav. 61 (2012) 320–330. [89] I.D. Neumann, R. Landgraf, Balance of brain oxytocin and vasopressin: implications for anxiety, depression, and social behaviors, Trends Neurosci. 35 (2012) 649–659. [90] S.L. Meddle, L.M. Romero, L.B. Astheimer, W.A. Buttemer, I.T. Moore, J.C. Wingfield, Steroid hormone interrelationships with territorial aggression in an Arcticbreeding songbird, Gambel's white-crowned sparrow, Zonotrichia leucophrys gambelii, Horm. Behav. 42 (2002) 212–221. [91] L.M. Romero, S.C. Dean, J.C. Wingfield, Neurally active stress peptide inhibits territorial defense in wild birds, Horm. Behav. 34 (1998) 239–247. [92] S.M. van Anders, K.L. Goldey, P.X. Kuo, The steroid/peptide theory of social bonds: integrating testosterone and peptide responses for classifying social behavioral contexts, Psychoneuroendocrinology 36 (2011) 1365–1375. [93] P.J. Zak, The physiology of moral sentiments, J. Econ. Behav. Organ. 77 (2011) 53–65. [94] A. Meyer-Lindenberg, G. Domes, P. Kirsch, M. Heinrichs, Oxytocin and vasopressin in the human brain: social neuropeptides for translational medicine, Nat. Rev. Neurosci. 12 (2011) 524–538.

163

[95] J.L. Goodson, Deconstructing sociality, social evolution, and relevant nonapeptide functions, Psychoneuroendocrinology 38 (2013) 465–478. [96] J.A. Bartz, J. Zaki, N. Bolger, K.N. Ochsner, Social effects of oxytocin in humans: context and person matter, Trends Cogn. Sci. 15 (2011) 301–309. [97] C.K. De Dreu, L.L. Greer, G.A. Van Kleef, S. Shalvi, M.J. Handgraaf, Oxytocin promotes human ethnocentrism, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 1262–1266. [98] C.K. De Dreu, L.L. Greer, M.J. Handgraaf, S. Shalvi, G.A. Van Kleef, M. Baas, et al., The neuropeptide oxytocin regulates parochial altruism in intergroup conflict among humans, Science 328 (2010) 1408–1411. [99] K.L. Bales, C.S. Carter, Developmental exposure to oxytocin facilitates partner preferences in male prairie voles (Microtus ochrogaster), Behav. Neurosci. 117 (2003) 854–859. [100] K.L. Bales, A.M. Perkeybile, O.G. Conley, M.H. Lee, C.D. Guoynes, G.M. Downing, et al., Chronic intranasal oxytocin causes long-term impairments in partner preference formation in male prairie voles, Biol. Psychiatry 74 (2012) 180–188. [101] L.J. Young, When too much of a good thing is bad: chronic oxytocin, development, and social impairments, Biol. Psychiatry 74 (2013) 160–161. [102] D. Wittfoth-Schardt, J. Grunding, M. Wittfoth, H. Lanfermann, M. Heinrichs, G. Domes, et al., Oxytocin modulates neural reactivity to children's faces as a function of social salience, Neuropsychopharmacology 37 (2012) 1799–1807. [103] K.L. Bales, J.A. van Westerhuyzen, A.D. Lewis-Reese, N.D. Grotte, J.A. Lanter, C.S. Carter, Oxytocin has dose-dependent developmental effects on pair-bonding and alloparental care in female prairie voles, Horm. Behav. 52 (2007) 274–279. [104] A. Thayananuphat, O.M. Youngren, S.W. Kang, T. Bakken, S. Kosonsiriluk, Y. Chaiseha, et al., Dopamine and mesotocin neurotransmission during the transition from incubation to brooding in the turkey, Horm. Behav. 60 (2011) 327–335. [105] J.D. Klatt, J.L. Goodson, Oxytocin-like receptors mediate pair bonding in a socially monogamous songbird, Proc. R. Soc. Lond. B Biol. Sci. 280 (2013) 20122396.