Testosterone facilitates nonreproductive, context-appropriate pro- and anti-social behavior in female and male Mongolian gerbils.

A growing body of literature suggests that testosterone (T) rapidly modulates behavior in a context-specific manner. However, the timescales in which T can rapidly mediate distinct types of behavior, such as pro- vs. anti- social responses, has not been studied. Thus, here we examined acute T influences on social behavior in male and female Mongolian gerbils in nonreproductive contexts. Females and males received an injection of either saline or T and were first tested in a social interaction test with a same-sex, familiar peer. 5 min after the peer interaction, subjects then underwent a resident-intruder test with a novel, same-sex conspecific. After another 5 min, gerbils were tested in a novel object task to test context-specificity (i.e., social vs. nonsocial) of T effects on behavior. Within 1 h, males and females injected with T exhibited more huddling with a peer but more active avoidance of and less time spent in proximity of an intruder than did animals injected with saline. T effects on behavior were specific to social contexts, such that T did not influence investigation of the novel object. Together these findings show that T rapidly promotes pro-social responses to a familiar peer and anti-social responses to an intruder in the same individuals within 5 min of experiencing these disparate social contexts. This demonstrates that T rapidly facilitates behavior in a context-appropriate manner outside the context of reproduction and reveals that rapid effects of T on behavior are not restricted to males.

Beyond sex and aggression: testosterone rapidly matches behavioural responses to social context and tries to predict the future.

Although androgens are widely studied in the context of aggression, androgenic influences on prosocial behaviours have been less explored. We examined testosterone's (T) influence on prosocial and aggressive responses in a positively valenced social context (interacting with a pairbond partner) and a negatively valenced context (interacting with an intruder) in socially monogamous Mongolian gerbils. T increased and decreased prosocial responses in the same individuals towards a pairbond partner and an intruder, respectively, both within 30 min, but did not affect aggression. T also had persistent effects on prosocial behaviour; males in which T initially increased prosocial responses towards a partner continued to exhibit elevated prosocial responses towards an intruder male days later until a second T injection rapidly eliminated those responses. Thus, T surges can rapidly match behaviour to current social context, as well as prime animals for positive social interactions in the future. Neuroanatomically, T rapidly increased hypothalamic oxytocin, but not vasopressin, cellular responses during interactions with a partner. Together, our results indicate that T can facilitate and inhibit prosocial behaviours depending on social context, that it can influence prosocial responses across rapid and prolonged time scales, and that it affects oxytocin signalling mechanisms that could mediate its context-dependent behavioural influences.

An updated field guide for snark hunting: Comparative contributions to behavioral neuroendocrinology in the era of model organisms.

Studying neuroendocrine behavioral regulatory mechanisms in a variety of species across vertebrate groups is critical for determining how they work in natural contexts, how they evolved, and ultimately what can be generalized from them, potentially even to humans. All of the above are difficult, at best, if work within our field is exclusively done in traditional laboratory organisms. The importance of comparative approaches for understanding the relationships between hormones and behavior has been recognized and advocated for since our field's inception through a series of papers centered upon a poetic metaphor of Snarks and Boojums, all of which have articulated the benefits that come from studying a diverse range of species and the risks associated with a narrow focus on "model organisms." This mini-review follows in the footsteps of those powerful arguments, highlighting some of the comparative work since the latest interactions of the metaphor that has shaped how we think about three major conceptual frameworks within our field, two of them formalized - the Organization/Activation Model of sexual differentiation and the Social Brain Network - and one, context-dependency, that is generally associated with virtually all modern understandings of how hormones affect behavior. Comparative approaches are broadly defined as those in which the study of mechanism is placed within natural and/or evolutionary contexts, whether they directly compare different species or not. Studies are discussed in relation to how they have either extended or challenged generalities associated with the frameworks, how they have shaped subsequent work in model organisms to further elucidate neuroendocrine behavioral regulatory mechanisms, and how they have stimulated work to determine if and when similar mechanisms influence behavior in our own species.

Rapid sex steroid effects on reproductive responses in male goldfish: Sensory and motor mechanisms.

Contribution to Special Issue on Fast effects of steroids. Although we have learned a great deal about the molecular mechanisms through which sex steroids rapidly affect cellular physiology, we still know little about the links between those mechanisms and behavioral output, nor about their functional consequences in natural contexts. In this review, we first briefly discuss the contexts associated with rapid effects of sex steroids on reproductive behaviors and their likely functional outcomes, as well the sensory, motor, and motivational mechanisms associated with those effects. We then discuss our recent studies on the rapid effects of testosterone in goldfish. Those studies indicate that testosterone, through its aromatization and the subsequent activation of estrogen receptors, rapidly stimulates physiological processes related to the release of milt/sperm through likely influences on motor pathways, as well as behavioral responses to female visual stimuli that may reflect, in part, influences on early stages of sensory processing. Such motor and sensory mechanism are likely important for sperm competition and mate detection / tracking, respectively, in competitive mating contexts. We also present preliminary data on rapid effects of testosterone on responses to pheromones that may not involve estrogen receptors, suggesting a dissociation in the receptor mechanisms that mediate behavioral responses in different sensory modalities. Lastly, we briefly discuss the implications of our work on unresolved questions about rapid sex steroid neuromodulation in fish.

Corrigendum: Dose-Dependent and Lasting Influences of Intranasal Vasopressin on Face Processing in Men.

[This corrects the article on p. 220 in vol. 8, PMID: 29018407.].

Dose-Dependent and Lasting Influences of Intranasal Vasopressin on Face Processing in Men.

Arginine vasopressin (AVP) and related peptides have diverse effects on social behaviors in vertebrates, sometimes promoting affiliative interactions and sometimes aggressive or antisocial responses. The type of influence, in at least some species, depends on social contexts, including the sex of the individuals in the interaction and/or on the levels of peptide within brain circuits that control the behaviors. To determine if AVP promotes different responses to same- and other-sex faces in men, and if those effects are dose dependent, we measured the effects of two doses of AVP on subjective ratings of male and female faces. We also tested if any influences persist beyond the time of drug delivery. When AVP was administered intranasally on an initial test day, 20 IU was associated with decreased social assessments relative to placebo and 40 IU, and some of the effects persisted beyond the initial drug delivery and appeared to generalize to novel faces on subsequent test days. In single men, those influences were most pronounced, but not exclusive, for male faces, whereas in coupled men they were primarily associated with responses to female faces. Similar influences were not observed if AVP was delivered after placebo on a second test day. In a preliminary analysis, the differences in social assessments observed between men who received 20 and 40 IU, which we suggest primarily reflect lowered social assessments induced by the lower dose, appeared most pronounced in subjects who carry what has been identified as a risk allele for the V1a receptor gene. Together, these results suggest that AVP's effects on face processing, and possibly other social responses, differ according to dose, depend on relationship status, and may be more prolonged than previously recognized.

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

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.

GPER/GPR30, a membrane estrogen receptor, is expressed in the brain and retina of a social fish (Carassius auratus) and colocalizes with isotocin.

Estradiol rapidly (within 30 minutes) influences a variety of sociosexual behaviors in both mammalian and nonmammalian vertebrates, including goldfish, in which it rapidly stimulates approach responses to the visual cues of females. Such rapid neuromodulatory effects are likely mediated via membrane-associated estrogen receptors; however, the localization and distribution of such receptors within the nervous system is not well described. To begin to address this gap, we identified GPER/GPR30, a G-protein-coupled estrogen receptor, in goldfish (Carassius auratus) neural tissue and used reverse-transcription polymerase chain reaction (RT-PCR) and in situ hybridization to test if GPR30 is expressed in the brain regions that might mediate visually guided social behaviors in males. We then used immunohistochemistry to determine whether GPR30 colocalizes with isotocin-producing cells in the preoptic area, a critical node in the highly conserved vertebrate social behavior network. We used quantitative (q)PCR to test whether GPR30 mRNA levels differ in males in breeding vs. nonbreeding condition and in males that were socially interacting with a female vs. a rival male. Our results show that GPR30 is expressed in the retina and in many brain regions that receive input from the retina and/or optic tectum, as well as in a few nodes in the social behavior network, including cell populations that produce isotocin. J. Comp. Neurol. 525:252-270, 2017. © 2016 Wiley Periodicals, Inc.

Pheromone exposure influences preoptic arginine vasotocin gene expression and inhibits social approach behavior in response to rivals but not potential mates.

The nonapeptides arginine vasotocin (AVT) and vasopressin mediate a variety of social behaviors in vertebrates. However, the effects of these peptides on behavior can vary considerably both between and within species. AVT, in particular, stimulates aggressive and courtship responses typical of dominant males in several species, although it can also inhibit social interactions in some cases. Such differential effects may depend upon AVT influences within brain circuits that differ among species or between males that adopt alternative reproductive phenotypes and/or upon the differential activation of those circuits in different social contexts. However, to date, very little is known about how social stimuli that promote alternative behavioral responses influence AVT circuits within the brain. To address this issue, we exposed adult male goldfish to androstenedione (AD), a pheromonal signal that is released by both males and females during the breeding season, and measured social approach responses of males towards same- and other-sex individuals before and after AD exposure. In a second experiment, we measured AD-induced AVT gene expression using in situ hybridization. We found that brief exposure to AD induces social avoidance in response to rival males, but does not affect the level of sociality exhibited in response to sexually receptive females. Exposure to AD also increases AVT gene expression in the preoptic area of male goldfish, particularly in the parvocellular population of the preoptic nucleus. Together, these data suggest that AD is part of a social signaling system that induces social withdrawal specifically during male-male interactions by activating AVT neurons.

An aggression-specific cell type in the anterior hypothalamus of finches.

The anterior hypothalamus (AH) is a major integrator of neural processes related to aggression and defense, but cell types in the AH that selectively promote aggression are unknown. We here show that aggression is promoted in a very selective and potent manner by dorsal AH neurons that produce vasoactive intestinal polypeptide (VIP). Fos activity in a territorial finch, the violet-eared waxbill (Estrildidae: Uraeginthus granatina) is positively related to aggression in the dorsal AH, overlapping a population of VIP-producing neurons. VIP is known to promote territorial aggression in songbirds, and thus we used antisense oligonucleotides to selectively block AH VIP production in male and female waxbills. This manipulation virtually abolishes aggression, reducing the median number of displacements in a 3-min resident-intruder test from 38 in control subjects to 0 in antisense subjects. Notably, most antisense and control waxbills exhibit an agonistic response such as a threat or agonistic call within 2 s of intrusion. Thus, antisense subjects clearly classify intruders as offensive, but fail to attack. Other social and anxiety-like behaviors are not affected and VIP cell numbers correlate positively with aggression, suggesting that these cells selectively titrate aggression. Additional experiments in the gregarious zebra finch (Estrildidae: Taeniopygia guttata) underscore this functional specificity. Colony-housed finches exhibit significant reductions in aggression (primarily nest defense) following AH VIP knockdown, but no effects are observed for social preferences, pair bonding, courtship, maintenance behaviors, or anxiety-like behaviors. To our knowledge, these findings represent a unique identification of an aggression-specific cell type in the brain.

Testosterone rapidly increases ejaculate volume and sperm density in competitively breeding goldfish through an estrogenic membrane receptor mechanism.

The social environment can have dramatic influences on reproductive behavior and physiology in many vertebrate species. In males, interactions with conspecifics affect physiological processes that increase an individual's ability to compete for mates. For example, in some species, males rapidly adjust the number of sperm they ejaculate in response to sociosexual cues from male and female conspecifics, however, little is known about the physiological mechanisms mediating this behavior. In goldfish, as in many vertebrates, social cues also drive transient surges of the gonadal hormone testosterone (T), which induces rapid effects on cellular processes via its conversion to estradiol (E2). We asked whether such surges rapidly influence ejaculate quantity and quality by experimentally manipulating peripheral levels of T and E2. We show that male goldfish injected with T increased ejaculate (milt) volume and sperm density within just 1 hr. Furthermore, increases in expressible milt were dependent on the conversion of T to E2 by the enzyme aromatase, required activation of estrogen receptors α and ß, and were also elicited by BSA-conjugated E2, which acts on cell membrane-bound estrogen receptors. Together, these findings represent a novel steroid mechanism for the social modulation of sperm output over the short time scales that characterize reproductive encounters, and thus demonstrate a previously undescribed functional consequence of rapid estrogen signaling mechanisms. We suggest that such mechanisms may play a critical role in the enhancement of physiological and behavioral processes that increase reproductive success in competitive mating contexts.

Vasotocin neurons and septal V1a-like receptors potently modulate songbird flocking and responses to novelty.

Previous comparisons of territorial and gregarious finches (family Estrildidae) suggest the hypothesis that arginine vasotocin (VT) neurons in the medial bed nucleus of the stria terminalis (BSTm) and V(1a)-like receptors in the lateral septum (LS) promote flocking behavior. Consistent with this hypothesis, we now show that intraseptal infusions of a V(1a) antagonist in male zebra finches (Taeniopygia guttata) reduce gregariousness (preference for a group of 10 versus 2 conspecific males), but have no effect on the amount of time that subjects spend in close proximity to other birds ("contact time"). The antagonist also produces a profound increase in anxiety-like behavior, as exhibited by an increased latency to feed in a novelty-suppressed feeding test. Bilateral knockdown of VT production in the BSTm using LNA-modified antisense oligonucleotides likewise produces increases in anxiety-like behavior and a potent reduction in gregariousness, relative to subjects receiving scrambled oligonucleotides. The antisense oligonucleotides also produced a modest increase in contact time, irrespective of group size. Together, these combined experiments provide clear evidence that endogenous VT promotes preferences for larger flock sizes, and does so in a manner that is coupled to general anxiolysis. Given that homologous peptide circuitry of the BSTm-LS is found across all tetrapod vertebrate classes, these findings may be predictive for other highly gregarious species.

Identification and characterization of mesotocin and V1a-like vasotocin receptors in a urodele amphibian, Taricha granulosa.

The cDNA sequences encoding the mesotocin receptor (MTR) and vasotocin 1a receptor (VTR-1a) were identified in a urodele amphibian, the rough-skinned newt, Taricha granulosa. Saturation binding of [(3)H]oxytocin (OT) to the Taricha MTR (tMTR) was best fit by a two-state model; a high affinity-low abundance site and a lower affinity-high abundance site. Competition-binding studies found the following rank-order affinities for the tMTR: mesotocin (MT)>OT≈vasotocin (VT)>vasopressin (VP)>isotocin (IT). Inositol phosphate (IP) accumulation studies demonstrated functional activity of both the tMTR and Taricha VTR-1a (tVTR-1a) in a heterologous cell culture system. The rank-order potencies for the tMTR were MT>OT>VT≈VP>IT. The combined binding and IP results indicate that VT may act as a partial agonist of the tMTR. Rank-order potencies for the tVTR-1a were VT>VP>MT≈OT>IT. For both receptors, stimulation of IP accumulation was blocked by d(CH(2))(5)[Tyr(Me)(2)]AVP (Manning compound) and d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2)]OVT (OTA). OTA was a more potent antagonist for the transiently expressed tMTR while Manning compound was relatively more potent at inhibiting IP accumulation in tVTR-1a expressing cells. In contradiction to earlier assumptions, the absolute IC(50) of Manning compound was lower for the tMTR (27nM±13) than the tVTR-1a (586nM±166) indicating its potential higher affinity for the tMTR, a finding with special relevance to interpretation of comparative studies investigating the behavioral and physiological actions of neurohypophysial peptides in non-mammalian species.

Nonapeptide mechanisms of social cognition, behavior and species-specific social systems.

Nonapeptide functions have been explored in a diverse literature that has burgeoned in recent years, particularly in relation to affiliation, bonding and human social cognition. However, brain distributions of the oxytocin-like and vasopressin-like peptides are fundamentally similar across all vertebrate animals, including many species that do not exhibit social bonds, grouping, or even parent-offspring interaction. Hence, unifying principles extend beyond, and may even constrain, nonapeptide effects on social cognition and behavior. Conversely, nonapeptide receptor distributions are highly species-specific, suggesting almost limitless functional variation. Drawing on the vast recent literature, we here present a phylogenetically integrated review of both ubiquitous vertebrate features and species diversity, highlighting important nonapeptide effects on socially relevant physiology, sensorimotor integration, assignment of valence, and functional connectivity.

Behavioral effects of hindbrain vasotocin in goldfish are seasonally variable but not sexually dimorphic.

We have previously demonstrated that centrally administered vasotocin (VT) inhibits social approach toward same-sex conspecifics in male and female goldfish, and that this behavioral effect is dependent upon VT projections to the hindbrain. We now show that there are no sex differences in sensitivity to the behavioral effects of VT, though differences do exist in responsiveness across seasons in both sexes. A central dose of 1 microg, but not 200 ng, inhibited social approach in goldfish in non-reproductive condition, whereas a dose as low as 40 ng inhibited social approach in fish in full reproductive condition. In males and females in full reproductive condition, social approach behavior was facilitated by central administration of 500 ng of a V(1A) specific antagonist. In addition, the behavioral effects of exogenously administered central VT were blocked by central administration of 1 microg of a V(1A) antagonist. These results demonstrate that the propensity to approach a conspecific, a simple behavior underlying many social interactions, is controlled by a V(1A)-like receptor, and that VT's behavioral effects depend on reproductive context. Quantitative real-time PCR showed that the seasonal changes in behavioral responsiveness to VT are associated with changes in the expression of a V(1A)-like receptor in the hindbrain, but not the mid- or forebrain, indicating that the seasonal regulation of social approach behavior likely depends on the local modulation of the expression of this receptor within a primitive peptide circuit in this species.

Rapid steroid influences on visually guided sexual behavior in male goldfish.

The ability of steroid hormones to rapidly influence cell physiology through nongenomic mechanisms raises the possibility that these molecules may play a role in the dynamic regulation of social behavior, particularly in species in which social stimuli can rapidly influence circulating steroid levels. We therefore tested if testosterone (T), which increases in male goldfish in response to sexual stimuli, can rapidly influence approach responses towards females. Injections of T stimulated approach responses towards the visual cues of females 30-45 min after the injection but did not stimulate approach responses towards stimulus males or affect general activity, indicating that the effect is stimulus-specific and not a secondary consequence of increased arousal. Estradiol produced the same effect 30-45 min and even 10-25 min after administration, and treatment with the aromatase inhibitor fadrozole blocked exogenous T's behavioral effect, indicating that T's rapid stimulation of visual approach responses depends on aromatization. We suggest that T surges induced by sexual stimuli, including preovulatory pheromones, rapidly prime males to mate by increasing sensitivity within visual pathways that guide approach responses towards females and/or by increasing the motivation to approach potential mates through actions within traditional limbic circuits.

Vasotocin immunoreactivity in goldfish brains: characterizing primitive circuits associated with social regulation.

The neuroanatomical characteristics of vasotocin (VT) and vasopressin (VP) systems have been described in numerous species from diverse vertebrate groups, including teleost fish. However, there are very few teleost species in which VT's effects on social behavior have been established and the neural circuits associated with that regulation fully described. We have previously shown that VT inhibits social approach behaviors in goldfish via actions in the hindbrain. Here we further describe that primitive VT circuit, as well as others throughout the goldfish brain that might contribute to social regulation in this species. In particular, we highlight forebrain projections to the dorsal telencephalon that have not been described previously in any teleost, projections to limbic forebrain areas, most notably the medial division of the dorsal telencephalon and the ventral telencephalon, and midbrain projections to the optic tectum and torus semicircularis that have rarely been described. However, the most dense VT projection in goldfish is to the hindbrain, particularly to motor divisions of the vagal complex and to area postrema, which we argue might influence social approach behaviors through autonomic regulatory mechanisms. Because hindbrain VT projections are some of the most primitive in vertebrates, we suggest they might represent an ancestral mechanism through which VT influenced social behavior.

Identification of mesotocin and vasotocin nucleotide sequences in two species of urodele amphibian.

We amplified and identified, for the first time in urodele amphibians, cDNA sequences that encode preprovasotocin (preproVT) and prepromesotocin (preproMT) from two distinct urodelian species, Taricha granulosa (the rough-skinned newt) and Plethodon shermanii (the spotted salamander). Each of these cDNA sequences encoded proteins that contained the characteristics of known neurohypophysial peptide precursors; each sequence consisting of (1) a signal peptide, (2) VT- or MT-like peptides, (3) neurophysin, and for the preproVTs, (4) copeptin. In T. granulosa, cDNA sequences encoded for the nine amino acids that define VT or MT. In P. shermani, cDNA sequences encoded for the VT peptide and a previously unidentified isoform of MT, ([Val(4)]-MT).

Peptide effects on social behavior: effects of vasotocin and isotocin on social approach behavior in male goldfish (Carassius auratus).

The authors measured the effects of centrally infused peptides on social approach behaviors in goldfish (Carassius auratus), a social teleost. Vasotocin (VT) inhibited approach responses toward the visual stimuli of conspecifics in the absence of aggressive or sexual olfactory contextual cues in males, and a V1 receptor antagonist stimulated such responses, at least in males that were not highly social in baseline conditions, as did isotocin (IT). In the absence of social stimuli, VT did not affect activity, therefore indicating that the inhibition was not the result of nonspecific effects on arousal or motor functioning. These experiments indicate that VT and IT induce opposite effects on social approach responses in male goldfish and that endogenous VT, at least, is associated with levels of sociality.

Testing the relationship between levels of endogenous testosterone and physiological responses to facial expressions in men: an experiment conducted by students in an undergraduate behavioral neuroscience class.

To determine if endogenous testosterone (T) is related to physiological responses to aggressive stimuli in human males, students in a behavioral neuroscience laboratory class conducted an experiment that determined if levels of salivary T in adult males are correlated with autonomic and/or somatic responses to angry facial expressions. Each student collected a saliva sample from one subject and, within 30 minutes of collecting the sample, measured heart rate (HR), skin conductance (SC), and corrugator supercilii electromyographic (EMG) responses to emotionally neutral, happy, and angry male facial expressions. Salivary T levels were analyzed by an enzyme-linked immunoassay. A significant, positive correlation was found between levels of salivary T and HR responses to angry and happy, but not neutral, male facial expressions. This laboratory experience not only provided students with the opportunity to design and conduct a scientific experiment, but it also generated preliminary data suggesting that levels of salivary T collected within 30 minutes of testing are related to autonomic responses to emotional social stimuli in humans. If verified by future experiments, this finding would be consistent with the hypothesis that fluctuations in circulating T might influence ongoing social behavior in human males by rapidly modulating autonomic responses to emotional social stimuli. The potential significance of such a general mechanism for the regulation of aggressive behavior is discussed.