From grouping and cooperation to menstruation: Spiny mice (Acomys cahirinus) are an emerging mammalian model for sociality and beyond.

While spiny mice are primarily used as a model for Type II diabetes and for studying complex tissue regeneration, they are also an emerging model for a variety of studies examining hormones, behavior, and the brain. We began studying the spiny mouse to take advantage of their highly gregarious phenotype to examine how the brain facilitates large group-living. However, this unique rodent can be readily bred and maintained in the lab and can be used to ask a wide variety of scientific questions. In this brief communication we provide an overview of studies that have used spiny mice for exploring physiology and behavior. Additionally, we describe how the spiny mouse can serve as a useful model for researchers interested in studying precocial development, menstruation, cooperation, and various grouping behaviors. With increasingly available technological advancements for non-traditional organisms, spiny mice are well-positioned to become a valuable organism in the behavioral neuroscience community.

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.

A test of the social behavior network reveals differential patterns of neural responses to social novelty in bonded, but not non-bonded, male prairie voles.

The social behavior network (SBN) has provided a framework for understanding the neural control of social behavior. The original SBN hypothesis proposed this network modulates social behavior and should exhibit distinct patterns of neural activity across nodes, which correspond to distinct social contexts. Despite its tremendous impact on the field of social neuroscience, no study has directly tested this hypothesis. Thus, we assessed Fos responses across the SBN of male prairie voles (Microtus ochrogaster). Virgin/non-bonded and pair bonded subjects were exposed to a sibling cagemate or pair bonded partner, novel female, novel male, novel meadow vole, novel object, or no stimulus. Inconsistent with the original SBN hypothesis, we did not find profoundly different patterns of neural responses across the SBN for different contexts, but instead found that the SBN generated significantly different patterns of activity in response to social novelty in pair bonded, but not non-bonded males. These findings suggest that non-bonded male prairie voles may perceive social novelty differently from pair bonded males or that SBN functionality undergoes substantial changes after pair bonding. This study reveals novel information about bond-dependent, context-specific neural responsivity in male prairie voles and suggests that the SBN may be particularly important for processing social salience. Further, our study suggests there is a need to reconceptualize the framework of how the SBN modulates social behavior.

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.

A consideration of brain networks modulating social behavior.

A primary goal of the field of behavioral neuroendocrinology is to understand how the brain modulates complex behavior. Over the last 20 years we have proposed various brain networks to explain behavioral regulation, however, the parameters by which these networks are identified are often ill-defined and reflect our personal scientific biases based on our area of expertise. In this perspective article, I question our characterization of brain networks underlying behavior and their utility. Using the Social Behavior Network as a primary example, I outline issues with brain networks commonly discussed in the field of behavioral neuroendocrinology, argue that we reconsider how we identify brain networks underlying behavior, and urge the future use of analytical tools developed by the field of Network Neuroscience. With modern statistical/mathematical tools and state of the art technology for brain imaging, we can strive to minimize our bias and generate brain networks that may more accurately reflect how the brain produces behavior.

Aggression: Perspectives from social and systems neuroscience.

Exhibiting behavioral plasticity in order to mount appropriate responses to dynamic and novel social environments is crucial to the survival of all animals. Thus, how animals regulate flexibility in the timing, duration, and intensity of specific behaviors is of great interest to biologists. In this review, we discuss how animals rapidly respond to social challenges, with a particular focus on aggression. We utilize a conceptual framework to understand the neural mechanisms of aggression that is grounded in Wingfield and colleagues' Challenge Hypothesis, which has profoundly influenced how scientists think about aggression and the mechanisms that allow animals to exhibit flexible responses to social instability. Because aggressive behavior is rooted in social interactions, we propose that mechanisms modulating prosocial behavior may be intricately tied to mechanisms of aggression. Therefore, in order to better understand how aggressive behavior is mediated, we draw on perspectives from social neuroscience and discuss how social context, species-typical behavioral phenotype, and neural systems commonly studied in relation to prosocial behavior (i.e., neuropeptides) contribute to organizing rapid responses to social challenges. Because complex behaviors are not the result of one mechanism or a single neural system, we consider how multiple neural systems important for prosocial and aggressive behavior (i.e., neuropeptides and neurosteroids) interact in the brain to produce behavior in a rapid, context-appropriate manner. Applying a systems neuroscience perspective and seeking to understand how multiple systems functionally integrate to rapidly modulate behavior holds great promise for expanding our knowledge of the mechanisms underlying social behavioral plasticity.

Mechanistic substrates of a life history transition in male prairie voles: Developmental plasticity in affiliation and aggression corresponds to nonapeptide neuronal function.

Although prairie vole (Microtus ochrogaster) social behavior is well-characterized in adults, surprisingly little is known about the development of social behavior in voles. Further, the overwhelming majority of studies in prairie voles examine social behavior in a reproductive context. Here, we examine developmental plasticity in affiliation and aggression and their underlying neural correlates. Using sexually naïve males, we characterized interactions with an age-matched, novel, same-sex conspecific in four different age groups that span pre-weaning to adulthood. We found that prosocial behavior decreased and aggression increased as males matured. Additionally, pre-weaning males were more prosocial than nonsocial, whereas post-weaning males were more nonsocial than prosocial. We also examined nonapeptide neural activity in response to a novel conspecific in brain regions important for promoting sociality and aggression using the immediate early gene cFos. Assessment of developmental changes in neural activity showed that vasopressin neurons in the medial bed nucleus of the stria terminalis exhibit functional plasticity, providing a potential functional mechanism that contributes to this change in sociality as prairie voles mature. This behavioral shift corresponds to the transition from a period of allopatric cohabitation with siblings to a period of time when voles disperse and presumably attempt to establish and defend territories. Taken together our data provide a putative mechanism by which brain and behavior prepare for the opportunity to pairbond (characterized by selective affiliation with a partner and aggression toward unfamiliar conspecifics) by undergoing changes away from general affiliation and toward selective aggression, accounting for this important life history event.

Hypothalamic oxytocin and vasopressin neurons exert sex-specific effects on pair bonding, gregariousness, and aggression in finches.

Antagonism of oxytocin (OT) receptors (OTRs) impairs the formation of pair bonds in prairie voles (Microtus ochrogaster) and zebra finches (Taenioypygia guttata), and also reduces the preference for the larger of two groups ("gregariousness") in finches. These effects tend to be stronger in females. The contributions of specific peptide cell groups to these processes remain unknown, however. This issue is complicated by the fact that OTRs in finches and voles bind not only forms of OT, but also vasopressin (VP), and >10 cell groups produce each peptide in any given species. Using RNA interference, we found that knockdown of VP and OT production in the paraventricular nucleus of the hypothalamus exerts diverse behavioral effects in zebra finches, most of which are sexually differentiated. Our data show that knockdown of VP production significantly reduces gregariousness in both sexes and exerts sex-specific effects on aggression directed toward opposite-sex birds (increases in males; decreases in females), whereas OT knockdown produces female-specific deficits in gregariousness, pair bonding, and nest cup ownership; reduces side-by-side perching in both sexes; modulates stress coping; and induces hyperphagia in males. These findings demonstrate that paraventricular neurons are major contributors to the effects of VP-OT peptides on pair bonding and gregariousness; reveal previously unknown effects of sex-specific peptide on opposite-sex aggression; and demonstrate a surprising lack of effects on same-sex aggression. Finally, the observed effects of OT knockdown on feeding and stress coping parallel findings in mammals, suggesting that OT modulation of these processes is evolutionarily conserved across the amniote vertebrate classes.

Social functions of individual vasopressin-oxytocin cell groups in vertebrates: what do we really know?

Vasopressin-oxytocin (VP-OT) nonapeptides modulate numerous social and stress-related behaviors, yet these peptides are made in multiple nuclei and brain regions (e.g., >20 in some mammals), and VP-OT cells in these areas often exhibit overlapping axonal projections. Furthermore, the magnocellular cell groups release peptide volumetrically from dendrites and soma, which gives rise to paracrine modulation in distal brain areas. Nonapeptide receptors also tend to be promiscuous. Hence, behavioral effects that are mediated by any given receptor type (e.g., the OT receptor) in a target brain region cannot be conclusively attributed to either VP or OT, nor to a specific cell group. We here review what is actually known about the social behavior functions of nonapeptide cell groups, with a focus on aggression, affiliation, bonding, social stress, and parental behavior, and discuss recent studies that demonstrate a diversity of sex-specific contributions of VP-OT cell groups to gregariousness and pair bonding.

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.

Functional significance of a phylogenetically widespread sexual dimorphism in vasotocin/vasopressin production.

Male-biased production of arginine vasotocin/vasopressin (VT/VP) in the medial bed nucleus of the stria terminalis (BSTm) represents one of the largest and most phylogenetically widespread sexual dimorphisms in the vertebrate brain. Although this sex difference was identified 30 years ago, the function of the dimorphism has yet to be determined. Because 1) rapid transcriptional activation of BSTm VT/VP neurons is observed selectively in response to affiliation-related stimuli, 2) BSTm VT/VP content and release correlates negatively with aggression, and 3) BSTm VT/VP production is often limited to periods of reproduction, we hypothesized that the sexual dimorphism serves to promote male-specific reproductive behaviors and offset male aggression in the context of reproductive affiliation. We now show that antisense knockdown of BSTm VT production in colony-housed finches strongly increases aggression in a male-specific manner and concomitantly reduces courtship. Thus, the widespread dimorphism may serve to focus males on affiliation in appropriate reproductive contexts (e.g., when courting) while concomitantly offsetting males' tendency for greater aggression relative to females.

Partner separation rescues pair bond-induced decreases in hypothalamic oxytocin neural densities.

Studies in prairie voles (Microtus ochrogaster) have shown that although formation of the pair bond is accompanied by a suite of behavioral changes, a bond between two voles can dissolve and individuals can form new pair bonds with other conspecifics. However, the neural mechanisms underlying this behavioral flexibility have not been well-studied. Here we examine plasticity of nonapeptide, vasopressin (VP) and oxytocin (OT), neuronal populations in relation to bonding and the dissolution of bonds. Using adult male and female prairie voles, animals were either pair bonded, co-housed with a same-sex sibling, separated from their pair bond partner, or separated from their sibling. We examined neural densities of VP and OT cell groups and observed plasticity in the nonapeptide populations of the paraventricular nucleus of the hypothalamus (PVN). Voles that were pair bonded had fewer PVN OT neurons, suggesting that PVN OT neural densities decrease with pair bonding, but increase and return to a pre-pair bonded baseline after the dissolution of a pair bond. Our findings suggest that the PVN nonapeptide cell groups are particularly plastic in adulthood, providing a mechanism by which voles can exhibit context-appropriate behavior related to bond status.

Biased brain and behavioral responses towards kin in males of a communally breeding species.

In complex social environments, individuals may interact with not only novel and familiar conspecifics but also kin and non-kin. The ability to distinguish between conspecific identities is crucial for most animals, yet how the brain processes conspecific type and how animals may alter behavior accordingly is not well known. We examined whether the communally breeding spiny mouse (Acomys cahirinus) responds differently to conspecifics that vary in novelty and kinship. In a group interaction test, we found that males can distinguish novel kin from novel non-kin, and preferentially spend time with novel kin over familiar kin and novel non-kin. To determine whether kinship and novelty status are differentially represented in the brain, we conducted immediate early gene tests, which revealed the dorsal, but not ventral, lateral septum differentially processes kinship. Neither region differentially processes social novelty. Further, males did not exhibit differences in prosocial behavior toward novel and familiar conspecifics but exhibited more prosocial behavior with novel kin than novel non-kin. These results suggest that communally breeding species may have evolved specialized neural circuitry to facilitate a bias to be more affiliative with kin, regardless of whether they are novel or familiar, potentially to promote prosocial behaviors, thereby facilitating group cohesion.

Early life social complexity shapes adult neural processing in the communal spiny mouse Acomys cahirinus.

RATIONALE: Early life social rearing has profound consequences on offspring behavior and resilience. Yet, most studies examining early life development in rodents use species whose young are born immobile and do not produce complex social behavior until later in development. Furthermore, models of rearing under increased social complexity, rather than deprivation, are needed to provide alternative insight into the development of social neural circuitry. OBJECTIVES: To understand precocial offspring social development, we manipulated early life social complexity in the communal spiny mouse Acomys cahirinus and assessed long-term consequences on offspring social behavior, exploration, and neural responses to novel social stimuli. METHODS: Spiny mouse pups were raised in the presence or absence of a non-kin breeding group. Upon adulthood, subjects underwent social interaction tests, an open field test, and a novel object test. Subjects were then exposed to a novel conspecific and novel group and neural responses were quantified via immunohistochemical staining in brain regions associated with social behavior. RESULTS: Early life social experience did not influence behavior in the test battery, but it did influence social processing. In animals exposed to non-kin during development, adult lateral septal neural responses toward a novel conspecific were weaker and hypothalamic neural responses toward a mixed-sex group were stronger. CONCLUSIONS: Communal species may exhibit robust behavioral resilience to the early life social environment. But the early life environment can affect how novel social information is processed in the brain during adulthood, with long-term consequences that are likely to shape their behavioral trajectory.

Distribution of Vasopressin 1a and Oxytocin Receptor Binding in the Basal Forebrain and Midbrain of Male and Female Mongolian Gerbils.

The nonapeptide system modulates a diversity of social behaviors, including aggression, parental care, affiliation, sexual behavior, and pair bonding. Such social behaviors are regulated through oxytocin and vasopressin activation of the oxytocin receptor (OXTR) and vasopressin V1a receptor (AVPR1A) in the brain. Nonapeptide receptor distributions have been mapped for several species, however, studies have demonstrated that there is substantial variation across species. Mongolian gerbils (Meriones unguiculatus) are an excellent organism for studying family dynamics, social development, pair bonding, and territorial aggression. Although an increasing number of studies are examining the neural mechanisms of social behavior in Mongolian gerbils, nonapeptide receptor distributions have yet to be characterized for this species. Here we conducted receptor autoradiography to map distributions of OXTR and AVPR1A binding throughout the basal forebrain and midbrain of female and male Mongolian gerbils. Further, we assessed whether gonadal sex influenced binding densities in brain regions important for social behavior and reward, however, we observed no effects of sex on OXTR or AVPR1A binding densities. These findings provide mapping distributions of nonapeptide receptors in male and female Mongolian gerbils, laying a foundation for future studies that seek to manipulate the nonapeptide system to examine nonapeptide-mediated social behavior.

Distribution of vasopressin 1a and oxytocin receptor protein and mRNA in the basal forebrain and midbrain of the spiny mouse (Acomys cahirinus).

The nonapeptide system modulates numerous social behaviors through oxytocin and vasopressin activation of the oxytocin receptor (OXTR) and vasopressin receptor (AVPR1A) in the brain. OXTRs and AVPR1As are widely distributed throughout the brain and binding densities exhibit substantial variation within and across species. Although OXTR and AVPR1A binding distributions have been mapped for several rodents, this system has yet to be characterized in the spiny mouse (Acomys cahirinus). Here we conducted receptor autoradiography and in situ hybridization to map distributions of OXTR and AVPR1A binding and Oxtr and Avpr1a mRNA expression throughout the basal forebrain and midbrain of male and female spiny mice. We found that nonapeptide receptor mRNA is diffuse throughout the forebrain and midbrain and does not always align with OXTR and AVPR1A binding. Analyses of sex differences in brain regions involved in social behavior and reward revealed that males exhibit higher OXTR binding densities in the lateral septum, bed nucleus of the stria terminalis, and anterior hypothalamus. However, no association with gonadal sex was observed for AVPR1A binding. Hierarchical clustering analysis further revealed that co-expression patterns of OXTR and AVPR1A binding across brain regions involved in social behavior and reward differ between males and females. These findings provide mapping distributions and sex differences in nonapeptide receptors in spiny mice. Spiny mice are an excellent organism for studying grouping behaviors such as cooperation and prosociality, and the nonapeptide receptor mapping here can inform the study of nonapeptide-mediated behavior in a highly social, large group-living rodent.

An AAV-CRISPR/Cas9 strategy for gene editing across divergent rodent species: Targeting neural oxytocin receptors as a proof of concept.

A major issue in neuroscience is the poor translatability of research results from preclinical studies in animals to clinical outcomes. Comparative neuroscience can overcome this barrier by studying multiple species to differentiate between species-specific and general mechanisms of neural circuit functioning. Targeted manipulation of neural circuits often depends on genetic dissection, and use of this technique has been restricted to only a few model species, limiting its application in comparative research. However, ongoing advances in genomics make genetic dissection attainable in a growing number of species. To demonstrate the potential of comparative gene editing approaches, we developed a viral-mediated CRISPR/Cas9 strategy that is predicted to target the oxytocin receptor (Oxtr) gene in >80 rodent species. This strategy specifically reduced OXTR levels in all evaluated species (n = 6) without causing gross neuronal toxicity. Thus, we show that CRISPR/Cas9-based tools can function in multiple species simultaneously. Thereby, we hope to encourage comparative gene editing and improve the translatability of neuroscientific research.

Evolving nonapeptide mechanisms of gregariousness and social diversity in birds.

Of the major vertebrate taxa, Class Aves is the most extensively studied in relation to the evolution of social systems and behavior, largely because birds exhibit an incomparable balance of tractability, diversity, and cognitive complexity. In addition, like humans, most bird species are socially monogamous, exhibit biparental care, and conduct most of their social interactions through auditory and visual modalities. These qualities make birds attractive as research subjects, and also make them valuable for comparative studies of neuroendocrine mechanisms. This value has become increasingly apparent as more and more evidence shows that social behavior circuits of the basal forebrain and midbrain are deeply conserved (from an evolutionary perspective), and particularly similar in birds and mammals. Among the strongest similarities are the basic structures and functions of avian and mammalian nonapeptide systems, which include mesotocin (MT) and arginine vasotocin (VT) systems in birds, and the homologous oxytocin (OT) and vasopressin (VP) systems, respectively, in mammals. We here summarize these basic properties, and then describe a research program that has leveraged the social diversity of estrildid finches to gain insights into the nonapeptide mechanisms of grouping, a behavioral dimension that is not experimentally tractable in most other taxa. These studies have used five monogamous, biparental finch species that exhibit group sizes ranging from territorial male-female pairs to large flocks containing hundreds or thousands of birds. The results provide novel insights into the history of nonapeptide functions in amniote vertebrates, and yield remarkable clarity on the nonapeptide biology of dinosaurs and ancient mammals. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.

Midbrain dopamine neurons reflect affiliation phenotypes in finches and are tightly coupled to courtship.

Mesolimbic dopamine (DA) circuits mediate a wide range of goal-oriented behavioral processes, and DA strongly influences appetitive and consummatory aspects of male sexual behavior. In both birds and mammals, mesolimbic projections arise primarily from the ventral tegmental area (VTA), with a smaller contribution from the midbrain central gray (CG). Despite the well known importance of the VTA cell group for incentive motivation functions, relationships of VTA subpopulations to specific aspects of social phenotype remain wholly undescribed. We now show that in male zebra finches (Estrildidae: Taeniopygia guttata), Fos activity within a subpopulation of tyrosine hydroxylase-immunoreactive (TH-ir; presumably dopaminergic) neurons in the caudal VTA is significantly correlated with courtship singing and coupled to gonadal state. In addition, the number of TH-ir neurons in this caudal subpopulation dichotomously differentiates courting from non-courting male phenotypes, and evolves in relation to sociality (flocking vs. territorial) across several related finch species. Combined, these findings for the VTA suggest that divergent social phenotypes may arise due to the differential assignment of "incentive value" to conspecific stimuli. TH-ir neurons of the CG (a population of unknown function in mammals) exhibit properties that are even more selectively and tightly coupled to the expression of courtship phenotypes (and appetitive courtship singing), both in terms of TH-ir cell number, which correlates significantly with constitutive levels of courtship motivation, and with TH-Fos colocalization, which increases in direct proportion to the phasic expression of song. We propose that these neurons may be core components of social communication circuits across diverse vertebrate taxa.

Protocol for multiplex fluorescent immunohistochemistry in free-floating rodent brain tissues.

Identifying multiple proteins within the same tissue allows for assessing protein colocalization, is cost effective, and maximizes efficiency. Here, we describe a protocol for multiplex immunolabeling of proteins in free-floating rodent brain sections. As opposed to slide-mounted immunohistochemistry, the free-floating approach results in less tissue loss and greater antibody penetration. Using distinct fluorophores for individual proteins, this protocol allows for visualization of three or more proteins within tissue sections. The protocol can be applied to other tissue types. For complete details on the use and execution of this protocol, please refer to Gonzalez Abreu et al. (2022).