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.

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.

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.

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.

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.

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.

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.

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.

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.

Behavioral trajectories of aging prairie voles (Microtus ochrogaster): Adapting behavior to social context wanes with advanced age.

Several studies using mice have examined the effects of aging on cognitive tasks, as well as sensory and motor functions. However, few studies have examined the influence of aging on social behavior. Prairie voles (Microtus ochrogaster) are a socially monogamous and biparental rodent that live in small family groups and are now among the most popular rodent models for studies examining social behavior. Although the social behavioral trajectories of early-life development in prairie voles have been well-studied, how social behavior may change throughout adulthood remains unknown. Here we examined behavior in virgin male and female prairie voles in four different age groups: postnatal day (PND) 60-80, 140-160, 220-240, and 300-320. All animals underwent testing in a novel object task, a dominance test, a resident-intruder test, and several iterations of social approach and social interaction tests with varying types of social stimuli (i.e., novel same-sex conspecific, novel opposite-sex conspecific, familiar same-sex sibling/cagemate, small group of novel same-sex conspecifics). We found that age influenced neophobia and dominance, but not social approach behavior. Further, we found that young adult, but not older adult, prairie voles adapt prosocial and aggressive behavior relative to social context, and that selective aggression occurs in relation to age even in the absence of a pair bond. Our results suggest that prairie voles calibrate social phenotype in a context-dependent manner in young adulthood and stop adjusting behavior to social context in advanced age, demonstrating that social behavior is plastic not only throughout early development, but also well into adulthood. Together, this study provides insight into age-related changes in social behavior in prairie voles and shows that prairie voles may be a viable model for studying the cognitive and physiological benefits of social relationships and social engagement in advanced age.

How inversion variants can shape neural circuitry: Insights from the three-morph mating tactics of ruffs.

Behavior polymorphisms underlying alternative mating tactics can evolve due to genetic inversions, especially when inversions capture sets of genes involved in hormonal regulation. In the three-morph system of the ruff (Calidris pugnax), two alternative morphs (Satellites and Faeders) with distinct behaviors and low circulating testosterone are genetically determined by an inverted region on an autosomal chromosome. Here, we discuss recent findings on the ruff and present novel insights into how an inversion that poses drastic constraints on testosterone production might lead to morph-specific differences in brain areas that regulate social behavior. A gene responsible for converting testosterone to androstenedione (HSD17B2) is located inside the inverted region and is a promising candidate. We identify a single missense mutation in the HSD17B2 gene of inverted alleles that is responsible for a 350-500% increase in testosterone to androstenedione conversion, when mutated in the human HSD17B2 protein. We discuss new evidence of morph differences in neural HSD17B2 expression in embryos and circulating androgens in sexually-immature juveniles. We suggest processes that shape morph differences in behavior likely begin early in ontogeny. We propose that the organization of behaviorally relevant neuron cell types that are canonically sexually dimorphic, such as subpopulations of aromatase and vasotocin neurons, should be particularly affected due to the life-long condition of low circulating testosterone in inversion morphs. We further emphasize how HSD17B2 catalytic activity extends beyond androgens, and includes estradiol oxidation into estrone and progesterone synthesis. Lastly, we underscore dimerization of HSD17B2 as an additional layer of complexity that merits consideration.

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).

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.

Species-typical group size differentially influences social reward neural circuitry during nonreproductive social interactions.

We investigated whether nonreproductive social interactions may be rewarding for colonial but not non-colonial species. We found that the colonial spiny mouse (Acomys cahirinus) is significantly more gregarious, more prosocial, and less aggressive than its non-colonial relative, the Mongolian gerbil (Meriones unguiculatus). In an immediate-early gene study, we examined oxytocin (OT) and tyrosine hydroxylase (TH) neural responses to interactions with a novel, same-sex conspecific or a novel object. The paraventricular nucleus of the hypothalamus (PVN) OT cell group was more responsive to interactions with a conspecific compared to a novel object in both species. However, the ventral tegmental area (VTA) TH cell group showed differential responses only in spiny mice. Further, PVN OT and VTA TH neural responses positively correlated in spiny mice, suggesting functional connectivity. These results suggest that colonial species may have evolved neural mechanisms associated with reward in novel, nonreproductive social contexts to promote large group-living.

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.

Support for the parental practice hypothesis: Subadult prairie voles exhibit similar behavioral and neural profiles when alloparenting kin and non-kin.

Parental care is critical for offspring survival in altricial species. Although parents are the most common caregivers, other individuals (e.g., older siblings) can also provide alloparental care. Some have argued that animals engage in alloparental behavior to practice providing care for their eventual offspring, whereas others have argued that alloparental behavior enhances indirect fitness. Proximate measures have the potential to test ultimate functions of behavior. A focus on neural expression of oxytocin and vasopressin (two neuropeptides modulating alloparental care) or neural activation following exposure to related and unrelated individuals could reveal whether practice or investment in indirect fitness explains alloparental behavior. This study examined alloparental behaviors and neural responses in prairie voles (Microtus ochrogaster), a species that engages in alloparental behavior. Subadult (independent, yet sexually immature) male prairie voles were exposed to one of four stimuli: same-age sibling, neonatal sibling, unrelated neonate, or inanimate neonate-sized object. We assessed alloparental behaviors and quantified cFos protein expression in oxytocin and vasopressin neuronal populations of the paraventricular nucleus of the hypothalamus and the supraoptic nucleus of the hypothalamus in response to stimulus exposure. We detected no differences in cFos and nonapeptide co-localization among stimulus groups. Subjects performed similar amounts of alloparental care toward related and unrelated neonates, but not other subadults or inanimate objects. Notably, caregiving did not differ based on kin-status. The lack of difference in alloparenting toward related and non-related neonates suggests that alloparental care in prairie voles primarily serves to provide subadults with parental practice.

Distribution of Vasopressin and Oxytocin Neurons in the Basal Forebrain and Midbrain of Spiny Mice (Acomys cahirinus).

The nonapeptides vasopressin (VP) and oxytocin (OT) are present in some form in most vertebrates. VP and OT play critical roles in modulating physiology and are well-studied for their influences on a variety of social behaviors, ranging from affiliation to aggression. Their anatomical distributions have been mapped for numerous species across taxa, demonstrating relatively strong evolutionary conservation in distributions throughout the basal forebrain and midbrain. Here we examined the distribution of VP-immunoreactive (-ir) and OT-ir neurons in a gregarious, cooperatively breeding rodent species, the spiny mouse (Acomys cahirinus), for which nonapeptide mapping does not yet exist. Immunohistochemical techniques revealed VP-ir and OT-ir neuronal populations throughout the hypothalamus and amygdala of males and females that are consistent with those of other rodents. However, a novel population of OT-ir neurons was observed in the median preoptic nucleus of both sexes, located dorsally to the anterior commissure. Furthermore, we found widespread sex differences in OT neuronal populations, with males having significantly more OT-ir neurons than females. However, we observed a sex difference in only one VP cell group - that of the bed nucleus of the stria terminalis (BST), a VP neuronal population that exhibits a phylogenetically widespread sexual dimorphism. These findings provide mapping distributions of VP and OT neurons in Acomys cahirinus. Spiny mice lend themselves to the study of mammalian cooperation and sociality, and the nonapeptide neuronal mapping presented here can serve as a basic foundation for the study of nonapeptide-mediated behavior in a group of highly social rodents.

Paternal deprivation impairs social behavior putatively via epigenetic modification to lateral septum vasopressin receptor.

Although it is well appreciated that the early-life social environment asserts subsequent long-term consequences on offspring brain and behavior, the specific mechanisms that account for this relationship remain poorly understood. Using a novel assay that forced biparental pairs or single mothers to prioritize caring for offspring or themselves, we investigated the impact of parental variation on adult expression of nonapeptide-modulated behaviors in prairie voles. We demonstrated that single mothers compensate for the lack of a co-parent. Moreover, mothers choose to invest in offspring over themselves when faced with a tradeoff, whereas fathers choose to invest in themselves. Furthermore, our study suggests a pathway whereby variation in parental behavior (specifically paternal care) may lead to alterations in DNA methylation within the vasopressin receptor 1a gene and gene expression in the lateral septum. These differences are concomitant with changes in social approach, a behavior closely associated with septal vasopressin receptor function.

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.