Genetic and neural mechanisms that inhibit Drosophila from mating with other species.

Genetically hard-wired neural mechanisms must enforce behavioral reproductive isolation because interspecies courtship is rare even in sexually naïve animals of most species. We find that the chemoreceptor Gr32a inhibits male D. melanogaster from courting diverse fruit fly species. Gr32a recognizes nonvolatile aversive cues present on these reproductively dead-end targets, and activity of Gr32a neurons is necessary and sufficient to inhibit interspecies courtship. Male-specific Fruitless (Fru(M)), a master regulator of courtship, also inhibits interspecies courtship. Gr32a and Fru(M) are not coexpressed, but Fru(M) neurons contact Gr32a neurons, suggesting that these genes influence a shared neural circuit that inhibits interspecies courtship. Gr32a and Fru(M) also suppress within-species intermale courtship, but we show that distinct mechanisms preclude sexual displays toward conspecific males and other species. Although this chemosensory pathway does not inhibit interspecies mating in D. melanogaster females, similar mechanisms appear to inhibit this behavior in many other male drosophilids.

Estrogen masculinizes neural pathways and sex-specific behaviors.

Sex hormones are essential for neural circuit development and sex-specific behaviors. Male behaviors require both testosterone and estrogen, but it is unclear how the two hormonal pathways intersect. Circulating testosterone activates the androgen receptor (AR) and is also converted into estrogen in the brain via aromatase. We demonstrate extensive sexual dimorphism in the number and projections of aromatase-expressing neurons. The masculinization of these cells is independent of AR but can be induced in females by either testosterone or estrogen, indicating a role for aromatase in sexual differentiation of these neurons. We provide evidence suggesting that aromatase is also important in activating male-specific aggression and urine marking because these behaviors can be elicited by testosterone in males mutant for AR and in females subjected to neonatal estrogen exposure. Our results suggest that aromatization of testosterone into estrogen is important for the development and activation of neural circuits that control male territorial behaviors.

Oxytocin receptor function regulates neural signatures of pair bonding and fidelity in the nucleus accumbens.

The formation of enduring relationships dramatically influences future behavior, promoting affiliation between familiar individuals. How such attachments are encoded to elicit and reinforce specific social behaviors in distinct ethological contexts remains unknown. Signaling via the oxytocin receptor (Oxtr) in the nucleus accumbens (NAc) facilitates social reward as well as pair bond formation between mates in socially monogamous prairie voles 1-9 . How Oxtr function influences activity in the NAc during pair bonding to promote affiliative behavior with partners and rejection of other potential mates has not been determined. Using longitudinal in vivo fiber photometry in wild-type prairie voles and those lacking Oxtr, we demonstrate that Oxtr function sex-specifically regulates pair bonding behaviors and associated activity in the NAc. Oxtr function influences prosocial behavior in females in a state-dependent manner. Females lacking Oxtr demonstrate reduced prosocial behaviors and lower activity in the NAc during initial chemosensory investigation of novel males. Upon pair bonding, affiliative behavior with partners and neural activity in the NAc during these interactions increase, but these changes do not require Oxtr function. Conversely, males lacking Oxtr display increased prosocial investigation of novel females. Using the altered patterns of behavior and activity in the NAc of males lacking Oxtr during their first interactions with a female, we can predict their future preference for a partner or stranger days later. These results demonstrate that Oxtr function sex-specifically influences the early development of pair bonds by modulating prosociality and the neural processing of sensory cues and social interactions with novel individuals, unmasking underlying sex differences in the neural pathways regulating the formation of long-term relationships.

Midline crossing by gustatory receptor neuron axons is regulated by fruitless, doublesex and the Roundabout receptors.

Although nervous system sexual dimorphisms are known in many species, relatively little is understood about the molecular mechanisms generating these dimorphisms. Recent findings in Drosophila provide the tools for dissecting how neurogenesis and neuronal differentiation are modulated by the Drosophila sex-determination regulatory genes to produce nervous system sexual dimorphisms. Here we report studies aimed at illuminating the basis of the sexual dimorphic axonal projection patterns of foreleg gustatory receptor neurons (GRNs): only in males do GRN axons project across the midline of the ventral nerve cord. We show that the sex determination genes fruitless (fru) and doublesex (dsx) both contribute to establishing this sexual dimorphism. Male-specific Fru (Fru(M)) acts in foreleg GRNs to promote midline crossing by their axons, whereas midline crossing is repressed in females by female-specific Dsx (Dsx(F)). In addition, midline crossing by these neurons might be promoted in males by male-specific Dsx (Dsx(M)). Finally, we (1) demonstrate that the roundabout (robo) paralogs also regulate midline crossing by these neurons, and (2) provide evidence that Fru(M) exerts its effect on midline crossing by directly or indirectly regulating Robo signaling.

Attachment across the lifespan: Examining the intersection of pair bonding neurobiology and healthy aging.

Increasing evidence suggests that intact social bonds are protective against age-related morbidity, while bond disruption and social isolation increase the risk for multiple age-related diseases. Social attachments, the enduring, selective bonds formed between individuals, are thus essential to human health. Socially monogamous species like the prairie vole (M. ochrogaster) form long-term pair bonds, allowing us to investigate the mechanisms underlying attachment and the poorly understood connection between social bonds and health. In this review, we explore several potential areas of focus emerging from data in humans and other species associating attachment and healthy aging, and evidence from prairie voles that may clarify this link. We examine gaps in our understanding of social cognition and pair bond behavior. Finally, we discuss physiologic pathways related to pair bonding that promote resilience to the processes of aging and age-related disease. Advances in the development of molecular genetic tools in monogamous species will allow us to bridge the mechanistic gaps presented and identify conserved research and therapeutic targets relevant to human health and aging.

Oxytocin receptor is not required for social attachment in prairie voles.

Prairie voles are among a small group of mammals that display long-term social attachment between mating partners. Many pharmacological studies show that signaling via the oxytocin receptor (Oxtr) is critical for the display of social monogamy in these animals. We used CRISPR mutagenesis to generate three different Oxtr-null mutant prairie vole lines. Oxtr mutants displayed social attachment such that males and females showed a behavioral preference for their mating partners over a stranger of the opposite sex, even when assayed using different experimental setups. Mothers lacking Oxtr delivered viable pups, and parents displayed care for their young and raised them to the weanling stage. Together, our studies unexpectedly reveal that social attachment, parturition, and parental behavior can occur in the absence of Oxtr signaling in prairie voles.

From mating to mama bear: Distinct VMHvl cell types drive female reproductive state-dependent behavior.

In this issue of Neuron, Liu et al. (2022) molecularly identify subsets of estrogen receptor-1-positive neurons within the female ventrolateral subdivision of the ventromedial hypothalamus activated during sexual receptivity versus agonistic behaviors in distinct reproductive states and demonstrate that these subsets control state-dependent changes in social behaviors.

Rethinking the Architecture of Attachment: New Insights into the Role for Oxytocin Signaling.

Social attachments, the enduring bonds between individuals and groups, are essential to health and well-being. The appropriate formation and maintenance of social relationships depend upon a number of affective processes, including stress regulation, motivation, reward, as well as reciprocal interactions necessary for evaluating the affective state of others. A genetic, molecular, and neural circuit level understanding of social attachments therefore provides a powerful substrate for probing the affective processes associated with social behaviors. Socially monogamous species form long-term pair bonds, allowing us to investigate the mechanisms underlying attachment. Now, molecular genetic tools permit manipulations in monogamous species. Studies using these tools reveal new insights into the genetic and neuroendocrine factors that design and control the neural architecture underlying attachment behavior. We focus this discussion on the prairie vole and oxytocinergic signaling in this and related species as a model of attachment behavior that has been studied in the context of genetic and pharmacological manipulations. We consider developmental processes that impact the demonstration of bonding behavior across genetic backgrounds, the modularity of mechanisms underlying bonding behaviors, and the distributed circuitry supporting these behaviors. Incorporating such theoretical considerations when interpreting reverse genetic studies in the context of the rich ethological and pharmacological data collected in monogamous species provides an important framework for studies of attachment behavior in both animal models and studies of human relationships.

Cupid's quiver: Integrating sensory cues in rodent mating systems.

In many animal species, males and females exploit different mating strategies, display sex-typical behaviors, and use distinct systems to recognize ethologically relevant cues. Mate selection thus requires mutual recognition across diverse social interactions based on distinct sensory signals. These sex differences in courtship and mating behaviors correspond to differences in sensory systems and downstream neural substrates engaged to recognize and respond to courtship signals. In many rodents, males tend to rely heavily on volatile olfactory and pheromone cues, while females appear to be guided more by a combination of these chemosensory signals with acoustic cues in the form of ultrasonic vocalizations. The mechanisms by which chemical and acoustic cues are integrated to control behavior are understudied in mating but are known to be important in the control of maternal behaviors. Socially monogamous species constitute a behaviorally distinct group of rodents. In these species, anatomic differences between males and females outside the nervous system are less prominent than in species with non-monogamous mating systems, and both sexes engage in more symmetric social behaviors and form attachments. Nevertheless, despite the apparent similarities in behaviors displayed by monogamous males and females, the circuitry supporting social, mating, and attachment behaviors in these species is increasingly thought to differ between the sexes. Sex differences in sensory modalities most important for mate recognition in across species are of particular interest and present a wealth of questions yet to be answered. Here, we discuss how distinct sensory cues may be integrated to drive social and attachment behaviors in rodents, and the differing roles of specific sensory systems in eliciting displays of behavior by females or males.

Male-specific fruitless specifies the neural substrates of Drosophila courtship behaviour.

Robust innate behaviours are attractive systems for genetically dissecting how environmental cues are perceived and integrated to generate complex behaviours. During courtship, Drosophila males engage in a series of innate, stereotyped behaviours that are coordinated by specific sensory cues. However, little is known about the specific neural substrates mediating this complex behavioural programme. Genetic, developmental and behavioural studies have shown that the fruitless (fru) gene encodes a set of male-specific transcription factors (FruM) that act to establish the potential for courtship in Drosophila. FruM proteins are expressed in approximately 2% of central nervous system neurons, at least one subset of which coordinates the component behaviours of courtship. Here we have inserted the yeast GAL4 gene into the fru locus by homologous recombination and show that (1) FruM is expressed in subsets of all peripheral sensory systems previously implicated in courtship, (2) inhibition of FruM function in olfactory system components reduces olfactory-dependent changes in courtship behaviour, (3) transient inactivation of all FruM-expressing neurons abolishes courtship behaviour, with no other gross changes in general behaviour, and (4) 'masculinization' of FruM-expressing neurons in females is largely sufficient to confer male courtship behaviour. Together, these data demonstrate that FruM proteins specify the neural substrates of male courtship.

Autism Spectrum Disorder Genetics and the Search for Pathological Mechanisms.

Recent progress in the identification of genes and genomic regions contributing to autism spectrum disorder (ASD) has had a broad impact on our understanding of the nature of genetic risk for a range of psychiatric disorders, on our understanding of ASD biology, and on defining the key challenges now facing the field in efforts to translate gene discovery into an actionable understanding of pathology. While these advances have not yet had a transformative impact on clinical practice, there is nonetheless cause for real optimism: reliable lists of risk genes are large and growing rapidly; the identified encoded proteins have already begun to point to a relatively small number of areas of biology, where parallel advances in neuroscience and functional genomics are yielding profound insights; there is strong evidence pointing to mid-fetal prefrontal cortical development as one nexus of vulnerability for some of the largest-effect ASD risk genes; and there are multiple plausible paths forward toward rational therapeutics development that, while admittedly challenging, constitute fundamental departures from what was possible prior to the era of successful gene discovery.

Cannabinoid receptor Type 1 densities reflect social organization in Microtus.

Across many species, endocannabinoids play an important role in regulating social play, reward, and anxiety. These processes are mediated through at least two distinct cannabinoid receptors (CB), CB1 and CB2. CB1 expression is found in appreciable densities across regions of the brain that integrate memory with socio-spatial information; many of these regions have been directly linked to the neurobiology of pair bonding in monogamous species. Using receptor autoradiography, we provide the first distributional map of CB1 within the brains of closely related monogamous prairie voles and promiscuous meadow voles, and compare receptor densities across sexes and species in limbic regions. We observe CB1-specific signal using [3H] CP-55,940 and [3H] SR141716A, though the latter exhibited a lower signal to noise ratio. We confirmed the presence of CB2 in prairie vole spleen tissue using [3H] CP-55,940. However, we found no evidence of CB2 in the brain using either [3H] CP-55,940 or [3H] A-836,339. The overall distribution of putative CB1 in the brain was similar across vole species and followed the pattern of CB1 expression observed in other species-high intensity binding within the telencephalon, moderate binding within the diencephalon, and mild binding within the mesencephalon and metencephalon (aside from the cerebellar cortex). However, we found profound differences in CB1 densities across species, with prairie voles having higher CB1 binding in regions implicated in social attachment and spatial memory (e.g., periaqueductal gray, hippocampus). These findings suggest that CB1 densities, but not distribution, correlate with the social systems of vole species.

Median bundle neurons coordinate behaviours during Drosophila male courtship.

Throughout the animal kingdom the innate nature of basic behaviour routines suggests that the underlying neuronal substrates necessary for their execution are genetically determined and developmentally programmed. Complex innate behaviours require proper timing and ordering of individual component behaviours. In Drosophila melanogaster, analyses of combinations of mutations of the fruitless (fru) gene have shown that male-specific isoforms (Fru(M)) of the Fru transcription factor are necessary for proper execution of all steps of the innate courtship ritual. Here, we eliminate Fru(M) expression in one group of about 60 neurons in the Drosophila central nervous system and observe severely contracted courtship behaviour, including rapid courtship initiation, absence of orienting and tapping, and the simultaneous occurrence of wing vibration, licking and attempted copulation. Our results identify a small group of median bundle neurons, that in wild-type Drosophila appropriately trigger the sequential execution of the component behaviours that constitute the Drosophila courtship ritual.

Sex and the Single Fly: A Perspective on the Career of Bruce S. Baker.

Bruce Baker, a preeminent Drosophila geneticist who made fundamental contributions to our understanding of the molecular genetic basis of sex differences, passed away July 1, 2018 at the age of 72. Members of Bruce's laboratory remember him as an intensely dedicated, rigorous, creative, deep-thinking, and fearless scientist. His trainees also remember his strong commitment to teaching students at every level. Bruce's career studying sex differences had three major epochs, where the laboratory was focused on: (1) sex determination and dosage compensation, (2) the development of sex-specific structures, and (3) the molecular genetic basis for sex differences in behavior. Several members of the Baker laboratory have come together to honor Bruce by highlighting some of the laboratory's major scientific contributions in these areas.

Blueprints for behavior: genetic specification of neural circuitry for innate behaviors.

Innate behaviors offer a unique opportunity to use genetic analysis to dissect and characterize the neural substrates of complex behavioral programs. Courtship in Drosophila involves a complex series of stereotyped behaviors that include numerous exchanges of multimodal sensory information over time. As we will discuss in this review, recent work has demonstrated that male-specific expression of Fruitless transcription factors (Fru(M) proteins) is necessary and sufficient to confer the potential for male courtship behaviors. Fru(M) factors program neurons of the male central and peripheral nervous systems whose function is dedicated to sexual behaviors. This circuitry seems to integrate sensory information to define behavioral states and regulate conserved neural elements for sex-specific behavioral output. The principles that govern the circuitry specified by Fru(M) expression might also operate in subcortical networks that govern innate behaviors in mammals.

Gene regulatory mechanisms underlying sex differences in brain development and psychiatric disease.

The sexual differentiation of the mammalian nervous system requires the precise coordination of the temporal and spatial regulation of gene expression in diverse cell types. Sex hormones act at multiple developmental time points to specify sex-typical differentiation during embryonic and early development and to coordinate subsequent responses to gonadal hormones later in life by establishing sex-typical patterns of epigenetic modifications across the genome. Thus, mutations associated with neuropsychiatric conditions may result in sexually dimorphic symptoms by acting on different neural substrates or chromatin landscapes in males and females. Finally, as stress hormone signaling may directly alter the molecular machinery that interacts with sex hormone receptors to regulate gene expression, the contribution of chronic stress to the pathogenesis or presentation of mental illness may be additionally different between the sexes. Here, we review the mechanisms that contribute to sexual differentiation in the mammalian nervous system and consider some of the implications of these processes for sex differences in neuropsychiatric conditions.

Spontaneous emergence of overgrown molar teeth in a colony of Prairie voles (Microtus ochrogaster).

Continuously growing incisors are common to all rodents, which include the Microtus genus of voles. However, unlike many rodents, voles also possess continuously growing molars. Here, we report spontaneous molar defects in a population of Prairie voles (Microtus ochrogaster). We identified bilateral protuberances on the ventral surface of the mandible in several voles in our colony. In some cases, the protuberances broke through the cortical bone. The mandibular molars became exposed and infected, and the maxillary molars entered the cranial vault. Visualisation upon soft tissue removal and microcomputed tomography (microCT) analyses confirmed that the protuberances were caused by the overgrowth of the apical ends of the molar teeth. We speculate that the unrestricted growth of the molars was due to the misregulation of the molar dental stem cell niche. Further study of this molar phenotype may yield additional insight into stem cell regulation and the evolution and development of continuously growing teeth.

Neural pathways for the detection and discrimination of conspecific song in D. melanogaster.

BACKGROUND: During courtship, male Drosophila melanogaster sing a multipart courtship song to female flies. This song is of particular interest because (1) it is species specific and varies widely within the genus, (2) it is a gating stimulus for females, who are sensitive detectors of conspecific song, and (3) it is the only sexual signal that is under both neural and genetic control. This song is perceived via mechanosensory neurons in the antennal Johnston's organ, which innervate the antennal mechanosensory and motor center (AMMC) of the brain. However, AMMC outputs that are responsible for detection and discrimination of conspecific courtship song remain unknown. RESULTS: Using a large-scale anatomical screen of AMMC interneurons, we identify seven projection neurons (aPNs) and five local interneurons (aLNs) that outline a complex architecture for the ascending mechanosensory pathway. Neuronal inactivation and hyperactivation during behavior reveal that only two classes of interneurons are necessary for song responses--the projection neuron aPN1 and GABAergic interneuron aLN(al). These neurons are necessary in both male and female flies. Physiological recordings in aPN1 reveal the integration of courtship song as a function of pulse rate and outline an intracellular transfer function that likely facilitates the response to conspecific song. CONCLUSIONS: These results reveal a critical pathway for courtship hearing in male and female flies, in which both aLN(al) and aPN1 mediate the detection of conspecific song. The pathways arising from these neurons likely serve as a critical neural substrate for behavioral reproductive isolation in D. melanogaster.

Neural control of sexually dimorphic behaviors.

All sexually reproducing animals exhibit gender differences in behavior. Such sexual dimorphisms in behavior are most obvious in stereotyped displays that enhance reproductive success such as mating, aggression, and parental care. Sexually dimorphic behaviors are a consequence of a sexually differentiated nervous system, and recent studies in fruit flies and mice reveal novel insights into the neural mechanisms that control these behaviors. In the main, these include a diverse array of novel sex differences in the nervous system, surprisingly modular control of various stereotyped dimorphic behavioral routines, and unanticipated sensory and central modulation of mating. We start with a brief overview to provide the appropriate conceptual framework so that the advances made by the newer studies discussed subsequently can be fully appreciated. We restrict our review to reporting progress in understanding the basis of mating and aggression in fruit flies and mice.