Hypothalamic neurons that mirror aggression.

Social interactions require awareness and understanding of the behavior of others. Mirror neurons, cells representing an action by self and others, have been proposed to be integral to the cognitive substrates that enable such awareness and understanding. Mirror neurons of the primate neocortex represent skilled motor tasks, but it is unclear if they are critical for the actions they embody, enable social behaviors, or exist in non-cortical regions. We demonstrate that the activity of individual VMHvlPR neurons in the mouse hypothalamus represents aggression performed by self and others. We used a genetically encoded mirror-TRAP strategy to functionally interrogate these aggression-mirroring neurons. We find that their activity is essential for fighting and that forced activation of these cells triggers aggressive displays by mice, even toward their mirror image. Together, we have discovered a mirroring center in an evolutionarily ancient region that provides a subcortical cognitive substrate essential for a social behavior.

A neural circuit for male sexual behavior and reward.

Male sexual behavior is innate and rewarding. Despite its centrality to reproduction, a molecularly specified neural circuit governing innate male sexual behavior and reward remains to be characterized. We have discovered a developmentally wired neural circuit necessary and sufficient for male mating. This circuit connects chemosensory input to BNSTprTac1 neurons, which innervate POATacr1 neurons that project to centers regulating motor output and reward. Epistasis studies demonstrate that BNSTprTac1 neurons are upstream of POATacr1 neurons, and BNSTprTac1-released substance P following mate recognition potentiates activation of POATacr1 neurons through Tacr1 to initiate mating. Experimental activation of POATacr1 neurons triggers mating, even in sexually satiated males, and it is rewarding, eliciting dopamine release and self-stimulation of these cells. Together, we have uncovered a neural circuit that governs the key aspects of innate male sexual behavior: motor displays, drive, and reward.

A functional cellular framework for sex and estrous cycle-dependent gene expression and behavior.

Sex hormones exert a profound influence on gendered behaviors. How individual sex hormone-responsive neuronal populations regulate diverse sex-typical behaviors is unclear. We performed orthogonal, genetically targeted sequencing of four estrogen receptor 1-expressing (Esr1+) populations and identified 1,415 genes expressed differentially between sexes or estrous states. Unique subsets of these genes were distributed across all 137 transcriptomically defined Esr1+ cell types, including estrous stage-specific ones, that comprise the four populations. We used differentially expressed genes labeling single Esr1+ cell types as entry points to functionally characterize two such cell types, BNSTprTac1/Esr1 and VMHvlCckar/Esr1. We observed that these two cell types, but not the other Esr1+ cell types in these populations, are essential for sex recognition in males and mating in females, respectively. Furthermore, VMHvlCckar/Esr1 cell type projections are distinct from those of other VMHvlEsr1 cell types. Together, projection and functional specialization of dimorphic cell types enables sex hormone-responsive populations to regulate diverse social behaviors.

Limbic Neurons Shape Sex Recognition and Social Behavior in Sexually Naive Males.

Sexually naive animals have to distinguish between the sexes because they show species-typical interactions with males and females without meaningful prior experience. However, central neural pathways in naive mammals that recognize sex of other individuals remain poorly characterized. We examined the role of the principal component of the bed nucleus of stria terminalis (BNSTpr), a limbic center, in social interactions in mice. We find that activity of aromatase-expressing BNSTpr (AB) neurons appears to encode sex of other animals and subsequent displays of mating in sexually naive males. Silencing these neurons in males eliminates preference for female pheromones and abrogates mating success, whereas activating them even transiently promotes male-male mating. Surprisingly, female AB neurons do not appear to control sex recognition, mating, or maternal aggression. In summary, AB neurons represent sex of other animals and govern ensuing social behaviors in sexually naive males.

Periodic Remodeling in a Neural Circuit Governs Timing of Female Sexual Behavior.

Behaviors are inextricably linked to internal state. We have identified a neural mechanism that links female sexual behavior with the estrus, the ovulatory phase of the estrous cycle. We find that progesterone-receptor (PR)-expressing neurons in the ventromedial hypothalamus (VMH) are active and required during this behavior. Activating these neurons, however, does not elicit sexual behavior in non-estrus females. We show that projections of PR+ VMH neurons to the anteroventral periventricular (AVPV) nucleus change across the 5-day mouse estrous cycle, with ∼3-fold more termini and functional connections during estrus. This cyclic increase in connectivity is found in adult females, but not males, and regulated by estrogen signaling in PR+ VMH neurons. We further show that these connections are essential for sexual behavior in receptive females. Thus, estrogen-regulated structural plasticity of behaviorally salient connections in the adult female brain links sexual behavior to the estrus phase of the estrous cycle.

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.

Sexually dimorphic neurons in the ventromedial hypothalamus govern mating in both sexes and aggression in males.

Sexual dimorphisms in the brain underlie behavioral sex differences, but the function of individual sexually dimorphic neuronal populations is poorly understood. Neuronal sexual dimorphisms typically represent quantitative differences in cell number, gene expression, or other features, and it is unknown whether these dimorphisms control sex-typical behavior exclusively in one sex or in both sexes. The progesterone receptor (PR) controls female sexual behavior, and we find many sex differences in number, distribution, or projections of PR-expressing neurons in the adult mouse brain. Using a genetic strategy we developed, we have ablated one such dimorphic PR-expressing neuronal population located in the ventromedial hypothalamus (VMH). Ablation of these neurons in females greatly diminishes sexual receptivity. Strikingly, the corresponding ablation in males reduces mating and aggression. Our findings reveal the functions of a molecularly defined, sexually dimorphic neuronal population in the brain. Moreover, we show that sexually dimorphic neurons can control distinct sex-typical behaviors in both sexes.

Modular genetic control of sexually dimorphic behaviors.

Sex hormones such as estrogen and testosterone are essential for sexually dimorphic behaviors in vertebrates. However, the hormone-activated molecular mechanisms that control the development and function of the underlying neural circuits remain poorly defined. We have identified numerous sexually dimorphic gene expression patterns in the adult mouse hypothalamus and amygdala. We find that adult sex hormones regulate these expression patterns in a sex-specific, regionally restricted manner, suggesting that these genes regulate sex typical behaviors. Indeed, we find that mice with targeted disruptions of each of four of these genes (Brs3, Cckar, Irs4, Sytl4) exhibit extremely specific deficits in sex specific behaviors, with single genes controlling the pattern or extent of male sexual behavior, male aggression, maternal behavior, or female sexual behavior. Taken together, our findings demonstrate that various components of sexually dimorphic behaviors are governed by separable genetic programs.

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 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.

Restriction of transient receptor potential vanilloid-1 to the peptidergic subset of primary afferent neurons follows its developmental downregulation in nonpeptidergic neurons.

Primary afferent "pain" fibers (nociceptors) are divided into subclasses based on distinct molecular and anatomical features, and these classes mediate noxious modality-specific contributions to behaviors evoked by painful stimuli. Whether the heat and capsaicin receptor transient receptor potential vanilloid-1 (TRPV1) is expressed heterogeneously across several sensory populations, or is selectively expressed by a unique nociceptor subclass, however, is unclear. Here we used two lines of Trpv1 reporter mice to investigate the primary afferent expression of TRPV1, both during development and in the adult. We demonstrate, using Cre-induced lineage tracing, that during development TRPV1 is transiently expressed in a wide range of dorsal root ganglion neurons, and that its expression is gradually refined, such that TRPV1 transcripts become restricted to a specific subset of peptidergic sensory neurons. Finally, the remarkable sensitivity that is characteristic of these reporter mice revealed an innervation of central and peripheral targets by TRPV1+ primary afferents in the adult that is considerably more extensive than has previously been appreciated.

Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells.

The heat and capsaicin receptor, TRPV1, is required for the detection of painful heat by primary afferent pain fibers (nociceptors), but the extent to which functional TRPV1 channels are expressed in the CNS is debated. Because previous evidence is based primarily on indirect physiological responses to capsaicin, here we genetically modified the Trpv1 locus to reveal, with excellent sensitivity and specificity, the distribution of TRPV1 in all neuronal and non-neuronal tissues. In contrast to reports of widespread and robust expression in the CNS, we find that neuronal TRPV1 is primarily restricted to nociceptors in primary sensory ganglia, with minimal expression in a few discrete brain regions, most notably in a contiguous band of cells within and adjacent to the caudal hypothalamus. We confirm hypothalamic expression in the mouse using several complementary approaches, including in situ hybridization, calcium imaging, and electrophysiological recordings. Additional in situ hybridization experiments in rat, monkey, and human brain demonstrate that the restricted expression of TRPV1 in the CNS is conserved across species. Outside of the CNS, we find TRPV1 expression in a subset of arteriolar smooth muscle cells within thermoregulatory tissues. Here, capsaicin increases calcium uptake and induces vasoconstriction, an effect that likely counteracts the vasodilation produced by activation of neuronal TRPV1.

Nursing behavior: remembrance of things past.

Successful suckling is vital to the survival of mammalian newborns. In many mammals, nursing behavior is triggered by maternally derived odors. Such odors may also promote the learned association of odorant cues present in the environment during nursing.

Deficits in sexual and aggressive behaviors in Cnga2 mutant mice.

Odors detected by the vomeronasal organ or the main olfactory epithelium (MOE) trigger social behaviors in many animals. It is unknown whether MOE neurons detect cues that initiate mating or aggression. We demonstrate that mice lacking functional CNGA2 (cyclic nucleotide-gated channel alpha2), which is required for odor-evoked MOE signaling, fail to mate or fight, suggesting a broad and essential role for the MOE in regulating these behaviors.

Evolution of Mechanisms that Control Mating in Drosophila Males.

Genetically wired neural mechanisms inhibit mating between species because even naive animals rarely mate with other species. These mechanisms can evolve through changes in expression or function of key genes in sensory pathways or central circuits. Gr32a is a gustatory chemoreceptor that, in D. melanogaster, is essential to inhibit interspecies courtship and sense quinine. Similar to D. melanogaster, we find that D. simulans Gr32a is expressed in foreleg tarsi, sensorimotor appendages that inhibit interspecies courtship, and it is required to sense quinine. Nevertheless, Gr32a is not required to inhibit interspecies mating by D. simulans males. However, and similar to its function in D. melanogaster, Ppk25, a member of the Pickpocket family, promotes conspecific courtship in D. simulans. Together, we have identified distinct evolutionary mechanisms underlying chemosensory control of taste and courtship in closely related Drosophila species.