Distinct afferent signatures onto anterior and posterior VMHvl subdomains and their progesterone receptor-expressing neurons

Abstract

Female sexual behavior is fundamentally coupled to reproductive state, requiring the dynamic and coordinated regulation of sexual receptivity and rejection across the estrous cycle. In mice, these opposing behaviors are controlled by distinct neuronal subpopulations within the anteroposterior subdomains of the ventrolateral ventromedial hypothalamus (VMHvl): the posterior VMHvl (pVMHvl) promotes sexual receptivity, whereas the anterior VMHvl (aVMHvl) drives sexual rejection. Although progesterone receptor-expressing (PR) neurons in both subregions are essential for these behaviors, the circuit mechanisms underlying this anteroposterior functional segregation remain poorly understood. Here, we investigated whether anterior and posterior VMHvl subdomains receive distinct afferent inputs that could support their divergent roles, and whether these inputs specifically target PR neurons. By combining retrograde tracing with whole-brain, machine learning-assisted quantification, we first mapped the afferent connectivity of the aVMHvl and pVMHvl. We found that while the two subdomains share the majority of their inputs, they also receive distinct and biased projections. The aVMHvl preferentially receives inputs from the anterior hypothalamic nucleus, paraventricular and peripeduncular thalamic nuclei, and the parabrachial nucleus, while the pVMHvl is preferentially innervated by the preoptic area, dorsal and ventral premammillary nuclei and anteroventral periventricular nucleus. To determine whether the identified afferents directly synapse onto the PR neurons, we performed monosynaptic rabies tracing from anterior and posterior VMHvl PR neurons. These experiments revealed that most anterior- and posterior-biased projections directly targeted PR VMHvl neurons. In addition, projections from the paraventricular thalamus, periaqueductal gray, and several pontine nuclei preferentially innervated aVMHvlPR neurons, whereas inputs from the mammillary nuclei mostly targeted pVMHvlPR neurons. Together, our results reveal distinct afferent architectures onto anterior and posterior PR VMHvl neurons, providing a circuit-level framework for understanding how hormonal state flexibly biases female sexual behavior toward receptivity or rejection.