Whole-Brain Monosynaptic Inputs to Parasubthalamic Calretinin Neurons in Mice

The parasubthalamic nucleus (PSTN) plays a crucial role in various physiological functions, including feeding, motivation, thermoregulation, exploration, sleep-wakefulness and cardiovascular system regulation. However, presynaptic partners that modulate PSTN activity are poorly understood. A significant proportion of PSTN neurons express calretinin (CR). This study employed a modified rabies virus method for cell-type-specific retrograde tracing in combination with a Cre/loxP gene-expression strategy to map the monosynaptic inputs to PSTN^CR neurons across the entire brain in mice. We identified 76 nuclei that target PSTN^CR neurons, distributed across five major brain regions: the telencephalon, diencephalon, midbrain, pons, and medulla. Among these, several nuclei implicated in both physiological and pathological processes, including the lateral hypothalamus (LH), central amygdaloid nucleus (CeA), ventral tegmental area (VTA), dorsal raphe (DR), and parabrachial nucleus (PB), exhibited dense projections to the PSTN^CR neurons. Further immunofluorescence experiments revealed that the inputs to PSTN^CR neurons were colocalized with neurons involved in regulating vital physiological functions. Specifically, we observed GABAergic neurons in the CeA and the bed nucleus of the stria terminalis (BST), dopamine neurons in the VTA and substantia nigra pars compacta (SNC), serotonergic neurons in the DR. Our findings provide anatomical evidence that advances our understanding of the neural circuits underlying the physiological functions of the PSTN. This study also clarifies how CR-expressing neurons in the PSTN integrate diverse signals to regulate various functions, potentially providing therapeutic strategies for neurological disorders.