Whole-Brain Monosynaptic Inputs and Outputs of Corticotropin-Releasing Hormone Neurons of the Paraventricular Nucleus of the Hypothalamus in Male Mice.

OBJECTIVE: The paraventricular nucleus of the hypothalamus (PVN) is a central hub integrating neuroendocrine, autonomic, and behavioral responses. Corticotropin-releasing hormone (CRH) neurons in the PVN (PVNCRH) play a pivotal role in stress responses, anxiety, and motivation. However, the brain-wide monosynaptic input-output architecture of PVNCRH neurons remains incompletely defined, limiting circuit-based functional dissection. METHODS: Using Cre-dependent modified rabies virus for retrograde monosynaptic tracing and adeno-associated viruses for anterograde tracing in CRH-Cre mice, we systematically mapped the whole-brain inputs and outputs of PVNCRH neurons. RESULTS: PVNCRH neurons received direct inputs from 55 brain regions, with a predominant contribution from diencephalon areas and an overall ipsilateral bias (61.4%). Notably, highly specific inputs originated from the ventral hippocampal CA1 and nucleus accumbens core. PVNCRH neurons also sent widespread axonal projections throughout the brain. These outputs converge onto multiple circuit modules, with dense projections to emotion-related nuclei and autonomic regulatory centers. CONCLUSION: This study establishes a comprehensive whole-brain monosynaptic input-output atlas of PVNCRH neurons. The resultant neuroanatomical framework delineates their extensive connectivity with stress-, emotion-, autonomic-, and respiration-related circuits, providing a structural basis for future circuit-level investigations into stress-related psychiatric disorders and systemic autonomic dysregulation.