Extended Data Fig. 18: Additional data supporting DPd’s projections to the hypothalamic neuroendocrine zone.

a, Additional evidence showing that DPd VGLUT2+ neurons directly innervate hypothalamic neuroendocrine cells. In VGLUT2 Cre mice, a Cre-dependent AAV-FLEX-RFP was injected into DPd (upper left), resulting in robust anterograde labelling in the hypothalamic neuroendocrine zone. FG was administered intravenously to reveal neuroendocrine cells⁴⁸. Neuroendocrine cells in the periventricular nucleus (PVpo) and PVH intermingle with dense AAV-RFP labelled axonal terminals from DP (Fig. 4d). Intermingling was also evident in the dorsomedial arcuate nucleus (ARH; b) and dorsal supraoptic nucleus (SO; c), indicating direct synaptic input from DPd VGLUT2+ neurons to diverse neuroendocrine cell populations. d, VGLUT2+ axons from the DPd closely appose OXY neurons in PVH (revealed with fluorescent immunostaining method). Close-up image shows putative synaptic appositions at 1 µm optical z-resolution. e, Monosynaptic rabies tracing reveals direct inputs from DPd to CRH-expressing neuroendocrine neurons in PVH. In CRH Cre transgenic mice, Cre-dependent helper virus (AAV8-hSyn-FLEX-TVA-P2A-GFP-2A-oG) was injected into the PVH to express TVA and rabies glycoprotein (RG), followed by injection of mCherry-expressing G-deleted rabies virus (left panel). This approach selectively labels neurons providing direct monosynaptic input to CRH neurons. Rabies-labelled neurons were observed in DPd (right panels), but not in other MPF areas, despite the known limitation of rabies virus in labelling deep-layer cortical neurons. These data provide anatomical evidence that DPd directly innervates CRH neuroendocrine neurons. f, BSTam PHAL/CTB coinjection labels neurons in DPd and ILA, as well as other structures (CEA, MEA, BMAa). Anterogradely labelled axons target multiple hypothalamic nuclei, including PVH, ARH, AHN, DMH, VMH, and LHA. These findings suggest that DPd and ILA modulate neuroendocrine, autonomic, and behavioural functions via their projections to BSTam. Scale bars = 500 µm.