Fig. 3: DP projections to brain structures regulating autonomic outputs.

a, The DPs innervates two parts of the PVHd (1–3), marked by VGLUT2 expression (2; Bright-field in situ hybridization images from the ABA (https://Brain-map.org)): the PVHlp and PVHf, which contain spinal-projecting neurons labelled by CTB spinal cord injection (4; see Extended Data Fig. 15a for PHAL injection). DPs axons also densely innervate the LHA (5), which contains CTB-labelled spinal-projecting neurons (6). fx, fornix; mtt, mammilothalamic tract; v3, third ventricle. b, In MORF3 mice, AAV-RFP was injected into the DP (1 and 2) and AAVretro-Cre was injected into the spinal cord to identify potential synaptic contacts between DP axons and hypothalamic spinal-projecting neurons. Cre-induced MORF3 expression revealed the morphology of spinal-projecting neurons (3). High-resolution light-sheet (1–4 and 7) and confocal (5, 6 and 8–12) images show close appositions between DP terminals and MORF3-labelled somas and dendrites in PVHd (6) and LHA (7–12). The blue arrowheads mark corresponding neurons across magnifications; the white arrowheads indicate putative axodendritic contacts (Supplementary Video 1). c, AAV1-Cre trans-synaptic tagging and Cre-dependent anterograde tracing confirmed the DP to LHA to IML column (sympathetic preganglionic neurons) pathway. AAV1-Cre DP injection (top left) transported Cre to postsynaptic LHA neurons, which were labelled through Cre-dependent AAV-GFP (bottom left). Middle, GFP-labelled axons in the IML (thoracic levels T1–T2). Right, magnified images showing terminal boutons apposed to neurons in the IML (1–3) and near the central canal (4). d, Pathways through which the DPs regulates vagal parasympathetic output. AAV1-Cre DPs injection (1) anterogradely transported Cre to the CEA, where Cre-dependent AAV-GFP was injected (2) to label axons from Cre-positive postsynaptic neurons (3–5). CEA starter cells co-expressed Cre and GFP (2). GFP-labelled axons in the DMX and NTS (5), confirming the DPs to CEA to DMX pathway. Projections targeted other structures that regulate autonomic function, for example, BST and SI (3), PB (4), PARN and IRN (5). Image 6 shows DMX/NTS labelling from non-selective CEA neurons. V, trigeminal motor nucleus. e, Schematic of the DPs, DPd and ILA in a cerebral network regulating autonomic outputs. f, Direct projections to Barrington’s nucleus (B) from the ILA (left) and DPd (right; green), indicating disynaptic MPF connections controlling lumbosacral parasympathetic outputs (DPd/ILA to Barrington’s nucleus to spinal cord). Note that PL also projects to Barrington’s nucleus (right; red). Scale bars, 5 μm (b (10 and 11)), 7 μm (b (12)), 10 μm (b (9)), 20 μm (c (right)), 30 μm (b (6)), 100 μm (c (top left, inset)), 200 μm (a and c (bottom left and middle)), 300 μm (b (2–5, 7 and 8)), 500 µm (d and f) and 1 mm (c (top left, main image)). A full list of abbreviations is provided in Supplementary Table 1.