Fig. 1: Morphological and functional classification of Dat1⁺ neurons in the PeVN.

a Immunohistochemical identification of Dat1⁺ A14 dopamine neurons co-expressing tyrosine hydroxylase (TH) and tdTomato (fluorescence marked by arrows) in the PeVN of Dat1-Ires-Cre:Ai14 mice (n = 10 animals). Scale bar = 50 μm. b and c Representative current-clamp recordings of Dat1⁺ neurons in the PeVN and their post-hoc morphological reconstruction, including subtype “1” (b) and “2” (c) cells. Top left: schematic illustration showing the rostrocaudal allocation of A14 subtypes across the PeVN. The proportion of each neuronal subtype is indicated. Middle left: Action potential (AP) responses to two rheobase current pulses (colored) and at maximal frequency (black). Bottom left: Phase-plane plots of APs rising from 2× rheobase current injection. Right: Morphological reconstruction of a representative biocytin-filled Dat1⁺ neuron for each subtype. Axons were colorized in cyan, whereas dendrites were pseudocolored in magenta. d Immunohistochemistry for neuromedin S in the SCN (n = 9 animals). Scale bar = 10 μm. e Immunohistochemical detection of neuromedin S⁺ terminals contacting biocytin-filled Dat1⁺ neurons in the PeVN (arrows). Open rectangle denotes the location of the high-resolution inset. Representative AP signature identifies the target as a subtype “1” neuron. The experiment was performed in triplicate and produced invariable results. Scale bars = 50 μm (left) and 10 μm (right). f Representative current-clamp recordings of neuromedin-S induced AP firing by subtype “1” (top, n = 17 cells with 14 responders) and “2” neurons (bottom, n = 21 cells; 16 responders). Data were obtained in n = 14 animals. g Schematic map of A14 neuronal responses to neuromedin S in the rostrocaudal domain of the PeVN. Circle sizes are proportional to response strength (a total of n = 30 neurons from 14 animals were mapped). 3V third ventricle, AH anterior hypothalamus, Arc arcuate nucleus, DMH dorsomedial hypothalamus, PVN paraventricular nucleus, ScN suprachiasmatic nucleus, VMH ventromedial hypothalamus. Biorender was used to style experimental arrangements and brain regions (in b, c, g).