a, We began by probing a marker for neurons of the parabrachial nucleus that signal water intake, as an index of general ingestion. Water intake in dehydrated mice produced robust expression of FOS, a neural activity marker, in the dorsal lateral subnucleus of the parabrachial nucleus (dl), where the gene Pdyn (which encodes prodynorphin) is highly expressed. FOS immunofluorescence images (top) and the expression pattern of Pdyn from Allen Brain Atlas49 (bottom) along the AP axis of the parabrachial nucleus. Brain slices were obtained after ad libitum water access following 46-h water restriction. Image credit: Allen Institute. b–d, To visualize PBPdyn neurons, we bred a knock-in mouse line expressing Cre recombinase at the Pdyn locus (Pdyncre/+ mice) with Cre-dependent tdTomato reporter (Ai14) mice. Water-deprived Pdyncre/+ Ai14 mice or Pdyncre/+ mice injected with a Cre-inducible AAV carrying mCherry were given water access (W-D + water) or not (W-D) before the FOS analysis (b). Representative confocal images (c) and quantification (d) of the overlap between immunolabelled FOS+ and genetically labelled Pdyn+ neurons in the PBdl. The majority of tdTomato-expressing Pdyn+ neurons were FOS+ (about 72%), whereas few FOS+ neurons were observed in the PBdl of control mice that remained dehydrated (about 12% of Pdyn+ neurons). Most FOS+ PBdl neurons were also Pdyn+ (about 80%), indicating that Pdyn as a useful genetic handle for water-intake-activated neurons in the parabrachial nucleus. We also obtained consistent results in a separate set of experiments using mice in which PBPdyn neurons were fluorescently labelled using AAV vectors. There were smaller number of FOS+ neurons in the control mice (n = 146 and 7 neurons for experiments using Ai14 mice and AAV vectors, respectively) than in the experimental group (n = 492 and 1,002 neurons for experiments using Ai14 mice and AAV vectors, respectively). e, Representative images and quantification of multicolour fluorescence in situ hybridization experiments. Pdyn+ neurons are essentially glutamatergic (Slc17a6+) (>99%) (left), partially overlapping with fluid-intake-regulating Oxtr+ neurons16 (about 23%) (middle), but are separate from Calca+ neurons that have previously been implicated in noxious visceral signalling18 and meal termination15 (0%) (right). Scale bars, 100 μm (a), 10 μm (e), 50 μm (g, i). Data are presented as mean ± s.e.m. For statistics, see Supplementary Table 1. Abbreviations are defined in ‘Abbreviations’ in Methods.