Fig. 4: Subtype “1” Dat1⁺ neurons in the anterior PeVN modulate neuronal activity in the LS.

a Experimental design of virus injections for the ex vivo pharmacogenetic mapping of the PeVN-LS circuitry. b–d CNO stimulation of Dat1⁺ terminals innervating the LS alters local Ca²⁺ oscillations. Colored traces (left) show representative examples of Ca²⁺ recordings at the single-cell level and their sensitivities to dopamine receptor antagonists. Day (sun symbol) and night (moon symbol) correspond to the timing of particular experiments (CT: 06:00–11:00 and 18:00–23:00, respectively). Right: Effect of D1 and D2 dopamine receptor antagonism by SCH23390 and sulpiride, respectively, on CNO-induced Ca²⁺ oscillations. Data were expressed as means ± s.e.m., with individual values also shown. Two-sided paired t-test was used for statistical analysis; *p < 0.05, **p < 0.01; n indicates the number of cells recorded from for each response type over 7 animals during the day and 3 animals during the night. In b at daytime: D1R inhibition: n = 4 cells, p = 0.0101; D2R inhibition: n = 4 cells, p = 0.1282; at nighttime: D1R inhibition: n = 5 cells, p = 0.0062, D2R inhibition: n = 3 cells, p = 0.668. In c for day recordings: D1R inhibition: n = 4 cells, p = 0.141; D2R inhibition: n = 7 cells, p = 0.0205; for night recordings: D1R inhibition: n = 11 cells, p = 0.915; D2R inhibition: n = 7 cells, p = 0.0306. In d during daytime: D1R inhibition: n = 4 cells, p = 0.1275; D2R inhibition: n = 15 cells, p = 0.0041. e Four neuronal populations were shown to exist in the LS, each responding differently to the chemogenetic activation of Dat1⁺ inputs from the PeVN. The distribution of functional groups differs during the day vs. night phases with an increased fraction of non-responsive neurons detected during the day. n states the number of independent experiments. f Levels of phospho-Ser⁴⁰-TH at day and night. Scale bar = 20 μm. Data were statistically evaluated by using two-sided unpaired t-test and presented as means ± s.e.m. p = 0.0119 (*p < 0.05); n = 4 animals for CT 09:00 and 5 animals for CT 21:00. 3V third ventricle, PeVN periventricular nucleus, PVN paraventricular nucleus. We used Biorender to visualize an experimental scheme.