(A) Left, top: an image of a horizontal brain section from a Slc6a3i-Cre; A14 mouse. A stimulation electrode (Stim) was placed ~100 μm away from the recording electrode (Record). Left, bottom: experimental protocol schematic. Right: example traces of evoked EPSCs from one VTA DA neuron during baseline and following 1 μM oxytocin application. Blue, baseline; orange, following oxytocin application. (B) The amplitude of evoked EPSC decreased during the application of 1 μM oxytocin. n = 15 neurons from 10 mice. Green dashed line, baseline EPSC amplitude average; shaded region, time of oxytocin application. (C) OxtR antagonist L368,899 blocked oxytocin-induced EPSC amplitude attenuation. Left: example traces from one neuron before and during oxytocin application. Right: summary data for n = 8 neurons from six mice. (D) Same as (B), but with a phospholipase C blocker U73122. n = 5 neurons from four mice. (E) Same as (B), but with a vasopressin receptor (V1aR) antagonist SR49059. n = 9 neurons from six mice. (F) Summary data for EPSC amplitude changes induced by oxytocin at different concentrations (0 nM, 10 nM, 100 nM, 500 nM, and 1 μM). For each concentration respectively, n = 8 neurons from five mice, 5 neurons from two mice, 8 neurons from four mice, 6 neurons from five mice, and 15 neurons from 10 mice. *p<0.05, ***p<0.001, Kruskal-Wallis test with Dunn’s Multiple Comparison post hoc test. (G) Summary data for oxytocin-induced EPSC amplitude changes in the presence of OxtR antagonist (L368,899), PLC blocker (U73122), and V1aR antagonist (SR49059). Wilcoxon signed rank test vs. baseline, ***p<0.001. (H) Left: Schematic of viral transduction strategy using AAV9-DIO-ChR2-mCherry to express ChR2 in PVN oxytocinergic neurons. Right, top: ChR2-mCherry (red) expression in PVN. Right, bottom: One example neuron identified as dopaminergic during whole-cell recording with Alexa Fluor 488 dye included in the internal solution (green). Post hoc immunofluorescent labeling against tyrosine hydroxylase (TH, grayscale) confirms its identity. (I) Left: Optical activation of oxytocin fibers in the VTA decreased evoked EPSC amplitude in DA neurons. Patterned light stimulation period is marked in blue. n = 9 neurons from six mice. Inset shows evoked EPSC traces from one VTA DA neuron before and after light stimulation, temporally matched to average data. Black dashed line shows EPSC amplitude of the example neuron at t = 120 s, 30 s after the end of optogenetic stimulation train. Right: Summary data for light stimulation-induced EPSC amplitude changes with and without OxtR antagonist (L368,899). n = 6 neurons from four mice in the presence of OxtR antagonist. **p<0.01, Wilcoxon signed rank test vs. baseline. ###p<0.001, Mann-Whitney test. Error bars reflect SEM. (J) Left: example traces of evoked EPSCs from one VTA DA neuron during baseline (blue) and after 30 s long light stimulation of PVN oxytocin fibers. Right: EPSC amplitudes recorded from one neuron (same as Left) during baseline and after light stimulation. (K) Summary data for light stimulation-induced EPSC amplitude changes 10–20 min and 20–30 min after light stimulation. *p<0.05, **p<0.01, Kruskal-Wallis test with Dunn’s Multiple Comparison post hoc test, n = 6 neurons from four mice.