Spatial distribution of VTA-projecting PAG inputs.

<p><b>A,</b> Schematic of the unilateral rabies injection protocol. <b>B-C,</b> Confocal images at low (B) and high (C) magnification showing the expression of TVA-mCherry (magenta) and RVΔG-EGFP (green) in the VTA seven days after the last injection. Scale bars, 500 μm (B) and 50 μm (C). <b>D,</b> Representative confocal images showing confined starter cell infection (i.e. TVA-mCherry and RVΔG-EGFP co-expression) in coronal sections rostral (top) or caudal (bottom) to the VTA of a DAT-Cre mouse. Antero-posterior coordinates (in mm) from Bregma are shown on the top right corner. Scale bars, 500 μm. ml, medial lemniscus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata. <b>E,</b> RVΔG-EGFP alone was not able to infect neurons without TVA-mCherry expression. Antero-posterior coordinates (in mm) from Bregma are shown on the top right corner. Scale bars, 500 μm. IPN, interpeduncular nucleus. <b>F,</b> RVΔG-EGFP-expressing retrogradely labeled input neurons across the rostral, central and caudal PAG segments. Scale bars, 200 μm. <b>G,</b> Average number of inputs to VTA DA (DAT, <i>n</i> = 5) and GABA (GAD, <i>n</i> = 5) neurons along the rostro-caudal axis of the PAG (two-way ANOVA: no interaction between the cell type factor and AP coordinate factor, <i>F</i>_11,120 = 0.7027, <i>p</i> > 0.05; main effect of cell type, <i>F</i>_1,120 = 18.2, <i>p</i> < 0.0001; main effect of AP coordinate, <i>F</i>_11,120 = 6.31, <i>p</i> < 0.0001; Bonferroni post-hoc test, * <i>p</i> < 0.05). Dashed lines denote the boundaries between rostral, central and caudal PAG. Inset shows the average number starter cells per ROI in DAT-Cre and GAD65-Cre mice (two-tailed <i>t</i> test: no difference between genotypes, <i>p</i> > 0.05). <b>H,</b> Relative contribution of different PAG subregions to the total inputs to VTA DA and GABA neurons (two-way ANOVA: no interaction between the cell type factor and subregion factor, <i>F</i>_5,48 = 0.5013, <i>p</i> > 0.05; no main effect of cell type, <i>F</i>_1,48 < 0.0001, <i>p</i> > 0.05; main effect of subregion, <i>F</i>_5,48 = 42.85, <i>p</i> < 0.0001; Bonferroni post-hoc test, * <i>p</i> < 0.05, **** <i>p</i> < 0.0001). Inset shows the degree of lateralization of the PAG inputs (two-tailed <i>t</i> test: no difference between genotypes, <i>p</i> > 0.05). <b>I,</b> Color-coded representation of the relative input contribution of ipsilateral and contralateral PAG subregions.</p

PAG afferents equally target VTA DA and GABA neurons.

<p><b>A,</b> Left, schematic of the injection protocol for patch clamp experiments. Right, example of image of ChR2-EYFP infection in the PAG. Scale bar, 500 μm. <b>B,</b> High magnification confocal images showing the colocalization or exclusion of mCherry and TH in DAT-Cre or GAD65-Cre mice, respectively. Scale bars, 50 μm. <b>C,</b> Mean amplitude of the light-evoked postsynaptic currents in VTA DA (<i>n</i> = 47) and GABA (<i>n</i> = 62) neurons plotted against the percentage of connected neurons (Mann Whitney <i>U</i> test: no difference in amplitudes, <i>p</i> > 0.05; Fisher’s exact test: no difference in connectivity, p > 0.05). Scale bars, 20 ms, 20 pA. <b>D,</b> Left, schematic of the patch clamp experiments: whole-cell recordings were performed from mCherry-expressing VTA neurons while PAG afferents inputs were light-stimulated (left). Right, excitatory currents were blocked with kynurenic acid (kyn), while kyn-resistant inhibitory currents were blocked with picrotoxin (PTX). Scale bars, 20 ms, 20 pA. <b>E,</b> Proportion of kyn-sensitive glutamate inputs and PTX-sensitive GABA inputs in VTA DA (<i>n</i> = 17) and GABA (<i>n</i> = 18) neurons (Fisher’s exact test: no difference between cell types, <i>p</i> > 0.05). <b>F,</b> Mean light-evoked current amplitude plotted against <i>I</i>_h amplitude (Spearman’s rank correlation: no correlation between the variables, <i>r</i> = 0.2702, <i>p</i> > 0.05) and comparison of <i>I</i>_h between connected and non-connected VTA DA (left) or GABA neurons (right) (Mann Whitney <i>U</i> test: * <i>p</i> < 0.05).</p