The effects of extracellular Ca2+ and Na+ on firing activity of the VTA DA neurons. A. Identification of the VTA DA neurons. i: confocal images showing the anatomical distribution of DA neurons in the VTA and SNc; the DA neurons were identified to be DAT-immunofluorescence positive (magnification,10X; scale bar, 100 µm). ii: single-cell PCR results; four cells showed the presence of genes indicated. B. The effect of replacing extracellular Ca2+ with Mg2+ on firing frequency of the VTA DA neurons. Example time-course (i) and traces (ii) of the spontaneous firing before and after replacement of extracellular Ca2+ (2 mM) by an equimolar amount of Mg2+. iii: Summarized data for ii. Firing of the VTA DA neurons were recorded using loose cell-attached patch from a brain slice of the VTA (n=6). C. The effect of replacing extracellular Ca2+ with Mg2+ on the resting membrane potential (RMP) of the VTA DA neurons, in the presence of 1 μM TTX. Example time-course (i) and summarized data (ii) for the resting membrane potential before and after replacement of extracellular Ca2+ (2 mM) by an equimolar amount of Mg2+ (n=6). D. i: Replacement of external Na+ by equimolar NMDG resulted in hyperpolarization of the resting membrane potential of the VTA DA neurons, in the presence of 1 μM TTX. ii: Summarized data for i (n=5). Paired-sample T test, *** P < 0.001, ** P < 0.01, * P < 0.05, n.s. P > 0.05. n is number of neurons recorded.

Gene expression profile of non-selective cation channels (NSCCs) in the VTA DA neurons. Single-cell RNA-Seq was performed on the VTA neurons projecting to five different brain regions of mice (NAc c, NAc ms, NAc ls, BLA, mPFC). A. Gene expression profile of markers for neuron subtypes from 45 VTA neurons; the neuron subtypes included: dopaminergic (Th, Ddc DAT), GABAergic (Gad1, Gad2, VGAT) and glutamatergic (VGluT2, VGluT1, VGluT3), which was arranged in different rows indicated in the right labels and in the left-colored vertical lines; projection targets of these neurons were indicated at the top by the colored lines and labels. Relative expression levels of these genes were indicated by the dark-blue color intensity which was transformed from the log2 values of the number of transcripts per million (FPKM) plus 1. B. Relative gene expression levels of TRP channels (i) and other NSCCs (ii) in 45 individual VTA DA neurons and the population average (mean, right columns). Each column of the individual neurons in (i) and (ii) corresponded to the columns in A. (iii) Bar graph of the mean log2 (FPKM +1) for the top 8 NSCCs in descending order. Error bars indicate SEM.C. Gene expression profile of aforementioned top 8 NSCCs from 45 VTA neurons.

NALCN contributes to subthreshold depolarization and spontaneous firing of the VTA DA neurons. A. Confocal images showing co-expression of NALCN (green) and DAT (red) representing DA neurons (scale bar, 10 µm). B. i: Single-cell PCR from the VTA DA neurons with the expression of NALCN. ii: Percentage of NALCN positive neurons from 15 DA neurons (with expression of DAT). C. Example time course (i) and summarized data (ii) showed that NALCN channel blockers (GdCl3) significantly hyperpolarized the resting membrane potential (RMP) (n=5). Paired-sample T test, * P < 0.05. D. Example time-course and traces (i) and summarized data (ii) of the effect of GdCl3 on spontaneous firing frequency in DA neurons (n=7). Paired-sample T test, ** P < 0.01. E. shRNA against NALCN carried by AAV virus (i) (NALCN-shRNA) was injected into the VTA of mice, the mRNA level in the shRNA-NALCN transfected VTA (NALCN-KD) and the scramble shRNA transfected VTA (Control) was analyzed using qPCR (ii) (n=6). Two-sample T test, ** P < 0.01. F. Confocal images showing the expression of AAV9-NALCN-shRNA-GFP (green) in the VTA DA neurons (DAT, red) (scale bar, 10 µm). G. Loose cell-attached current clamp recordings of the spontaneous firing of the VTA DA neurons transfected with either NALCN-shRNA (n=12) or scramble-shRNA (Con, n=10) (both GFP and DAT positive). Examples of firing traces (i) and summarized data (ii) were shown. Two-sample T test, **** P < 0.0001. H. Whole-cell current clamp recordings of the resting membrane potential (RMP) of the VTA DA neurons transfected with either NALCN-shRNA (NALCN-KD, n=18) or scramble-shRNA (Con, n=15) (both GFP and DATpositive). Examples of firing traces (i) and summarized data (ii) were shown. Two-sample T test, **** P < 0.0001. n is number of neurons recorded.

TRP channels contributes to subthreshold depolarization and spontaneous firing of the VTA DA neurons. A. The expression of TRP channels in VTA DA neurons. i: Single-cell PCR from 5 VTA cells (C1-C5). ii: Percentage of TRP channels (C3, C6 and V2) positive neurons in the VTA DA neurons (DAT positive). B and C. Example time-course (i), traces (ii) and the summarized data (iii) for the effect of nonselective cation channel blockers 2-APB (100 μM, n=12) (B), FFA (100 μM, n=13) (C) on the spontaneous firing frequency in the VTA DA neurons. B: Wilcoxon matched-pairs signed rank test, C: Paired-sample T test, *** P < 0.001. n is number of neurons recorded. D and E. Example time-course (i) and the summarized data (ii) for the effect of nonselective cation channel blockers 2-APB (n=6) (D), FFA (n=6) (E) in the resting membrane potential (RMP) of the VTA DA neurons. Paired-sample T test, ** P< 0.01. n is number of neurons recorded.

TRPC6 contributes to subthreshold depolarization and spontaneous firing of the VTA DA neurons. A. The normalized expression profile of TRPC6 in NAc c and mPFC-projecting VTA DA neurons was verified using single cell-qPCR, (NAc c: n=28; mPFC: n=25, Both TH positive) Mann-Whitney U test, ** P <0.01. B. Confocal images showing co-expression of TRPC6 (green) and TH (red) representing DA neurons (scale bar, 10 µm). C. The efficiency of shRNA knockdown of TRPC6 in the VTA verified by qPCR. Two-sample T test, ** P <0.01. shRNA against TRPC6 carried by AAV virus (i) (TRPC6-shRNA) was injected into the VTA of mice, the mRNA level in the shRNA-TRPC6 transfected VTA (TRPC6-KD) and the scramble shRNA transfected VTA (Con) was analyzed using qPCR (ii). D. Immunofluorescence labeling showing the expression of AAV9-shRNA(TRPC6)-GFP (green) and DAT (red) in the VTA (scale bar, 10 µm). E. Loose cell-attached current clamp recordings of the spontaneous firing of the VTA DA neurons from the mice transfected with either TRPC6-shRNA (TRPC6-KD, n=9) or scramble-shRNA (Con, n=9) (both GFP and DAT positive). Examples of firing traces (i) and summarized data (ii) were shown. Two-sample T test, ** P < 0.01. F. Example time-course (i) and summarized data (ii) showed that shRNA knockdown of TRPC6 in the VTA decreased the 2-APB-inhibited firing responses of the VTA DA neurons. (n=7, both GFP and DAT positive), Wilcoxon matched-pairs signed rank test, n.s. P > 0.05. G. Whole-cell current clamp recordings of the resting membrane potential (RMP) of the VTA DA neurons transfected with either TRPC6-shRNA (TRPC6-KD, n=10) or scramble-shRNA (Con, n=10) (both GFP and DAT positive). Examples of firing traces (i) and summarized data (ii) were shown. Two-sample T test, *** P < 0.001.

CMUS depression mice have a decreased firing activity and downregulated TRPC6 expression in the VTA DA neurons. A. Experimental procedure timeline. B. i: Sucrose preference test for CMUS mice. Mann-Whitney U test, *** P < 0.001, compared with control mice. ii: Tail suspension test for CMUS mice, Two-sample T test, *** P < 0.001, compared with control mice. C. Loose cell-attached current clamp recordings of the spontaneous firing of the VTA DA neurons from the CMUS (n = 28 cells, DAT positive) and the control (n = 18 cells, DAT positive) mice. Mann-Whitney U test, **P < 0.01. D. Representative Western blot assay (i) and summarized data (ii) showing the expression of TRPC6 and GAPDH in the VTA of the control (Con) and the CMUS mice. Two-sample T test, ** P < 0.01. n is the number of mice used. E. i and ii: Example time-course (up) and traces (bottom) showing the characteristic firing responses of 2-APB-inhibited mPFC-projecting VTA DA neurons in Con (i) and CMUS (ii) groups. iii. The effects of TRP channel inhibitor (2-APB) on the spontaneous firing activity of the mPFC-projecting VTA DA neurons in the control (n = 12 cells, DAT positive) and CMUS (n = 15 cells, DAT positive) mice. One-way repeated measures ANOVA with Dunnett’s multiple comparisons test, n.s. P > 0.05, * P < 0.05, *** P < 0.001. iv. The inhibition rate (%) on firing rate by 2-APB in the control group (n = 12) and the CMUS group (n = 15). Mann-Whitney U test, *** P < 0.001.

Selective knockdown of TRPC6 in the VTA DA neurons produces depression-like and anxiety-like behaviors and selective overexpression of TRPC6 in the VTA DA neurons rescues the TRPC6-knockdown-mediated depression-like and anxiety-like behaviors. A. shRNA against TRPC6 carried by AAV-loxp virus (TRPC6-shRNA) was injected into the VTA of the DAT-Cre mice. B. The mRNA level in the shRNA-TRPC6 transfected VTA (TRPC6-cKD) and the scramble shRNA transfected VTA (Con) was analyzed using qPCR (Con: n=4; TRPC6-cKD: n=6). Mann-Whitney U test, * P < 0.05. C. Immunofluorescence labeling showing the expression of AAV9-hSyn-DIO-shRNA(TRPC6)-RFP (red) and DAT (green) in the VTA of the DAT-Cre C57BL/6 mice (scale bar, 10 µm). D. The effects of Cre-induced conditional knockdown of TRPC6 in the VTA on the behaviors of mice in the sucrose preference test (i), the tail suspension test (ii), and the elevated plus-maze test (iii) (Con: n=9; TRPC6-cKD: n=13). Two-sample T test, ** P < 0.01, *** P < 0.001. E. AAV9-DIO-TRPC6-GFP was injected into the VTA of the DAT-Cre mice, 7 days after injection of AAV9-hSyn-DIO-shRNA(TRPC6)-RFP. F. The protein level in the AAV9-DIO-TRPC6-GFP transfected TRPC6-cKD VTA (TRPC6-cKD+TRPC6-cOE) and the scramble RNA transfected Con and TRPC6-cKD VTA (Con and TRPC6-cKD) was analyzed using Western blot (Con: n=7; TRPC6-cKD: n=8; TRPC6-cKD+TRPC6-cOE: n=7). Kruslal-Wallis-H test with Dunnett’s multiple comparisons test, **P < 0.01. G. Immunofluorescence labeling showing the expression of AAV9-hSyn-DIO-shRNA(TRPC6)-RFP (red), AAV9-DIO-TRPC6-GFP (green) and DAT (blue) in the VTA of the DAT-Cre C57BL/6 mice (scale bar, 10 µm). H. The effects of Cre-induced conditional overexpression of TRPC6 in the VTA on the selective-knockdown-TRPC6-induced depression-like behaviors of mice in the sucrose preference test (i), the tail suspension test (ii), and the elevated plus-maze test (iii) (Con: n=11; TRPC6-cKD: n=11; TRPC6-cKD+TRPC6-cOE: n=11). One-way ANOVA with Dunnett’s multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001.