The effects of extracellular Ca2+ and Na+ on firing activity of the male and female 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 (scale bar, 100 µm). ii: left panel: single-cell PCR results; four cells (C1-C4) showed the presence of genes indicated. right panel: Percentage of neuronal markers (DA neuron: TH, DAT, D2R, Girk2; Glu neuron: VGluT2 and GABA neuron: GAD1) positive neurons in the VTA neurons (GAPDH positive). n = 102 cells, N = 22. B. The effect of replacing extracellular Ca2+ with Mg2+ on firing frequency of the male and female 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+.The inset on the top of ii shows a map of a coronal midbrain slice indicating the location of neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (red dots). 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 = 32, N = 16). Wilcoxon matched-pairs signed rank test, W = 526.0, ****P < 0.0001. C. The effect of replacing extracellular Ca2+ with Mg2+ on the resting membrane potential (RMP) of the male and female 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 = 25, N = 15). The inset on the top of ii shows a map of a coronal midbrain slice indicating the location of neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (red dots). Paired-sample T test, t = 2.368, df = 24, 95% CI: 0.1454 to 2.119, *P = 0.0263. D. Replacement of external Na+ by equimolar NMDG resulted in hyperpolarization of the resting membrane potential of the male and female 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 Na+ (151 mM) with an equimolar amount of NMDG (n = 17, N = 10). The inset on the top of ii shows a map of a coronal midbrain slice indicating the location of neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (red dots). Paired-sample T test, t = 7.103, df = 16, 95% CI: −15.35 to −8.293, ****P < 0.0001. * P < 0.05, **** P < 0.0001. n is number of neurons recorded and N is number of mice used.

Gene expression profile of non-selective cation channels (NSCCs) in the male VTA DA neurons.

Single-cell RNA-Seq was performed on the VTA neurons projecting to five different brain regions of male 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 (C1-C5) 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 blocker (GdCl3) significantly hyperpolarized the resting membrane potential (RMP) (n = 7, N = 7) of male VTA DA neurons. The inset on the top of Cii shows a map of a coronal midbrain slice indicating the location of neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (red dots). Paired-sample T test, t = 11.38, df = 6, 95% CI: −24.30 to −15.70, ****P < 0.0001. D. Example time-course and traces (i) and summarized data (ii) of the effect of GdCl3 on spontaneous firing frequency in male VTA DA neurons (n = 7, N = 3). The inset on the top of Dii shows a map of a coronal midbrain slice indicating the location of neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (red dots). Paired-sample T test, t = 5.711, df = 6, 95% CI: −0.8183 to −0.3274, **P = 0.0012. E. Example time-course (i) and summarized data (ii) showed that NALCN channel blocker (L703,606) significantly hyperpolarized the resting membrane potential (RMP) (n = 6, N = 4) of VTA DA neurons in male mice. The inset on the top of Eii shows a map of a coronal midbrain slice indicating the location of neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (red dots). Paired-sample T test, t = 14.95, df = 5, 95% CI: −5.498 to −3.885, ****P < 0.0001. F. Example time-course and traces (i) and summarized data (ii) of the effect of L703,606 on spontaneous firing frequency in DA neurons of both sexes (n = 20, N = 10). The inset on the top of Fii shows a map of a coronal midbrain slice indicating the location of neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (red dots). Wilcoxon matched-pairs signed rank test, W = −210.0, ****P < 0.0001.G. 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, N = 6) and the scramble shRNA transfected VTA (Control, N = 6) was analyzed using qPCR (ii). Two-sample T test, t = 3.714, df = 10, 95% CI: −1.200 to −0.3000, **P = 0.0040. H. Confocal images showing the expression of AAV9-NALCN-shRNA-GFP (green) in the VTA DA neurons (DAT, red) (scale bar, 10 µm). I. Loose cell-attached current clamp recordings of the spontaneous firing of the male and female VTA DA neurons transfected with either NALCN-shRNA (ii, n = 35, N = 8) or scramble-shRNA (Con, i, n = 30, N = 8) (both GFP and DAT positive). Examples of firing traces in the middle of i and ii and summarized data in the bottom of i were shown. The inset on the top of i and ii shows a map of a coronal midbrain slice indicating the location of GFP+ neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (i: Con, grey dots; ii: NALCN-KD, red dots). Mann-Whitney U test, U = 0, ****P < 0.0001. J. Whole-cell current clamp recordings of the resting membrane potential (RMP) of the male VTA DA neurons transfected with either NALCN-shRNA (NALCN-KD, ii, n = 18, N = 5) or scramble-shRNA (Con, i, n = 15, N = 5) (both GFP and DAT positive). Examples of RMP traces in the middle of i and ii and summarized data in the bottom of i were shown. The inset on the top of i and ii shows a map of a coronal midbrain slice indicating the location of GFP+ neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (i: Con, grey dots; ii: NALCN-KD, red dots). Mann-Whitney U test, U = 0, ****P < 0.0001. * P < 0.05, ** P < 0.01, **** P < 0.0001. n is number of neurons recorded and N is number of mice used.

TRP channels contribute 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 a nonselective cation channel blocker 2-APB (100 μM, n = 12, N = 5 for male mice) (B) and a potent TRPC6 inhibitor LA (10 μM, n = 14, N = 8 for male and female mice) (C) on the spontaneous firing frequency in the VTA DA neurons. The inset on the top of Bii and Cii shows a map of a coronal midbrain slice indicating the location of neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (red dots). Wilcoxon matched-pairs signed rank test, B: W = −78.00, ***P = 0.0005. C: W = −105.0, ***P = 0.0001. D and E. Example time-course (i) and the summarized data (ii) for the effect of a nonselective cation channel blocker 2-APB (n = 10, N = 9) (D) and a potent TRPC6 inhibitor LA (n = 9, N = 7) (E) on the resting membrane potential (RMP) of the male and female VTA DA neurons. Paired-sample T test, D: t = 8.082, df = 9, 95% CI: −11.90 to −6.693, ****P < 0.0001, E: t = 6.431, df = 8, 95% CI: −5.993 to −2.829, ***P = 0.0002. *** P < 0.001, **** P < 0.0001.n is number of neurons recorded and N is number of mice used.

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, N = 7; mPFC: n = 25, N=8, both TH positive), Mann-Whitney U test, U = 173, **P = 0.0013. 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. 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, N = 5) and the scramble shRNA transfected VTA (Con, N = 5) was analyzed using qPCR (ii). Two-sample T test, t = 4.223, df = 8, 95% CI: −0.8194 to −0.2406, **P = 0.0029. D. Immunofluorescence labelling 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 of both sexes transfected with either TRPC6-shRNA (TRPC6-KD, ii, n = 37, N = 12) or scramble-shRNA (Con, i, n = 27, N = 11) (both GFP and DAT positive). Examples of firing traces in the middle of i and ii and summarized data in the bottom of i were shown. The inset on the top of i and ii shows a map of a coronal midbrain slice indicating the location of GFP+ neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (i: Con, grey dots; ii: TRPC6-KD, red dots). Mann-Whitney U test, U = 48, ****P < 0.0001. F. Example time-course (i) and summarized data (ii) showed that shRNA knockdown of TRPC6 in the VTA of male mice decreased the 2-APB-inhibited firing responses of the VTA DA neurons. The inset on the top of ii shows a map of a coronal midbrain slice indicating the location of GFP+ neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (TRPC6-KD, red dots). (n = 7, N = 5, both GFP and DAT positive), Wilcoxon matched-pairs signed rank test, W = −18.00, P = 0.1563. G. Whole-cell current clamp recordings of the resting membrane potential (RMP) of the male mice VTA DA neurons transfected with either TRPC6-shRNA (TRPC6-KD, ii, n = 10, N = 6) or scramble-shRNA (Con, i, n = 10, N = 6) (both GFP and DAT positive). Examples of RMP traces in the middle of i and ii and summarized data in the bottom of i were shown. The inset on the top of i and ii shows a map of a coronal midbrain slice indicating the location of GFP+ neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (i: Con, grey dots; ii: TRPC6-KD, red dots). Mann-Whitney U test, U = 8, ***P = 0.0007. H. Example time-course (i) and summarized data (ii) showed that shRNA knockdown of TRPC6 in the male mice VTA decreased the LA-inhibited firing responses of the VTA DA neurons. The inset on the top of ii shows a map of a coronal midbrain slice indicating the location of GFP+ neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (TRPC6-KD, red dots). (n = 8, N = 5, both GFP and DAT positive), Paired-sample T test, t = 0.000, df = 7, 95% CI: −0.05740 to 0.05740, P > 0.9999. n.s. P > 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001. n is number of neurons recorded and N is number of mice used.

CMUS depression mice have a decreased firing activity and down-regulated TRPC6 expression in the VTA DA neurons.

A. Experimental procedure timeline. B. i: Sucrose preference test for CMUS male mice (Con: N = 10; CMUS: N = 10). Two-sample T test, t = 3.569, df = 18, 95% CI: −20.73 to −5.369, **P = 0.0022, compared with control mice. ii: Tail suspension test for CMUS male mice (Con: N = 10; CMUS: N = 10), Two-sample T test, t = 5.826, df = 18, 95% CI: 23.21 to 49.39, ****P < 0.0001, compared with control mice. C. Loose cell-attached current clamp recordings of the spontaneous firing of the VTA DA neurons from the CMUS (n = 68, N = 33, DAT positive) and the control (n = 53, N = 31, DAT positive) mice of both sexes. The inset on the top of i and ii shows a map of a coronal midbrain slice indicating the location of neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (i: Con, grey dots; ii: CMUS, red dots). Examples of firing traces in the bottom of i as well as in the bottom left of ii and summarized data in the bottom right of ii were shown. Mann-Whitney U test, U = 415, ****P < 0.0001. D. Representative Western blot assay (i) and summarized data (ii) showing the expression of TRPC6 and GAPDH in the VTA of the control (Con, N = 10) and the CMUS (N = 11) mice of both sexes. Two-sample T test, t = 5.134, df = 19, 95% CI: −0.7480 to −0.3147, ****P < 0.0001. E. i and ii: Example traces (up) and time-course (bottom) of firing frequency of mPFC-projecting VTA DA neurons, effect of 2-APB. (i) Con and (ii) CMUS of both sexes. iii. Summarized effect of 2-APB in the control (n = 22 cells, N = 15, DAT positive) and CMUS (n = 25 cells, N = 15, DAT positive) mice. Kruskal-Wallis-H test with Dunnett’s multiple comparisons test, Kruskal-Wallis statistic = 66.03, Con-ACSF vs. CMUS-ACSF, **P = 0.0056; Con-2-APB vs. CMUS-2-APB, **P = 0.0016; Con-ACSF vs. Con-2-APB, ****P < 0.0001; CMUS-ACSF vs. CMUS-2-APB, P > 0.9999. iv. The inhibition rate (%) on firing rate by 2-APB in the control group (n = 22, N = 15) and the CMUS group (n = 25, N = 15). Mann-Whitney U test, U = 7, ****P < 0.0001. The inset on the top of iii and iv shows a map of a coronal midbrain slice indicating the location of mPFC-projecting neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (iii: Con, grey dots; iv: CMUS, red dots). F. i and ii: Example traces (up) and time-course (bottom) of firing frequency of mPFC-projecting VTA DA neurons, effects of LA. (i) Con and (ii) CMUS of both sexes. iii. Summarized effect of LA on the control (n = 25 cells, N = 16, DAT positive) and CMUS (n = 30 cells, N = 18, DAT positive) mice. Kruskal-Wallis-H test with Dunnett’s multiple comparisons test, Kruskal-Wallis statistic = 79.59, Con-ACSF vs. CMUS-ACSF: ****P < 0.0001, CON-LA vs. CMUS-LA: **P = 0.0026, Con-ACSF vs. Con-LA: ****P < 0.0001, CMUS-ACSF vs. CMUS-LA: P > 0.9999. iv. The inhibition rate (%) on firing rate by LA in the control group (n = 25, N = 16) and the CMUS group (n = 30, N = 18). Mann-Whitney U test, U = 2, ****P < 0.0001. The inset on the top of iii and iv shows a map of a coronal midbrain slice indicating the location of mPFC-projecting neurons that were recorded and subsequently identified as DA neurons which were DAT positive with single cell-PCR (iii: Con, grey dots; iv: CMUS, red dots). n.s. P > 0.05, **P < 0.01, ****P < 0.0001.n is number of neurons recorded and N is the number of mice used.

Selective knockdown of TRPC6 in the VTA DA neurons confers the male mice with depression-like and anxiety-like behaviors which are reversed by overexpression of TRPC6.

A. shRNA against TRPC6 carried by AAV-loxp virus (TRPC6-shRNA) was injected into the VTA of the DAT-Cre male 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 = 8; TRPC6-cKD: N = 6). Mann-Whitney U test, U = 0, ***P = 0.0003. C. Immunofluorescence labelling showing the expression of AAV9-hSyn-DIO-shRNA(TRPC6)-RFP (red) and DAT (green) in the VTA of the DAT-Cre mice (scale bar, 10 µm). D. The effects of Cre-induced conditional knockdown of TRPC6 in the VTA DA neurons on the behaviors of mice in the sucrose preference test (i), the tail suspension test (ii), and the elevated plus-maze test (iii, iv) (Con: N = 10; TRPC6-cKD: N = 10). Sucrose preference test: Two-sample T test, t = 4.172, df = 18, 95% CI: −17.07 to −5.634, ***P = 0.0006; Tail suspension test: Two-sample T test, t = 3.377, df = 18, 95% CI: 7.785 to 33.41, **P = 0.0034; Elevated plus-maze test: Mann-Whitney U test, open arm, U = 2, ****P < 0.0001, closed arm, U = 13, **P = 0.0039. E. The effects of TRPC6 over-expression in the TRPC6-cKD-induced depression-like behavior. AAV9-DIO-TRPC6-GFP (TRPC6-cOE) was injected into the VTA of the male DAT-Cre mice, 7 days after injection of AAV9-hSyn-DIO-shRNA(TRPC6)-RFP (TRPC6-cKD). F. The protein level TRPC6 of VTA (Con: the scramble shRNA for TRPC6, N = 7; C6-cKD: TRPC6-cKD, N = 10; C6-cKD+cOE: TRPC6-cKD+TRPC6-cOE, N = 7). Kruskal-Wallis H test with Dunnett’s multiple comparisons test, Kruskal-Wallis statistic = 16.92, Con vs. C6-cKD: ***P = 0.0009, C6-cKD vs. C6-cKD+cOE: **P = 0.0034. G. Immunofluorescence labelling 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 mice (scale bar, 10 µm). H. The effects of TRPC6 over-expression on depression-like behaviors of TRPC6-cKD male mice: the sucrose preference test (i), the tail suspension test (ii), and the elevated plus-maze test (iii) (Con: N = 10; C6-cKD: N = 10; C6-cKD+ cOE: N= 10). the sucrose preference test: Kruskal-Wallis H test with Dunnett’s multiple comparisons test, Kruskal-Wallis statistic = 12.96, Con vs. C6-cKD: **P = 0.0025, C6-cKD vs. C6-cKD+cOE: *P = 0.0139; tail suspension test: One-way ANOVA with Dunnett’s multiple comparisons test, F (DFn, DFd) = 0.8336(2, 27), Con vs. C6-cKD: **P = 0.0011, C6-cKD vs. C6-cKD+cOE: ***P = 0.0003; elevated plus-maze test: One-way ANOVA with Dunnett’s multiple comparisons test, F(DFn, DFd) = 4.884(2, 27), Con vs. C6-cKD: ****P < 0.0001, C6-cKD vs. C6-cKD+cOE: **P = 0.0023. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.n is number of neurons recorded and N is the number of mice used.

Selective knockdown of TRPC6 in the mPFC-projecting VTA DA neurons produces depression-like and anxiety-like behaviors.

A. shRNA against TRPC6 carried by retro-AAV-loxp virus (TRPC6-shRNA) was injected into the mPFC of the DAT-Cre male mice. B. The mRNA level in the shRNA-TRPC6 transfected mPFC-projecting VTA (TRPC6-rcKD) DA neurons and the scramble shRNA transfected mPFC-projecting VTA DA neurons (Con) was analyzed using single-cell quantitative PCR (scqPCR)(Con: n = 15, N = 6; TRPC6-rcKD: n = 16, N = 6). Mann-Whitney U test, U =8.500, ****P < 0.0001.C. Immunofluorescence labelling showing the expression of AAV-retro-hSyn-DIO-mCherry-shRNA(TRPC6) (red) and DAT (green) in the VTA of the DAT-Cre mice (scale bar, 10 µm). D. The effects of Cre-induced conditional knockdown of TRPC6 in mPFC-projecting VTA DA neurons on the behaviors of mice: (i) the sucrose preference test, (ii) the tail suspension test, and (iii) the elevated plus-maze test (Con: N = 10; TRPC6-rcKD: N = 10). Two-sample T test, sucrose preference test: t = 3.592, df = 18, 95% CI: −11.48 to −3.008, **P = 0.0021; tail suspension test: t = 3.707, df = 18, 95% CI: 10.79 to 39.01, **P = 0.0016; elevated plus-maze test: open arm t = 3.756, df = 18, 95% CI: −8.099 to −2.288, **P = 0.0014. **P < 0.01, ****P < 0.0001.n is number of neurons recorded and N is the number of mice used.