The cation channel mechanisms of subthreshold inward depolarizing currents in the mice VTA dopaminergic neurons and their roles in the chronic-stress-induced depression-like behavior
- Department of Pharmacology, Hebei Medical University, China
- Department of Chinese Medicinal Chemistry, Hebei University of Chinese Medicine, China
- The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, China
- The Key Laboratory of New Drug Pharmacology and Toxicology, Hebei Medical University, China
- Yiling Pharmaceutical Company, China
- Department of Clinical Pharmacy, Xingtai Ninth Hospital, China
- College of Chemical Engineering, Shijiazhuang University, China
- Shijiazhuang Key Laboratory of Targeted Drugs Research and Efficacy Evaluation, China
- Department of Pharmacy, Handan First Hospital, China
- Department of Psychiatry, The First Hospital of Hebei Medical University, Mental Health Institute of Hebei Medical University, China
Figures
Figure 1
The effects of extracellular Ca2+ and Na+ on the firing activity of the male and female ventral tegmental area (VTA) dopaminergic (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 dopamine transporter (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, Slc6a3, Drd2, Kcnj6; Glu neuron: Slc17a6 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 the 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 Slc6a3 positive with single cell-PCR (red dots). (iii) Summarized data for (ii) Firing of the VTA DA neurons was recorded using the 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 Slc6a3 positive with single cell-PCR (red dots). Paired-sample t-test, t=2.368, df = 24, 95% CI: 0.1454–2.119, *p=0.0263. (D) Replacement of external Na+ by equimolar N-methyl-d-glucamine (NMDG) resulted in hyperpolarization of the resting membrane potential of the male and female VTA DA neurons, in the presence of 1 μM Tetrodotoxin (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 Slc6a3 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 the number of neurons recorded and N is the number of mice used.
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Figure 1—source data 1
Confocal image for the anatomical distribution of dopaminergic (DA) neurons in the ventral tegmental area (VTA) and SNc for Figure 1Ai.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig1-data1-v1.pdf
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Figure 1—source data 2
Data and PDF file containing original gels for Figure 1Aii, indicating the relevant bands and groups.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig1-data2-v1.zip
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Figure 1—source data 3
Original files for gels of single-cell PCR are displayed in Figure 1Aii.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig1-data3-v1.zip
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Figure 1—source data 4
Data of the effect of extracellular Ca2+ on the firing frequency of the VTA DA neurons for Figure 1B.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig1-data4-v1.xlsx
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Figure 1—source data 5
Data of the effect of extracellular Ca2+ on the resting membrane potential (RMP) of the ventral tegmental area dopaminergic (VTA DA) neurons for Figure 1C.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig1-data5-v1.xlsx
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Figure 1—source data 6
Data of the effect of Na+ in resting membrane potential (RMP) of the ventral tegmental area dopaminergic (VTA DA) neurons for Figure 1D.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig1-data6-v1.xlsx
Figure 2 with 4 supplements
Gene expression profile of non-selective cation channels (NSCCs) in the male ventral tegmental area dopaminergic (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, Slc6a3), GABAergic (Gad1, Gad2, Slc32a1), and glutamatergic (Slc17a6, Slc17a7, Slc17a8), 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 transient receptor potential (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 eight NSCCs in descending order. Error bars indicate SEM. (C) Gene expression profile of aforementioned top eight NSCCs from 45 VTA neurons.
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Figure 2—source data 1
Gene expression data for Figure 2.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
The targeted regions for ventral tegmental area dopaminergic (VTA DA) projection.
(A and B) Injection sites (red colored) in sagittal (A) and coronal plane (B) of mouse brain with DAPI (405 nm)-counterstained sections (80 μm) showing locations of retrobeads (546 nm, yellow). From top to bottom: mPFC (bregma +2.22 mm), BLA (bregma –1.58 mm), NAc ms (bregma +1.10 mm), NAc c (bregma +0.98 mm), NAc ls (bregma +0.86 mm).
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Figure 2—figure supplement 1—source data 1
PDF file containing confocal images for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig2-figsupp1-data1-v1.pdf
Figure 2—figure supplement 2
The subregions of projection-specific ventral tegmental area dopaminergic (VTA DA) neurons.
(A) Confocal images showing the anatomical distribution of retrobeads (red) in the VTA after dopamine transporter (DAT)-immunofluorescence (green) staining at different magnifications (i, 20x; ii, 40x); individual labeled cells are shown in (iii). Scale bars i=100 μm, ii=20 μm and iii=10 μm. (B) and (C) average retrogradely labeled cell distributions in the VTA.
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Figure 2—figure supplement 2—source data 1
PDF file containing confocal images for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig2-figsupp2-data1-v1.pdf
Figure 2—figure supplement 3
Blocking HCN channels does not affect the spontaneous firing of the ventral tegmental area dopaminergic (VTA DA) neurons in adult male mice.
(A and B) Effects of HCN channel inhibitors CsCl (A) and ZD7288 (B) on the sag membrane potential of adult male VTA DA neurons. The sag membrane potential was recorded using the whole-cell current-clamp method, and sag was calculated as the difference between the peak negative voltage and steady-state negative voltage responding to hyperpolarizing current injection (–100 pA). Ai and Bi: CsCl (3 mM, n=4, N=4) and ZD7288 (60 µM, n=4, N=4) significantly decreased the sag potentials. (Aii) and (Bii) Summary for CsCl and ZD7288 induced inhibition on sag potentials. Paired-sample t-test, CsCl: t=4.246, df=3, 95% CI: –22.03 to –3.155, *p=0.0239; ZD7288: t=5.017, df=3, 95% CI: –24.83 to –5.555, *p=0.0152. (C) and (D) effects of HCN channel inhibitors CsCl (C, n=7, N=4) and ZD7288 (D, n=13, N=5) on the spontaneous firing activity of the VTA DA neurons (Slc6a3 positive). The spontaneous firing of the VTA DA neurons was recorded using the loose cell-attached current clamp method on the brain slice of the VTA. The example timecourses (i) and the summarized data (ii) for the effects of CsCl and ZD7288 were shown. Paired-sample t-test, CsCl: t=0.7171, df=6, 95% CI: –0.4325–0.7911, p=0.5003; ZD7288: t=1.349, df=12, 95% CI: –0.4033–0.09486, p=0.2022, n.s. p>0.05, *p<0.05. n is the number of neurons recorded and N is the number of mice.
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Figure 2—figure supplement 3—source data 1
Data of the effects of CsCl on the sag membrane potential of ventral tegmental area dopaminergic (VTA DA) neurons for Figure 2—figure supplement 3A.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig2-figsupp3-data1-v1.xlsx
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Figure 2—figure supplement 3—source data 2
Data of the effects of ZD7288 on the sag membrane potential of ventral tegmental area dopaminergic (VTA DA) neurons for Figure 2-figure supplement 3B.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig2-figsupp3-data2-v1.xlsx
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Figure 2—figure supplement 3—source data 3
Data of the effects of CsCl on the excitability of ventral tegmental area dopaminergic (VTA DA) neurons for Figure 2—figure supplement 3C.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig2-figsupp3-data3-v1.xlsx
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Figure 2—figure supplement 3—source data 4
Data of the effects of ZD7288 on the excitability of ventral tegmental area dopaminergic (VTA DA) neurons for Figure 2—figure supplement 3D.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig2-figsupp3-data4-v1.xlsx
Figure 2—figure supplement 4
HCN channel blocker does not affect the spontaneous firing of the adult male ventral tegmental area dopaminergic (VTA DA) neurons projecting to NAc lateral shell but blocks the spontaneous firing of the VTA DA neurons bathed in a low extracellular K+ which hyperpolarized the resting membrane potential.
(A and B) Effects of HCN channel inhibitors (CsCl A, ZD7288 B) on the spontaneous firing activity of the VTA DA neurons projecting to NAc lateral shell. The spontaneous firing of VTA DA neurons was recorded using the loose cell-attached current clamp method on the brain slice of the VTA. The example time-courses (i), traces (ii), and the summarized data (iii) for the effects of CsCl (A, n=5, N=4, Red and Slc6a3 positive), ZD7288 (B, n=5, N=5, Red and Slc6a3 positive) were shown. Paired-sample t-test, CsCl: t=0.4798, df=4, 95% CI: –0.7601–0.5361, p=0.6564; ZD7288: t=0.4421, df=4, 95% CI: –0.4368–0.3168, p=0.6813. (iv): a map of a coronal midbrain slice indicating the location of red neurons that were recorded and subsequently identified as DA neurons which were Slc6a3 positive with single cell-PCR (red dots). (C) (i) Example time course for hyperpolarization of the resting membrane potential (RMP) induced by low extracellular K+ (low [K+]e). (ii) Summarized data for experiments shown in Ci (n=7, N=7), Paired-sample t-test, t=6.373, df = 6, 95% CI: –18.69 to –8.318, ***p=0.0007. (iii) A map of a coronal midbrain slice indicating the location of neurons that were recorded and subsequently identified as DA neurons which were Slc6a3 positive with single cell-PCR (red dots). (D) ZD7288 inhibited the spontaneous firing frequency of VTA DA neurons only in low [K+]e condition. Example time courses of firing rates (i), traces (ii) and summarized data (iii) were shown (n=7, N=7, Slc6a3 positive). Wilcoxon matched-pairs signed rank test, W=–28.00, *p=0.0156. (E) Example time-courses (i), traces (ii), and the summarized data (iii) for the effects of ZD7288 on the spontaneous firing of juvenile (less than 15 d postnatal) male VTA DA neurons were shown (n=9, N=5, Slc6a3 positive). Paired-sample t-test, t=3.345, df=8, 95% CI: –1.487 to –0.2734, *p=0.0101, n.s. p>0.05, *p<0.05, ***p<0.001. n is the number of neurons recorded and N is the number of mice.
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Figure 2—figure supplement 4—source data 1
Data of the effects of CsCl on the excitability of ventral tegmental area (VTA) NAc ls-projecting dopaminergic (DA) neurons for Figure 2—figure supplement 4A.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig2-figsupp4-data1-v1.xlsx
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Figure 2—figure supplement 4—source data 2
Data of the effects of ZD7288 on the excitability of VTA NAc ls-projecting dopaminergic (DA) neurons for Figure 2—figure supplement 4B.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig2-figsupp4-data2-v1.xlsx
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Figure 2—figure supplement 4—source data 3
Data of the effect of low extracellular K+ on the resting membrane potential (RMP) of ventral tegmental area dopaminergic (VTA DA) neurons for Figure 2—figure supplement 4C.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig2-figsupp4-data3-v1.xlsx
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Figure 2—figure supplement 4—source data 4
Data of the effect of ZD7288 on the excitability of ventral tegmental area dopaminergic (VTA DA) neurons in low [K+]e condition for Figure 2—figure supplement 4D.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig2-figsupp4-data4-v1.xlsx
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Figure 2—figure supplement 4—source data 5
Data of the effects of ZD7288 on the excitability of juvenile male ventral tegmental area dopaminergic (VTA DA) neurons for Figure 2—figure supplement 4E.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig2-figsupp4-data5-v1.xlsx
Figure 3
NALCN contributes to subthreshold depolarization and spontaneous firing of the ventral tegmental area dopaminergic (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 Slc6a3). (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 Slc6a3 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 Slc6a3 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 Slc6a3 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 Slc6a3 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) 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 Slc6a3 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 Slc6a3 positive with single cell-PCR (i: Con, gray 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 Slc6a3 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 Slc6a3 positive with single cell-PCR (i: Con, gray 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 the number of neurons recorded and N is the number of mice used.
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Figure 3—source data 1
PDF file containing confocal images for Figure 3A and H.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig3-data1-v1.pdf
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Figure 3—source data 2
PDF file containing original gels of single-cell PCR for Figure 3Bi, indicating the relevant bands and groups.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig3-data2-v1.zip
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Figure 3—source data 3
Original files for gels of single-cell PCR are displayed in Figure 3Bi.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig3-data3-v1.zip
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Figure 3—source data 4
Data on the effect of GdCl3 on the resting membrane potential (RMP) of ventral tegmental area dopaminergic (VTA DA) neurons is displayed in Figure 3C.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig3-data4-v1.xlsx
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Figure 3—source data 5
Data on the effect of GdCl3 on the firing frequency of ventral tegmental area dopaminergic (VTA DA) neurons is displayed in Figure 3D.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig3-data5-v1.xlsx
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Figure 3—source data 6
Data on the effect of L703,606 on the resting membrane potential (RMP) of ventral tegmental area dopaminergic (VTA DA) neurons is displayed in Figure 3E.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig3-data6-v1.xlsx
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Figure 3—source data 7
Data on the effect of L703,606 on the firing frequency of ventral tegmental area dopaminergic (VTA DA) neurons is displayed in Figure 3F.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig3-data7-v1.xlsx
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Figure 3—source data 8
Data of the expression level of Nalcn RNA in the shRNA-Nalcn transfected ventral tegmental area (VTA) and the scramble shRNA transfected VTA for Figure 3G.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig3-data8-v1.xlsx
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Figure 3—source data 9
Data of excitability of ventral tegmental area dopaminergic (VTA DA) neurons transfected with either Nalcn-shRNA or scramble-shRNA for Figure 3I.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig3-data9-v1.xlsx
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Figure 3—source data 10
Data of resting membrane potential (RMP) of ventral tegmental area dopaminergic (VTA DA) neurons transfected with either Nalcn-shRNA or scramble-shRNA for Figure 3J.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig3-data10-v1.xlsx
Figure 4 with 1 supplement
Transient receptor potential (TRP) channels especially TRPC6 contribute to subthreshold depolarization and spontaneous firing of the ventral tegmental area dopaminergic (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 (Trpc3, Trpc6 and Trpv2) positive neurons in the VTA DA neurons (Slc6a3 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 Slc6a3 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. The inset on the top of Dii and 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 Slc6a3 positive with single cell-PCR (red dots). 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 the number of neurons recorded and N is the number of mice used.
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Figure 4—source data 1
Data and a PDF file containing original gels of single-cell PCR for Figure 4A, indicating the relevant bands and groups.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for gels of single-cell PCR are displayed in Figure 4A.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig4-data2-v1.zip
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Figure 4—source data 3
Data on the effect of 2-APB on the firing frequency of ventral tegmental area dopaminergic (VTA DA) neurons are displayed in Figure 4B.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig4-data3-v1.xlsx
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Figure 4—source data 4
Data on the effect of LA on the firing frequency of ventral tegmental area dopaminergic (VTA DA) neurons are displayed in Figure 4C.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig4-data4-v1.xlsx
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Figure 4—source data 5
Data on the effect of 2-APB on the resting membrane potential (RMP) of ventral tegmental area dopaminergic (VTA DA) neurons are displayed in Figure 4D.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig4-data5-v1.xlsx
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Figure 4—source data 6
Data on the effect of LA on the resting membrane potential (RMP) of ventral tegmental area dopaminergic (VTA DA) neurons are displayed in Figure 4E.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig4-data6-v1.xlsx
Figure 4—figure supplement 1
Effects of TRPV channel blocker ruthenium red (RR) on the spontaneous firing of the male ventral tegmental area dopaminergic (VTA DA) neurons.
(A) The inset on the top of i shows a map of a coronal midbrain slice indicating the location of neurons that were recorded and subsequently identified as DA neurons which were Slc6a3 positive with single cell-PCR (red dots). The bottom panel of (i) summarizes (n=9, N=6) the percentage of RR-activated and -inhibited VTA DA cells. (ii) The summarized data for the effects of RR on the spontaneous firing frequency of VTA DA neurons. Paired-sample t-test, t=0.4486, df=8, 95% CI: –0.7621–0.5139, p=0.6656. (B and C) Example time-course (i) and traces (ii) show the characteristic responses of firing activity to RR-activated (B) and -inhibited (C) neurons. n.s. p>0.05, n is the number of neurons recorded and N is the number of mice used.
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Figure 4—figure supplement 1—source data 1
Data of the effects of ruthenium red (RR) on the spontaneous firing of the male ventral tegmental area dopaminergic (VTA DA) neurons.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig4-figsupp1-data1-v1.xlsx
Figure 5 with 1 supplement
TRPC6 contributes to subthreshold depolarization and spontaneous firing of the ventral tegmental area dopaminergic (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 was 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 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 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 Slc6a3 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 Slc6a3 positive with single cell-PCR ((i) Con, gray 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 Slc6a3 positive with single cell-PCR (TRPC6-KD, red dots). (n=7, N=5, both GFP and Slc6a3 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 Slc6a3 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 Slc6a3 positive with single cell-PCR ((i) Con, gray 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 Slc6a3 positive with single cell-PCR (TRPC6-KD, red dots). (n=8, N=5, both GFP and Slc6a3 positive), Paired-sample t-test, t=0.000, df = 7, 95% CI: –0.05740–0.05740, p>0.9999. n.s. p>0.05, **p<0.01, ***p<0.001, ****p<0.0001. n is the number of neurons recorded and N is the number of mice used.
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Figure 5—source data 1
Data of the expression profile of Trpc6 in NAc c- and medial prefrontal cortex (mPFC)-projecting ventral tegmental area dopaminergic (VTA DA) neurons by single cell-qPCR is displayed in the Figure 5A.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig5-data1-v1.xlsx
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Figure 5—source data 2
PDF file containing confocal images for Figure 5B and D.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig5-data2-v1.pdf
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Figure 5—source data 3
Data of the expression level of Trpc6 RNA in the shRNA-Trpc6 transfected ventral tegmental area (VTA) and the scramble shRNA transfected VTA for Figure 5C.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig5-data3-v1.xlsx
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Figure 5—source data 4
Data of excitability of ventral tegmental area dopaminergic (VTA DA) neurons transfected with either Trpc6-shRNA or scramble-shRNA for Figure 5E.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig5-data4-v1.xlsx
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Figure 5—source data 5
Data of the effect of shRNA knockdown of Trpc6 on the 2-APB-inhibited firing responses of the ventral tegmental area dopaminergic (VTA DA) neurons for Figure 5F.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig5-data5-v1.xlsx
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Figure 5—source data 6
Data of the effect of shRNA knockdown of Trpc6 on the RMP of the ventral tegmental area dopaminergic (VTA DA) neurons for Figure 5G.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig5-data6-v1.xlsx
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Figure 5—source data 7
Data of the effect of shRNA knockdown of Trpc6 on the LA-inhibited firing responses of the ventral tegmental area dopaminergic (VTA DA) neurons for Figure 5H.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig5-data7-v1.xlsx
Figure 5—figure supplement 1
The expression of Trpc4 and Trpc7 channels in male ventral tegmental area (VTA) Trpc6+ dopaminergic (DA) neurons.
(A) Single-cell PCR from 7 VTA Trpc6+Slc6a3+ cells (C1–C7) and control. (B) (i) and (ii) Percentage of transient receptor potential (TRP) channels (Trpc4 and Trpc7) positive neurons in the VTA Trpc6+Slc6a3+ DA neurons.
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Figure 5—figure supplement 1—source data 1
Data and a PDF file containing original gels for Figure 5—figure supplement 1, indicating the relevant bands and groups.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Original files for gels of single-cell PCR are displayed in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig5-figsupp1-data2-v1.zip
Figure 6 with 4 supplements
Chronic mild unpredictable stress (CMUS) depression mice have a decreased firing activity and down-regulated TRPC6 expression in the ventral tegmental area dopaminergic (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: 23.21–49.39, ****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, Slc6a3 positive) and the control (n=53, N=31, Slc6a3 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 Slc6a3 positive with single cell-PCR ((i) Con, gray 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, the 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, Slc6a3 positive) and CMUS (n=25 cells, N=15, Slc6a3 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, ****p0.0001; 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 Slc6a3 positive with single cell-PCR ((iii) Con, gray dots; (iv) CMUS, red dots). (F) (i and ii) Example traces (up) and time-course (bottom) of firing frequency ofmedial prefrontal cortex (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, Slc6a3 positive) and CMUS (n=30 cells, N=18, Slc6a3 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 medial prefrontal cortex (mPFC)-projecting neurons that were recorded and subsequently identified as DA neurons which were Slc6a3 positive with single cell-PCR ((iii) Con, gray dots; (iv) CMUS, red dots). n.s. p>0.05, **p<0.01, ****p<0.0001. n is the number of neurons recorded and N is the number of mice used.
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Figure 6—source data 1
Data of the behavior test for chronic mild unpredictable stress (CMUS) male mice in Figure 6B.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig6-data1-v1.xlsx
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Figure 6—source data 2
Data of excitability of ventral tegmental area dopaminergic (VTA DA) neurons from the chronic mild unpredictable stress (CMUS) and the control mice for Figure 6C.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig6-data2-v1.xlsx
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Figure 6—source data 3
Data and a PDF file containing original western blots for Figure 6D, indicating the relevant bands and groups.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig6-data3-v1.zip
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Figure 6—source data 4
Original files for western blot analysis are displayed in Figure 6D.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig6-data4-v1.zip
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Figure 6—source data 5
Data of the effect of 2-APB on the firing frequency of medial prefrontal cortex (mPFC)-projecting ventral tegmental area dopaminergic (VTA DA) neurons between Con and chronic mild unpredictable stress (CMUS) for Figure 6E.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig6-data5-v1.xlsx
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Figure 6—source data 6
Data of the effect of LA on the firing frequency of medial prefrontal cortex (mPFC)-projecting ventral tegmental area dopaminergic (VTA DA) neurons between Con and chronic mild unpredictable stress (CMUS) for Figure 6F.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig6-data6-v1.xlsx
Figure 6—figure supplement 1
Body weight and behavior of the male mice subjected to chronic mild unexpected stress (CMUS).
(A) Changes in body weight of the CMUS mice from week 2–6 (Con: N=10; CMUS: N=10), Two-sample t-test, t=6.284, df=18, 95% CI: –3.002 to –1.498, ****p<0.0001, compared with the control mice. (B) The total travel distance of the CMUS mice in the open-field test (Con: N=10; CMUS: N=10), Two-sample t-test, t=4.798, df=18, 95% CI: –1767 to –690.9, ***p=0.0001, compared with control mice. (C) The time of the CMUS mice stayed in the center zone in the open-field test (Con: N=10; CMUS: N=10), Two-sample t-test, t=4.733, df = 18, 95% CI: –27.29 to –10.51, ***p=0.0002, compared with control mice. (D) The percentage of time of CMUS mice stayed in open arm in the elevated plus-maze test (Con: N=10; CMUS: N=10), Two-sample t-test, t=4.440, df=18, 95% CI: –12.36 to –4.421, ***p=0.0003, compared with control mice. (E) The percentage of time of CMUS mice stayed in closed arms in the elevated plus-maze test (Con: N=10; CMUS: N=10), Mann-Whitney U test, U=14, **p=0.0052, compared with control mice. (F) The immobility time of mice in the forced-swimming test (Con: N=10; CMUS: N=10), Two-sample t-test, t=4.570, df=18, 95% CI: 17.56–47.44, ***p=0.0002, compared with control mice. (G) The time of latency to fall in the rotarod test (Con: N=10; CMUS: N=10), Two-sample t-test, t=0.3323, df=18, 95% CI: –11.18–15.38, p=0.7435, compared with control mice. n.s. p>0.05, **p<0.01, ***p<0.001, ****p<0.0001. N is the number of mice used.
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Figure 6—figure supplement 1—source data 1
Data of body weight and behavior tests of the male mice between chronic mild unpredictable stress (CMUS) and Con.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig6-figsupp1-data1-v1.xlsx
Figure 6—figure supplement 2
Body weight and behaviors of the female mice subjected to chronic mild unexpected stress (CMUS).
(A) The change in body weight of the CMUS female mice (Con: N=10; CMUS: N=10), Two-sample t-test, t=4.191, df=18, 95% CI: –1.802 to –0.5984, ***p=0.0005, compared with control mice. (B) Sucrose preference test for the CMUS female mice (Con: N=10; CMUS: N=10), Two-sample t-test, t=4.100, df = 18, 95% CI: –18.85 to –6.075, ***p=0.0007, compared with control mice. (C) The total travel distance of the CMUS female mice in the open-field test (Con: N=10; CMUS: N=10), Two-sample t-test, t=5.552, df=18, 95% CI: –1287 to –580.3, ****p<0.0001, compared with control mice. (D) The time of the CMUS female mice stayed in the center zone in the open-field test (Con: N=10; CMUS: N=10), Two-sample t-test, t=3.183, df=18, 95% CI: –37.44 to –7.665, **p=0.0052, compared with control mice. (E) The percentage of time of the CMUS female mice stayed in open arms in the elevated plus-maze test (Con: N=10; CMUS: N=10), Two-sample t-test, t=3.773, df = 18, 95% CI: –12.22 to –3.478, **p=0.0014, compared with control mice. (F) The percentage of time of the CMUS female mice stayed in closed arm in the elevated plus-maze test (Con: N=10; CMUS: N=10), Two-sample t-test, t=2.966, df=18, 95% CI: 3.143–18.40, **p=0.0083, compared with control mice. (G) The time of latency to fall in the rotarod test (Con: N=10; CMUS: N=10), Two-sample t-test, t=0.7688, df = 18, 95% CI: –14.18–6.584, p=0.4520, compared with control mice. (H) The immobility time of female mice in the forced-swimming test (Con: N=10; CMUS: N=10), Two-sample t-test, t=5.350, df = 18, 95% CI: 21.44–49.16, ****p<0.0001, compared with control mice. (I) The immobility time of female mice in the tail suspension test (Con: N=10; CMUS: N=10), Two-sample t-test, t=5.308, df = 18, 95% CI: 22.23–51.37, ****p<0.0001, compared with control mice. n.s. p>0.05, **p<0.01, ***p<0.001, ****p<0.0001. N is the number of mice used.
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Figure 6—figure supplement 2—source data 1
Data of body weight and behavior tests of the female mice between chronic mild unpredictable stress (CMUS) and Con.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig6-figsupp2-data1-v1.xlsx
Figure 6—figure supplement 3
Expression of NALCN protein in the ventral tegmental area (VTA) of the chronic mild unpredictable stress (CMUS) male mice.
(A and B) Western blot was used to measure the protein level of NALCN in the CMUS mice and control mice. Representative western blot assay showing the expression of NALCN and GAPDH in the VTA of the CMUS (N=12) and control (Con, N=9) mice (A), and the summarized data (B). Two-sample t-test, t=0.2724, df=19, 95% CI: –0.2703–0.3512, p=0.7883, n.s. p>0.05. N is the number of mice used.
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Figure 6—figure supplement 3—source data 1
Data and a PDF file containing original western blots for Figure 6—figure supplement 3, indicating the relevant bands and groups.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig6-figsupp3-data1-v1.zip
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Figure 6—figure supplement 3—source data 2
Original files for western blot analysis are displayed in Figure 6—figure supplement 3.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig6-figsupp3-data2-v1.zip
Figure 6—figure supplement 4
Body weight and behaviors of the male mice subjected to chronic restraint stress (CRS).
(A) Experimental procedure timeline for CRS. (B) The change in body weight of the CRS mice (Con: N=10; CRS: N=10), Two-sample t-test, t=5.481, df = 18, 95% CI: –2.144 to –0.9559, ****p<0.0001, compared with control mice. (C) Sucrose preference test for the CRS mice (Con: N=10; CRS: N=10), Two-sample t-test, t=4.653, df=18, 95% CI: –19.05 to –7.199, ***p=0.0002, compared with control mice. (D) The total travel distance of the CRS mice in the open-field test (Con: N=10; CRS: N=10), Two-sample t-test, t=4.680, df=18, 95% CI: –1385 to –526.6, ***p=0.0002, compared with control mice. (E) The time of the CRS mice stayed in the center zone in the open-field test (Con: N=10; CRS: N=1p), Mann-Whitney U test, U=8, ***p=0.0007, compared with control mice. (F) The percentage of time of the CRS mice stayed in open arms in the elevated plus-maze test (Con: N=10; CRS: N=10), Mann-Whitney U test, U=9, ***p=0.0010, compared with control mice. (G) The percentage of time of the CRS mice stayed in closed arms in the elevated plus-maze test (Con: N=10; CRS: N=10), Two-sample t-test, t=4.852, df = 18, 95% CI: 11.62–29.35, ***p=0.0001, compared with control mice. (H) The tail suspension test for the CRS mice (Con: N=10; CRS: N=10), Two-sample t-test, t=3.624, df=18, 95% CI: 10.93–41.07, **p=0.0019, compared with control mice. (I) The time of latency to fall in the rotarod test (Con: N=10; CRS: N=10), Two-sample t-test, t=0.2178, df = 18, 95% CI: –11.24–13.84, p=0.8300, compared with control mice. (J) Representative Western blot assay (i) and summarized data (ii) showing the expression of TRPC6 and GAPDH in the VTA of the CRS mice (Con: N=10; CRS: N=14). Two-sample t-test, t=7.369, df=22, 95% CI: –0.8006 to –0.4489, ****p<0.0001. **p<0.01, ***p<0.001, ****p<0.0001. N is the number of mice used.
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Figure 6—figure supplement 4—source data 1
Data of body weight and behavior tests of the male mice between CRS and Con for Figure 6—figure supplement 4B-I.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig6-figsupp4-data1-v1.xlsx
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Figure 6—figure supplement 4—source data 2
Data and a PDF file containing original western blots for Figure 6—figure supplement 4J, indicating the relevant bands and groups.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig6-figsupp4-data2-v1.zip
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Figure 6—figure supplement 4—source data 3
Original files for western blot analysis are displayed in Figure 6—figure supplement 4J.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig6-figsupp4-data3-v1.zip
Figure 7
Selective knockdown of TRPC6 in the ventral tegmental area dopaminergic (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-DIO virus was injected into the VTA of the Slc6a3-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 labeling showing the expression of AAV9-hSyn-DIO-shRNA(Trpc6)-RFP (red) and DAT (green) in the VTA of the Slc6a3-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: 7.785–33.41, **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) AAV9-DIO-Trpc6-GFP (TRPC6-cOE) was injected into the VTA of the male Slc6a3-Cre mice, 7 d 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 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 Slc6a3-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 the number of neurons recorded and N is the number of mice used.
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Figure 7—source data 1
Data of the relative mRNA level of Trpc6 in the ventral tegmental area (VTA) between TRPC6-cKD and Con for the Figure 7B.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig7-data1-v1.xlsx
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Figure 7—source data 2
PDF file containing confocal images for Figure 7C and G.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig7-data2-v1.pdf
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Figure 7—source data 3
Data of behavior tests between TRPC6-cKD and Con for Figure 7D.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig7-data3-v1.xlsx
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Figure 7—source data 4
Data and a PDF file containing original western blots for Figure 7F, indicating the relevant bands and groups.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig7-data4-v1.zip
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Figure 7—source data 5
Original files for western blot analysis are displayed in Figure 7F.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig7-data5-v1.zip
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Figure 7—source data 6
Data of the effects of TRPC6 over-expression on depression-like behaviors of TRPC6-cKD mice in Figure 7H.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig7-data6-v1.xlsx
Figure 8
Selective knockdown of TRPC6 in the mPFC-projecting ventral tegmental area dopaminergic (VTA DA) neurons produces depression-like and anxiety-like behaviors.
(A) shRNA against Trpc6 carried by the retro-AAV-DIO vector was injected into the mPFC of the Slc6a3-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 labeling showing the expression of AAV-retro-hSyn-DIO-mCherry-shRNA(Trpc6)(red) and dopamine transporter (DAT) (green) in the VTA of the Slc6a3-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: 10.79–39.01, **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 the number of neurons recorded and N is the number of mice used.
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Figure 8—source data 1
Data of the relative mRNA level of Trpc6 in the ventral tegmental area (VTA) mPFC-projecting dopaminergic (DA) neurons between TRPC6-rcKD and Con for the Figure 8B.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig8-data1-v1.xlsx
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Figure 8—source data 2
PDF file containing confocal images for Figure 8C.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig8-data2-v1.pdf
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Figure 8—source data 3
Data of behavior tests for Figure 8D.
- https://cdn.elifesciences.org/articles/88319/elife-88319-fig8-data3-v1.xlsx
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The cation channel mechanisms of subthreshold inward depolarizing currents in the mice VTA dopaminergic neurons and their roles in the chronic-stress-induced depression-like behavior
eLife 12:RP88319.
https://doi.org/10.7554/eLife.88319.4
