Article Figures & Data
Figures
- Figure 1.
Anatomic analysis of Grp and Neurod6-expressing DA neurons in the midbrain. A, Confocal images of multiplex fluorescent in situ hybridization using probes against Grp (left), Neurod6 (middle), and tyrosine hydroxylase (Th, right) mRNA. Inset boxes show higher-magnification images of the boxed regions. B, Schematic showing the location of Grp + (blue), Neurod6+ (red), and Grp+/Neurod6+ (purple) DA neurons in the midbrain. C, High-magnification confocal images showing Th-positive VTA neurons expressing Grp (blue circles), Neurod6 (red circles), Grp and Neurod6 (purple circles), or neither marker (white circles). D, Charts show the distribution of Grp+ or Neurod6 + neurons across different midbrain regions, expressed as a percentage of the total Grp+ or Neurod6 + population. Only DA neurons, defined by expression of Th mRNA, were included in the analysis, n = 4377 cells quantified from 8 mice (4 male and 4 female). E, Quantification of the number of Th + DA neurons that coexpress Grp or Neurod6 in subregions of the VTA and SNc. Bars represent mean ± SEM, dots represent the values from individual mice, n = 4377 Th + cells quantified from 4 male and 4 female mice. Grp one-way ANOVA, p < 0.0001; Tukey’s post hoc tests revealed significant (p < 0.0001) differences for each subpopulation compared to all others except the PBP versus SNc-V (p = 0.9998). Neurod6 one-way ANOVA, p < 0.0001; Tukey’s post hoc tests revealed significant (p < 0.0001) differences for each subpopulation compared to all others except the IF versus PN/PIF (p = 0.0842) and SNc-V versus SNc-D (p = 0.9699). F, Venn diagrams display the extent of overlap between the Grp and Neurod6-expressing DA neuron populations across four midbrain subregions. 1833 cells were quantified from 8 mice (4 male and 4 female). G, H, Graphs display the mean ± SEM percentage of Grp+ (G) or Neurod6 + (H) DA neurons in the VTA at 2, 8, and 16 wk. Statistical comparisons were made with a one-way ANOVA with Tukey’s post hoc test; Grp 8 wk (34.9%) vs. 16 wk (36.9%), *, p = 0.0316, Neurod6 2 wk (25.2%) vs. 16 wk (27.6%), *, p = 0.0201. Dots represent values from individual mice, n = 2053–2324 cells from 2 male and 2 female mice per time point. I, J, Graphs display the mean ± SEM percentage of Grp+ (I) or Neurod6 + (J) DA neurons across subregions of the VTA and SNc in male (M) and female (F) mice. Unpaired t tests between males and females for each region revealed no significant sex differences. Dots represents values from individual mice, n = 3586 cells from 6 male mice and 3424 cells from 6 female mice. IF, interfascicular nucleus; PN/PIF, paranigral/parainterfascicular nuclei; PBP, parabrachial pigmented nucleus; SNc-V, substantia nigra pars compacta-ventral portion; SNc-D, substantia nigra pars compacta-dorsal portion. See also Fig. 1-1.
- Figure 2.
Projection targets of Grp + or Neurod6+ DA neurons. A, Schematic of retrobead injection sites in sagittal view. Numbers correspond to the coronal section schematics below showing the eight projection target sites. mPFC, medial prefrontal cortex; DLS, dorsolateral striatum; DMS, dorsomedial striatum; LSh, lateral shell; C, core; MSh, medial shell; LS, lateral septum; BLA, basolateral amygdala. B, Image showing a green fluorescent retrobead injection into the nucleus accumbens (NAc) medial shell with DAPI staining in blue. C, Image of the VTA showing fluorescent in situ hybridization (FISH) for Slc6a3 (DAT) mRNA (white), Neurod6 mRNA (red), and green retrobeads from a NAc medial shell injection. D, Image showing a green retrobead injection into the dorsomedial striatum (DMS) with DAPI staining in blue. E, FISH image of the midbrain showing Th mRNA (white), Grp mRNA (red), and green retrobeads from a DMS injection. Insets boxes in C and E show higher-magnification images of the boxed regions. Red circles identify Neurod6+ (C) or Grp + (E) neurons, green circles define bead-positive neurons, and yellow circles show bead-positive neurons expressing Neurod6 (C) or Grp (E). White circles identify neurons expressing Th only. F, Quantification of the percentage of Th +/bead+ midbrain neurons that coexpressed Grp mRNA for each of the projection sites. Bars represent mean ± SEM, each dot represents one mouse. G, Summary of the projection targets of Grp + DA neurons expressed as a percentage of total bead+/Grp +/Th + neurons. H, Quantification of the percentage of DAT +/bead+ midbrain neurons that coexpressed Neurod6 mRNA for each of the projection sites. Bars represent mean ± SEM, each dot represents one mouse. I, Summary of the projection targets of Neurod6 + DA neurons expressed as a percentage of total bead+/Neurod6 +/DAT + neurons. For panels F–I: NAc MSh n = 850 cells from 2 male and 2 female mice, NAc Core n = 858 cells from 2 males and 2 females, NAc LSh n = 1215 cells from 3 males and 1–2 females, DMS n = 637 cells from 3 males and 2 females, DLS n = 1038 cells from 2–4 males and 2–3 females, mPFC n = 211 cells from 3–4 males and 1 female, BLA n = 297 cells from 3 males, and LS n = 345 cells from 2 males and 4 females. See also Fig. 2-1.
- Figure 3.
Neurod6-expressing DA neurons have noncanonical physiological properties. A, Cre-dependent tdTomato-expressing virus (AAV-Flex-tdTomato) was injected unilaterally into the VTA of a NEX-Cre mouse. Right panel shows tdTomato-labeled NEX-Cre+ neurons in the ventromedial VTA. Tyrosine hydroxylase (TH) staining is in blue. B, Confocal image of the nucleus accumbens (NAc) showing tdTomato-labeled projections from midbrain NEX-Cre+ neurons. MSh, medial shell; LSh, lateral shell. C, Confocal images of VTA neurons from NEX-Cre;Ai9 mice with fluorescent in situ hybridization (FISH) for tyrosine hydroxylase (Th) and Neurod6 mRNA. The tdTomato Cre-reporter is expressed in NEX-Cre+ neurons. NEX-Cre+/Neurod6 + DA neurons are circled in red, NEX-Cre-/Neurod6+ DA neurons are circled in white. D, E, Left panels show representative recording sites of NEX-Cre+ VTA (D) and NEX-Cre- SNc neurons (E). Circles show the locations of recorded DA neurons. Right panels show examples of recorded, neurobiotin-filled neurons positive for TH. F–I, Graphs display the mean ± SEM membrane potential (F, Vm, **, p = 0.0075), membrane resistance (G, Rm, ***, p < 0.0001), capacitance (H, Cm, ***, p < 0.0001), and afterhyperpolarization (I, AHP, ***, p = 0.0002) for NEX-Cre+ and SNc neurons. J, K, Representative action potential traces and phase plots (mVms−1/mV) from a NEX-Cre+ (J) and SNc (K) neuron. L, M, Representative responses to a 500 ms negative current step from a NEX-Cre+ (–25 pA, L) and SNc (–150 pA, M) neuron. N, Quantification of the mean ± SEM sag component in NEX-Cre+ and SNc neurons (***, p < 0.0001). O, Quantification of the mean ± SEM rebound depolarization from neurons hyperpolarized to –100 ± 7 mV (***, p < 0.0001). An unpaired t test was used for all comparisons. For all panels, dots represent values from individual cells, NEX-Cre+ n = 7 male and 7 female mice, NEX-Cre-/SNc n = 4 male mice and 1 female mouse. See Fig. 3-1 for a complete summary of the electrophysiology results.
- Figure 4.
Neurod6-expression affects susceptibility to 6-OHDA–induced degeneration. A, Confocal image of a midbrain section stained with a tyrosine hydroxylase (TH) antibody 4 wk after unilateral saline (left) or 6-OHDA (right) injection into the medial forebrain bundle. B, Image of a striatal (Str) section showing loss of tdTomato+ axon terminals 4 wk following unilateral 6-OHDA injection in a DATIRESCre;Ai9 mouse. NAc, nucleus accumbens. C, Schematic showing the location of DA neurons in the saline and 6-OHDA injected hemispheres labeled with different markers by fluorescent in situ hybridization (FISH): blue circles are Grp+, red circles are Neurod6+, and purple circles are Grp+/Neurod6+. D, Schematic showing the location of Lypd1+ (green), Neurod6+ (red), and Lypd1+/Neurod6+ (yellow) DA neurons in the midbrain following saline and 6-OHDA injection. E, F, Confocal images of FISH for the indicated markers. E, Images show Grp+ (blue circle), Neurod6+ (red circles), and Grp+/Neurod6+ (purple circles) DA neurons in the VTA following saline and 6-OHDA injection. F, Images show Lypd1+ (green circles), Neurod6+ (red circles), and Lypd1+/Neurod6+ (yellow circle) DA neurons in the VTA following saline and 6-OHDA injection. G, Box-and-whisker plots (min to max) show the percentage of VTA DAT neurons expressing each set of markers in the 6-OHDA–injected hemisphere compared to the saline-injected hemisphere. Dotted line represents the percentage of all VTA DA neurons (Th+) surviving in the 6-OHDA hemisphere. Dots represent data from individual mice, n = 4 female mice. A paired one-way ANOVA with Dunnett’s multiple comparisons test was used to compare each subpopulation to all Th+ VTA DA neurons (n = 4376 saline and 1352 6-OHDA cells): Grp–/Neurod6+ (n = 184 saline and 151 6-OHDA cells), ** p = 0.0022; Grp+/Neurod6+ (n = 763 saline and 207 6-OHDA cells), p = 0.3169; Grp+/Neurod6– (n = 749 saline and 165 6-OHDA cells), * p = 0.0246; Lypd1+/Neurod6– (n = 1056 saline and 171 6-OHDA cells), * p = 0.0313; Nd6 = Neurod6. H, Bar graphs display the percentage of Lypd1 + VTA DA neurons that coexpress Neurod6 mRNA in the saline-injected (n = 102/3750 cells) and 6-OHDA–injected (n = 16/904 cells) hemispheres. Bars represent mean ± SEM, dots represent the values from individual mice, n = 4 female mice; paired t test, n.s., not significant, p = 0.8091.
Tables
Location Data structure Type of test CI/power P value Comparison a Normally distributed One-way ANOVA 0.97 <0.0001 Grp expression in midbrain subregions Normally distributed Tukey’s post hoc 6.508 to 17.74 <0.0001 Grp IF vs. PN/PIF Normally distributed Tukey’s post hoc 34.77 to 46 <0.0001 Grp IF vs. PBP Normally distributed Tukey’s post hoc 35.1 to 46.33 <0.0001 Grp IF vs. SNc-V Normally distributed Tukey’s post hoc 57.26 to 68.49 <0.0001 Grp IF vs. SNc-D Normally distributed Tukey’s post hoc 22.65 to 33.88 <0.0001 Grp PN/PIF vs. PBP Normally distributed Tukey’s post hoc 22.97 to 34.2 <0.0001 Grp PN/PIF vs. SNc-V Normally distributed Tukey’s post hoc 45.13 to 56.37 <0.0001 Grp PN/PIF vs. SNc-D Normally distributed Tukey’s post hoc –5.292 to 5.942 0.9998 Grp PBP vs. SNc-V Normally distributed Tukey’s post hoc 16.87 to 28.1 <0.0001 Grp PBP vs. SNc-D Normally distributed Tukey’s post hoc 16.55 to 27.78 <0.0001 Grp SNc-V vs. SNcD b Normally distributed One-way ANOVA 0.96 <0.0001 Neurod6 expression in midbrain subregions Normally distributed Tukey’s post hoc –0.4177 to 9.918 0.0842 Neurod6 IF vs. PN/PIF Normally distributed Tukey’s post hoc 16.28 to 26.62 <0.0001 Neurod6 IF vs. PBP Normally distributed Tukey’s post hoc 34.69 to 45.03 <0.0001 Neurod6 IF vs. SNc-V Normally distributed Tukey’s post hoc 35.82 to 46.16 <0.0001 Neurod6 IF vs. SNc-D Normally distributed Tukey’s post hoc 11.53 to 21.87 <0.0001 Neurod6 PN/PIF vs. Normally distributed Tukey’s post hoc PBP Normally distributed Tukey’s post hoc 29.94 to 40.28 <0.0001 Neurod6 PN/PIF vs. SNc-V Normally distributed Tukey’s post hoc 31.07 to 41.41 <0.0001 Neurod6 PN/PIF vs. SNc-D Normally distributed Tukey’s post hoc 13.24 to 23.58 <0.0001 Neurod6 PBP vs. SNc-V Normally distributed Tukey’s post hoc 14.37 to 24.71 <0.0001 Neurod6 PBP vs. SNc-D Normally distributed Tukey’s post hoc –4.043 to 6.293 0.9699 Neurod6 SNc-V vs. SNc-D c Normally distributed One-way ANOVA 0.522 0.036 Grp expression by age Normally distributed Tukey’s post hoc –1.112 to 2.462 0.5636 Grp 2 wk vs. 8 wk Normally distributed Tukey’s post hoc –3.087 to 0.4872 0.1603 Grp 2 wk vs. 16 wk Normally distributed Tukey’s post hoc –3.762 to –
0.18780.0316 Grp 8 wk vs. 16 wk d Normally distributed One-way ANOVA 0.56 0.0236 Neurod6 expression by age Normally distributed Tukey’s post hoc –2.859 to 1.109 0.4655 Neurod6 2 wk vs. 8 wk Normally distributed Tukey’s post hoc –4.384 to –
0.41620.0201 Neurod6 2 wk vs. 16 wk Normally distributed Tukey’s post hoc –3.509 to 0.4588 0.1351 Neurod6 8 wk vs. 16 wk e Normally distributed Unpaired t test –7.68 to 3.147 0.3728 Grp IF M vs. F f Normally distributed Unpaired t test –12.78 to 0.4155 0.0634 Grp PN/PIF M vs. F g Normally distributed Unpaired t test –1.924 to 4.89 0.3549 Grp PBP M vs. F h Normally distributed Unpaired t test –7.321 to 5.188 0.7119 Grp SNCv M vs. F i Normally distributed Unpaired t test –0.6657 to 1.199 0.5383 Grp SNCd M vs. F j Normally distributed Unpaired t test –13.48 to 4.412 0.2852 Neurod6 IF M vs. F k Normally distributed Unpaired t test –8.615 to 3.682 0.3924 Neurod6 PN/PIF M vs. F l Normally distributed Unpaired t test –1.028 to 2.095 0.4642 Neurod6 PBP M vs. F m Normally distributed Unpaired t test –2.251 to 0.1514 0.0801 Neurod6 SNCv M vs. F n Negative binomial Likelihood ratio test n/a 0.5113 Neurod6 M vs. F RNASeq data o Negative binomial Likelihood ratio test n/a 0.6835 Grp M vs. F RNASeq data p Normally distributed Unpaired t test –9.156 to –1.532 0.0075 Vm – SNc vs. NEXCre q Normally distributed Unpaired t test –1222 to –729.4 <0.0001 Rm – SNc vs. NEXCre r Normally distributed Unpaired t test 23.67 to 43.99 <0.0001 Cm – SNc vs. NEXCre s Normally distributed Unpaired t test 11.3 to 21.88 <0.0001 AP height – SNc vs. NEX-Cre t Normally distributed Unpaired t test 3.372 to 9.524 0.0002 AHP – SNc vs. NEXCre u Normally distributed Unpaired t test 6.564 to 10.49 <0.0001 Sag – SNc vs. NEXCre v Normally distributed Unpaired t test 4.295 to 7.773 <0.0001 Rebound – SNc vs. NEX-Cre w Normally distributed One-way ANOVA 0.85 0.0002 Cylinder test Normally distributed Tukey’s post hoc 6.33 to 55.07 0.0163 Saline vs. 6-OHDA paw Normally distributed Tukey’s post hoc –55.27 to –6.53 0.0157 Saline vs. both paws Normally distributed Tukey’s post hoc –85.97 to –37.23 0.0002 6-OHDA vs. both paws x Normally distributed Paired one-way ANOVA 0.96 <0.0001 Cell population survival Normally distributed Dunnett’s post hoc –69.31 to –34.69 0.0022 All TH+ vs. Grp-/ Neurod6 + Normally distributed Dunnett’s post hoc –6.01 to 15.16 0.3169 All TH+ vs. Grp +/ Neurod6 + Normally distributed Dunnett’s post hoc 2.07 to 15.78 0.0246 All TH+ vs. Grp+/ Neurod6- Normally distributed Dunnett’s post hoc 0.6149 to 15.14 0.0403 All TH+ vs. Lypd1 +/Neurod6- y Normally distributed Paired t test –3.593 to 3.043 0.8091 Lypd1+/Neurod6 + saline vs. 6-OHDA
Figure 1-1
Single-cell RNA-sequencing defines genetic subpopulations of mouse midbrain dopamine neurons. A, Confocal image of a midbrain section from a DATIRESCre;Ai9 mouse showing tdTomato Cre-reporter expression in midbrain dopamine (DA) neurons. B, t-distributed stochastic neighborhood embedding (tSNE) plot shows genetically defined clusters of DA neurons. 232 cells were analyzed from 8 mice. C, Heatmap displays a selection of differentially expressed genes in cluster 8 (highlighted by the dashed box). Individual genes are along the Y-axis and individual cells are along the X-axis. Colored bars at the top indicate the assigned cell cluster. D, Heatmap of cell coclustering. The RSEC clustering method is based on consensus clustering over many parameters. To assess the robustness of the final clustering, we generated a coclustering matrix where, for each pair of cells, we recorded the proportion of times in which two cells clustered together. The cells that failed to cluster at least 50% of the time were dropped from the analysis. The plot can be used to assess the robustness of the clustering procedure. Clusters 1, 2, 3, 7, and 8 are very robust, as almost all of the cells cluster together 100% of the time. Clusters 4, 5, and 6 are less robust, meaning that their boundaries are not well defined. Both the X- and Y-axis correspond to cells, ordered according to hierarchical clustering; colored bars at the top indicate the final nine cluster labels. E, Heatmap of the top 151 differentially expressed genes. Individual genes are along the Y-axis and individual cells are along the X-axis (n = 232 cells). Colored bars at the top indicate cell cluster and sex of the animal. Differentially expressed genes were obtained by performing all pairwise comparisons among the nine defined clusters with the R package limma. Genes were sorted by p-value, and the top 10 genes per comparison were selected for visualization (with nine clusters there are a total of 36 pairwise comparisons). If a gene was identified as differentially expressed in more than one comparison, it was reported in the heatmap only once. This procedure resulted in 151 distinct differentially expressed genes. F, Table summarizing selected markers and the anatomical location of each genetically defined cluster. Anatomical location was determined by in situ hybridization of marker genes and expression data from the Allen Brain Atlas. Marker genes were included in the table if there was significant differential expression from at least three other clusters. Download Figure 1-1, TIF file.
Table 1-1
Differential gene expression by cluster Excel table reports gene names, expression values, and p values for the genes used to generate the heatmap in Figure 1-1E. Cluster comparison = Clusters that are compared where cluster n is expressed as Xn. Gene = differentially expressed gene between the two clusters compared in “Cluster comparison”. Log Fold Change = log-fold change of gene expression between the two clusters in “Cluster comparison”. Negative value indicates increased expression in the second cluster (e.g. a negative value in X1-X2 means the gene is higher expressed in X2 relative to X1). Average Expression = Average expression of the gene across all cells. T = moderated t-statistic from the limma DE test. P.Value = probability that the gene is differentially expressed between the two clusters. Adj.P.Val = p value adjusted for multiple testing using the Benjamini-Hochberg method. B = the log-odds that the gene is differentially expressed. Download Table 1-1, XLSX file.
Figure 2-1
Retrobead injection sites and location of Grp + DA neurons projecting to the dorsomedial striatum or NAc medial shell. A, Schematics of coronal brain sections from the indicated anterior/posterior (A/P) positions from bregma. Colored regions represent retrobead injection sites from individual mice, NAc MSh n = 2 male and 2 female mice, NAc Core n = 2 male and 2 female mice, NAc LSh n = 3 male and 2 female mice, DMS n = 3 male and 2 female mice, DLS n = 4 male and 3 female mice, mPFC n = 4 male and 1 female mouse, BLA n = 3 male mice, and LS n = 2 male and 3 female mice. B, C, Schematics showing the locations of Grp +/TH +/bead+ cells in the midbrain that project to the dorsomedial striatum (B) or nucleus accumbens (NAc) medial shell (C). Schematics were generated from representative single FISH images. Download Figure 2-1, TIF file.
Figure 3-1
Summary of electrophysiology data. Extended Data table supporting Fig. 3. Table reports sample size, group means ± SEM, and unpaired t-test p values for each parameter measured. M, male mice; F, female mice. Download Figure 3-1, DOCX file.
Extended Data 1
Computer code for single cell RNA-sequencing analysis. Download Extended Data 1, TXT file.
In this issue
