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A B C Supplementary Figure 1. Experimental Workflow And a c Ion channel activity Midbrain microdissection Cacna1c Cav1.2 Collected material Cacna1d Cav1.3 Cacna1g Cav3.1 SNc Hcn2 HCN2 SNr VTA Hcn4 HCN4 Scn2a1 Nav1.2 + Scn5a Nav1.5 TaqMan assays Scn8a Nav1.6 Kcna2 Kv1.2 Dissociated midbrain neurons Kcnb1 Kv2.1 Kcnd2 Kv4.2 Kcnd3 Kv4.3 Targeted reverse transcription Kcnip3 KCHIP3 and preamplification Kcnj11 Kir6.2 Abcc8 SUR1 Abcc9 SUR2B Fluorescence imaging Kcnj5 GIRK4 Kcnj6 GIRK2 GFP Kcnn3 SK3 DA metabolism & signaling Non-GFP Microfluidic quantitative PCR Th TH Slc6a3 DAT Assays Samples Slc18a2 VMAT2 Pipette harvesting Drd2 D2R Glia-specific markers Gfap GFAP Aldh1l1 FDH Calcium-ion-binding Calb1 CB Pvalb PV Other neuronal markers b 40 Slc17a6 VGLUT2 30 Gad1 GAD67 20 Gad2 GAD65 10 Chat CHAT Cell count 0 Penk ENK 16 GFP Neuronal structure Non-GFP 14 Ncam2 NCAM2 WT 12 Map2 MAP2 10 Nefm NEF3 Th (TH) 8 Neuronal activation x E Creb1 CREB 2 6 g DA neurons Fos C-FOS o 4 L (n=111) Bdnf BDNF 2 nDA neurons Housekeeping/ 0 (n=37) transcriptional factors 0 2 4 6 8 10 12 14 16 0 10 20 30 40 Hprt HGPRT Tbp TBP Log2Ex Slc6a3 (DAT) Cell count Tbx3 TBX3 Supplementary Figure 1. Experimental workflow and classification of DA and nDA neurons. a, schematic showing the workflow for the single-cell gene profiling. Left, neurons were manually collected after acute dissociation from microdissected midbrain slices containing the substantia nigra pars compacta and reticulata (SNc, SNr) and part of the ventral tegmental area (VTA) obtained from TH-GFP mice. Right, specific targeted reverse transcription and amplification was then performed in the same tube, and microfluidic qPCR was run by using 96.96 Dynamic arrays on a BiomarkTM HD System. b, DA and nDA were classified based on the expression levels of Th (TH) and Slc6a3 (DAT), nDA neurons corresponding to neurons expressing 0 Th and/or 0 Slc6a3. c, list of the genes selected for the single-cell study with the gene names (left) and corresponding protein names (right). Genes are classified based on the function of the corresponding proteins (colored text). a b acute slices neuron P GF acute dissociation neuron P non-GF c *** 2.5 n=10 n=9 ** -30 30 *** 80 2.0 -35 20 1.5 -40 d (mV) 60 l o litude (mV) h -45 p 10 1.0 s e m r half-width (ms) 40 h a -50 acute slices t P P 0.5 P A A Sag amplitude (mV) 0 A -55 20 0.0 ** n=10 n=10 n=10 n=9 n=10 n=9 -60 * 2.5 n=13 n=8 * -30 30 *** 80 2.0 -35 20 -40 1.5 d (mV) 60 l o h litude (mV) -45 1.0 s p 10 e r m 40 h -50 half-width (ms) t a 0.5 P P P A A 0 A Sag amplitude (mV) -55 20 0.0 acute dissociation n=12 n=5 n=13 n=8 n=13 n=8 -60 ** Supplementary figure 2. Electrophysiological properties of GFP and non-GFP midbrain neurons in acute dissociation and in acute slices. a, bright field and fluorescence images corresponding to recorded GFP (top pictures) and non-GFP neurons (bottom pictures). b, representative recordings obtained from GFP (green traces) and non-GFP midbrain neurons (dark red traces) in acute slices (top traces) and after acute dissociation (bottom traces). Please note the differences in sag amplitude and rebound kinetics (left traces) and the significantly broader action potential (right traces) in GFP neurons. c, bar and scatter plots summarizing the electrophysiological differences observed between GFP and non-GFP neurons in acute slices (top row) and after acute dissociation (bottom row). As expected for midbrain DA neurons, GFP neurons were characterized by significantly larger sag amplitude (left plot), smaller and broader action potential (middle plots), and more depolarized action potential threshold (right plot). Scale bars in b: 20mV, 500ms for left traces; 20mV, 2ms for right traces. Dotted lines indicate -60mV. Asterisks in c indicate statistical significance (Mann-Whitney). * p<0.05, ** p<0.01, *** p<0.001. 60 Scn2a1 (Nav1.2) 60 Kcnj6 (GIRK2) 60 Kcna2 (Kv1.2) 60 Slc17a6 (VGLUT2) 60 Ncam2 (NCAM2) 40 40 40 40 40 20 20 20 20 20 Cell count (%) 0 0 0 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 60 Scn8a (Nav1.6) 60 Kcnj11 (Kir6.2) 60 Kcnb1 (Kv2.1) 60 Gad1 (GAD67) 60 Map2 (MAP2) 40 40 40 40 40 20 20 20 20 20 Cell count (%) 0 0 0 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 60 Scn5a (Nav1.5) 60 Abcc8 (SUR1) 60 Kcnn3 (SK3) 60 Gad2 (GAD65) 60 Nefm (NEF3) 40 40 40 40 40 20 20 20 20 20 Cell count (%) 0 0 0 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 60 Cacna1c (Cav1.2) 60 Abcc9 (SUR2B) 60 Th (TH) 60 Penk (PENK) 60 Creb1 (CREB) 40 40 40 40 40 20 20 20 20 20 Cell count (%) 0 0 0 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 60 Cacna1d (Cav1.3) 60 Kcnd2 (Kv4.2) 60 Slc6a3 (DAT) 60 Calb1 (CB) 60 Fos (C-FOS) 40 40 40 40 40 20 20 20 20 20 Cell count (%) 0 0 0 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 60 Cacna1g (Cav3.1) 60 Kcnd3_2 (Kv4.3) 60 Slc18a2 (VMAT2) 60 Pvalb (PV) 60 Bdnf (BDNF) 40 40 40 40 40 20 20 20 20 20 Cell count (%) 0 0 0 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 60 Hcn2 (HCN2) 60 Kcnd3_1 (Kv4.3) 60 Drd2 (D2R) 60 Gfap (GFAP) 60 Hprt (HGPRT) 40 40 40 40 40 20 20 20 20 20 Cell count (%) 0 0 0 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Log2Ex Log2Ex 60 Hcn4 (HCN4) 60 Kcnip3 (KCHIP3) 60 Aldh1l1 (FDH) 40 40 40 20 20 20 Cell count (%) 0 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 DA neurons nDA neurons Log2Ex Log2Ex Log2Ex Supplementary Figure 3. Gene expression profiles in DA and nDA neurons. Graphs showing the frequency distribution of gene expression levels in DA (green) and nDA (dark red) neurons. The three genes with the lowest expression (Tbp, Kcnj5 and Tbx3) are not represented. The Y-axes correspond to normalized cell count (%) and the X-axes to relative gene expression levels in logarithmic (Log2Ex) scale. DA neurons 16 16 16 r=0.867, p<0.001, n=111 r=0.855, p<0.001, n=111 r=0.777, p<0.001, n=111 14 14 r= 0.783, p<0.001, n=111 14 r=-0.145, p=0.622, n=14 r=0.235, p=0.382, n=16 12 12 12 10 10 10 8 8 8 6 6 6 4 4 4 Kcnd3_2(Kv4.3) Slc6a3 (DAT) 2 r=0.695, p< 0.001, n=105 2 2 0 0 Kcnd3_2 (Kv4.3) 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Th (TH) Kcnn3 (SK3) Slc6a3 (DAT) Kcnj6 (GIRK2) Scn2a1 (Nav1.2) 16 16 16 r=0.852, p<0.001, n=111 r=0.706, p<0.001, n=106 r=0.786, p<0.001, n=105 r=0.721, p<0.001, n=105 14 14 n=1 n=2 14 r=0.542, p=0.055, n=13 r=-0.07, p=0.816, n=14 12 12 12 10 10 10 8 8 8 6 6 6 4 4 4 Kcnj6 (GIRK2) Kcnn3 (SK3) 2 2 Kcnj6a (GIRK2) 2 r=0.768, p<0.001, n=109 0 0 0 r=-0.521, p=0.1235, n=10 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Slc6a3 (DAT) Drd2 (D2R) Kcnj6 (GIRK2) Scn2a1 (Nav1.2) Slc18a2 (VMAT2) 16 16 16 r=0.785, p<0.001, n=110 r=0.745, p<0.001, n=111 r=0.783, p<0.001, n=106 r=0.711, p<0.001, n=106 14 14 14 r=0.686, p< 0.001, n=30 r=0.38, p=0.024, n=35 r=0.37, p=0.044, n=30 r=0.451, p=0.046, n=20 12 12 12 10 10 10 2R) 8 D 8 8 6 6 6 4 Drd2( 4 4 Kcnd2 (Kv4.2) Scn2a1 (Nav1.2) 2 2 2 0 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Map2 (MAP2) Ncam2 (NCAM2) Th (TH) Slc6a3 (DAT) Slc17a6 (VGLUT2) 16 16 16 r= 0.757, p<0.001, n=111 r=0.736, p<0.001, n=109 r=0.708, p<0.001, n=109 r=-0.848, p=0.033, n=6 14 14 14 n=3 n=1 n=3 12 12 12 10 10 10 8 8 8 6 6 6 4 4 4 r= 0.775, p<0.001, n=111 Kcnj6 (GIRK2) Slc18a2 (VMAT2) 2 2 r=0.436, p=0.013, n=32 2 Cacna1d (Cav1.3) 0 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Scn2a1 (Nav1.2) Th (TH) Slc6a3 (DAT) Th (TH) Scn8a (Nav1.6) nDA neurons 16 16 16 r=-0.054, p=0.653, n=71 r=0.107, p=0.362, n=74 r=0.285, p=0.045, n=50 r=0.306, p=0.003, n=92 r=0.405, p<0.001, n=92 14 14 14 r=-0.784, p=0.007, n=10 r=0.710, p=0.048, n=8 r=0.735, p=0.004, n=13 r=0.631, p<0.001, n=31 r=0.609, p<0.001, n=30 12 12 12 10 10 10 8 8 8 6 6 6 4 4 4 Hcn2 (HCN2) Kcna2 (Kv1.2) Kcnd3_2 (Kv4.3) 2 2 2 0 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Calb1 (CB) Kcnj11 (Kir6.2) Abcc8 (SUR1) Nefm (NEF3) Nefm (NEF3) 16 16 16 r=0.366, p=0.008, n=51 r=0.331, p<0.001, n=101 r=-0.401, p=0.001, n=62 r=0.48, p<0.001,n=51 r= 0.492, p<0.001, n=62 14 14 14 r=0.628, p=0.016, n=14 r=0.622, p<0.001, n=26 r= 0.616, p<0.001, n=28 r=0.647, p=0.012, n=14 r=-0.634, p=0.002, n=23 12 12 12 10 10 10 8 8 8 6 6 6 4 4 4 Map2 (MAP2) 2 2 2 Scn2a1 (Nav1.2) Ncam2 (NCAM2) 0 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Abcc8 (SUR1) Kcnb1 (Kv2.1) Slc17a6 (VGLUT2) Abcc8 (SUR1) Slc17a6 (VGLUT2) 16 16 16 r=0.501, p<0.001, n=95 r=0.376, p=0.006, n=51 r=0.308, p=0.028, n=51 r=0.164, p=0.295, n=43 r=0.36, p=0.006, n=58 14 14 14 r=0.616, p=0.002, n=23 r=0.653, p=0.016, n=13 r=0.875, p=0.010, n=7 r=0.621, p=0.023, n=13 r=0.663, p=0.01, n=14 12 12 12 10 10 10 8 8 8 6 6 6 4 4 4 Hcn2 (HCN2) 2 Abcc8 (SUR1) 2 2 Ncam2 (NCAM2) 0 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Fos (C-FOS) Abcc8 (SUR1) Kcnd3_2 (Kv4.3) Scn8a (Nav1.6) Creb1 (CREB) 16 16 16 r=0.663, p<0.001, n=38 r=0.078, p=0.521, n=69 14 14 r=0.668, p=0.049, n=9 14 r=0.61, p=0.009, n=17 12 12 12 10 10 10 8 8 8 6 6 6 4 4 4 Nefm (NEF3) Gad2 (GAD2) r= 0.55, p<0.001, n=47 r=0.53, p<0.001, n=54 2 2 Kcnj6 (GIRK2) 2 r=-0.676, p=0.008, n=14 r=0.662, p=0.007, n=15 r=-0.084, p=0.517, n=62 0 0 0 r=0.645, p<0.001, n=29 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Fos (C-FOS) Gad1 (GAD1) Creb1 (CREB) Hcn4 (HCN4) Slc17a6 (VGLUT2) Supplementary Figure 4.
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