Article Figures & Data
Figures
- Figure 1.
D1+ and D2+ cells are distinctly distributed through layers of the motor cortex. A, Coronal sections obtained from a Drd1aCre mouse injected with DIO-mCherry virus and a Drd2Cre::Ai14 mouse showing laminar distribution of D1+ and D2+ neurons across layers of M1. Laminar boundaries are designated with white dashed lines. WM, white matter. Scale bar, 100 µm (B, C) Laminar distribution of fluorescently labeled D1+ and D2+ neurons. B, Somatic distance is measured in normalized units (0, pia; 1, white matter), cell number per unit is normalized to a total number of fluorescently labeled cells within the section. C, Distribution in layers I, II/III, and V–VI represented as a percentage of fluorescently labeled neurons in all layers. D, A coronal section obtained from a double transgenic Drd1a-tdTomato::Drd2Cre mouse injected with DIO-EYFP virus. Insets show single- (tdTomato or EYFP) and double-labeled cells. Scale bar, main image 100 µm, inset 50 µm. E, Venn diagram showing the overlap between Drd1a (tdTomato-positive) and Drd2 (EYFP-positive) expressing neurons. B, C, E, Slices were obtained from Drd1aCre, Drd2Cre::Ai14, and Drd1a-tdTomato::Drd2Cre mice (n = 3 mice per group, n = 4 slices per animal). B, C, Results are displayed as mean ± SEM. C, Bonferroni's post hoc test; **p < 0.01, ***p < 0.001.
- Figure 2.
D1+ and D2+ neurons are primarily glutamatergic and GABAergic neuronal populations. A, Coronal sections obtained from a Drd1a-tdTomato and Drd2Cre::Ai14 mouse showing tdTomato mRNA expression across layers of M1. Laminar boundaries are designated with white dashed lines. WM, white matter. Scale bar, 100 µm. Area-proportional Euler diagrams represent proportions and relationships between clusters of neurons identified based on mRNA expression. B, Representative images showing colocalization of tdTomato (pink), vGlut1 (blue) or vGAT1 (yellow), and either D1 (green) or D2 (turquoise) receptors mRNA within an individual neuron. Scale bar, 10 µm. C, Proportion of neurons coexpressing vGlut1, vGAT1, or mRNAs for both transporters represented as a percentage of all tdTomato+ labeled neurons. D, Proportion of neurons coexpressing vGlut1, vGAT1, or mRNAs for both transporters represented as a percentage of tdTomato+ neurons found in layer II/III (left) or layers V–VI (right). C, D, Slices are obtained from Drd1a-tdTomato and Drd2Cre::Ai14 mice (n = 2 mice per group, n = 2 slices per animal). Results are displayed as mean ± SEM. Bonferroni's post hoc test; ***p < 0.001, **p < 0.01, *p < 0.05.
- Figure 3.
Layer V D1+ and D2+ pyramidal neurons exhibit unique electrophysiological properties. A, An example of layer V D1+ and D2+ pyramidal neurons and recording patch-clamp pipette in differential interference contrast (DIC) and fluorescence modes. Slices were obtained from Drd1a-tdTomato (top panels) and Drd2Cre::Ai14 (bottom panels) mice. Scale bar, 10 µm. B, Stimulation protocols (top panels) and corresponding current and voltage responses (bottom panels) of exemplary layer V pyramidal neuron. Voltage (left) and current (middle) steps protocols and ramp current protocol (right). C, The current–voltage (I-V) relationship of D1+ and D2+ neurons, measured from the steady-state current responses to voltage step pulses shown in B (left panel). D1+ and D2+ neurons displayed highly linear steady-state I–V relationship (R2 = 0.95 for both groups). D, Membrane properties of D1+ and D2+ neurons. Membrane potential was recorded in 0 current-clamp mode. Resistance, capacitance, and time constant (tau) were measured from the voltage response to a −50 pA hyperpolarizing current pulse. Voltage sag was measured from the voltage response to a −250 pA hyperpolarizing current step marked in red in B (middle panel). E, The input–output (I–O) relationship reflecting the excitability of D1+ and D2+ neurons, quantified by measuring the number of AP elicited by incremental current pulses (B, middle panel). D1+ and D2+ neurons displayed a highly linear I–O relationship (R2 = 0.99 for D1+ and 0.97 for D2+). F, D1+ and D2+ neurons exemplary waveforms of a single, evoked AP elicited from membrane potential of −75 mV, with a 0.5 ms rectangle current injection. G, AP properties of D1+ and D2+ neurons. C–G, Overall, n = 18 D1+ (from n = 9 mice) and n = 19 D2+ (from n = 7 mice) neurons were tested during patch-clamp recordings. Results are displayed as mean ± SEM. Bonferroni's post hoc test or unpaired t test; ***p < 0.001, **p < 0.01, *p < 0.05.
- Figure 4.
Layer V D2+ pyramidal neurons have more complex basal dendrite morphology. A, B, Representative images of biocytin-filled layer V pyramidal D1+ and D2+ neurons (left panels) and 2D reconstruction of their basal dendrites (right panels). Scale bar, 50 µm. C, D, Representative image of layer V pyramidal cell soma and measured widths of the shafts of n = 5 D1+ (from n = 4 mice) and n = 7 D2+ (from n = 4 mice) neuron dendrites. Scale bar, 10 µm. E, Representation of the Sholl analysis—quantification made by superimposing a series of concentric circles of gradually increasing radius around the soma. The Sholl intersection profile, obtained by counting the number of dendritic branches at a given distance from the soma. F, Morphological parameters of n = 6 D1+ (from n = 6 mice) and n = 6 D2+ (from n = 5 mice) neurons. D–F, Results are displayed as mean ± SEM. Fisher's LSD post hoc test or unpaired t test; **p < 0.01, *p < 0.05.
- Figure 5.
D1+ and D2+ neurons have extensive projection patterns. A–F, Coronal sections obtained from Drd1aCre and Drd2Cre mice injected with DIO-mCherry, red arrows indicate the injection site (replicated in n = 3 animals from each strain). A, Forebrain (0.50 from Bregma): DLS, dorsolateral striatum; M1, primary motor cortex. Scale bar, 500 µm. B, Forebrain (−1.34 from Bregma): S2, secondary somatosensory cortex; THAL, thalamus (posterior and ventral thalamic nuclear groups); IC, internal capsule. Scale bar, 500 µm. C, Midbrain (−2.70 from Bregma): CP, cerebral peduncle. Scale bar, 500 µm. D, Pons (−4.24 from Bregma): PN, pontine nucleus. Scale bar, 200 µm. E, Medulla (−5.80 from Bregma): PY, pyramidal tract. Scale bar, 100 µm. F, Medulla (−8.12 from Bregma): PYX, pyramidal decussation. Scale bar, 200 µm (left panel), 100 µm (right panel).
- Figure 6.
Chemogenetic inhibition of D2+ cells disrupts skilled grasping. A, Timeline for the injection of inhibitory DREADDs (DIO-hM4Di-mCherry) and motor skill training experiment. B, Representative images showing the pellet reaching sequence. Color circles indicate labeled body parts: nose, trained forelimb (contralateral to the inhibited motor cortex), and food pellet, which were further used for trajectory analysis. C, Coronal sections showing exemplary expression of hM4Di-mCherry in D1+ and D2+ neurons in M1. Scale bar, 100 µm. D, Success rate expressed as a percentage of trials in which the pellet was successfully retrieved. E, Reach failure represented as a percentage of reach outcomes (“no-grab,” “miss,” or “drop”) calculated from the total number of reaching attempts. F, Trajectory analysis of the first reach attempts in the trial, from the moment the paw was lifted from the ground until it touched the pellet. Kinematic variables analyzed: paw distance to pellet and velocity. G, Analysis of the distance traveled and velocity during the exploration of the open field. D–G, Drd1aCre n = 5, wild-type n = 7; Drd2Cre n = 7, wild-type n = 7 (WT = wild-type). Results are displayed as mean ± SEM. Bonferroni's post hoc test; **p < 0.01. Extended Data Figure 6-1 shows additional details on the validation of chemogenetic inhibition of neuronal activity in Drd1aCre and Drd2Cre mice.
Tables
Figure Type of test Statistical values Fig. 1C 1-way ANOVA D1+: F(2,33) = 1,219, p < 0.001
D2+: F(2,33) = 76.61, p < 0.001
Fig. 2C 1-way ANOVA D1+: F(2,9) = 660.7, p < 0.001
D2+: F(2,9) = 134.4, p < 0.001
Fig. 2D, II/III 1-way ANOVA D1+: F(2,9) = 3.74, p = 0.066
D2+: F(2,9) = 199, p < 0.001
Fig. 2D, V-VI 1-way ANOVA D1+: F(2,9) = 933, p < 0.001
D2+: F(2,9) = 88.7, p < 0.001
Fig. 3C Linear regression D1+: R2 = 0.95, F(1,5) = 91.34, p = 0.0002
D2+: R2 = 0.95, F(1,5) = 104.5, p = 0.0002
slopes difference: F(1,234) = 13.08, p = 0.0004
Fig. 3C 2-way RM ANOVA cell type: F(1,32) = 9.35, p = 0.005
voltage: F(6,192) = 132.1, p < 0.001
cell type x voltage: F(6,192) = 3.25, p = 0.005
Fig. 3D, Potential Unpaired t test t(18) = 1.19, p = 0.249 Fig. 3D, Resistance Unpaired t test t(28) = 0.78, p = 0.439 Fig. 3D, Capacitance Unpaired t test t(27) = 1.32, p = 0.178 Fig. 3D, Tau Unpaired t test t(28) = 1.62, p = 0.116 Fig. 3D, Sag Unpaired t test t(28) = 2.06, p = 0.049 Fig. 3D, Rheobase Unpaired t test t(26) = 2.53, p = 0.018 Fig. 3E Linear regression D1+: R2 = 0.99, F(1,10) = 1856, p < 0.001
D2+: R2 = 0.97, F(1,11) = 316.1, p < 0.001
slopes difference: F(1,238) = 6.50, p = 0.011
Fig. 3G, Half width Unpaired t test t(22) = 3.27, p = 0.004 Fig. 3G, AHP Unpaired t test t(29) = 2.87, p = 0.007 Fig. 3G, 10-90 rise time Unpaired t test t(22) = 1.18, p = 0.253 Fig. 3G, Amplitude Unpaired t test t(22) = 0.27, p = 0.789 Fig. 3G, Peak to AHP Unpaired t test t(29) = 1, p = 0.324 Fig. 3G, Threshold Unpaired t test t(22) = 0.1, p = 0.922 Fig. 4D Unpaired t test t(10) = 1.3, p = 0.222 Fig. 4E 2-way RM ANOVA cell type: F(1,10) = 9.48, p = 0.012
distance: F(23,230) = 11.10, p < 0.001
cell type x distance: F(23,230) = 1.95, p = 0.007
Fig. 4F, No. of primary dendrites Unpaired t test t(10) = 2.35, p = 0.041 Fig. 4F, No. of branches Unpaired t test t(10) = 2.08, p = 0.064 Fig. 4F, No. of bifurcations Unpaired t test t(10) = 1.85, p = 0.095 Fig. 4F, No. of dendritic tips Unpaired t test t(10) = 2.27, p = 0.047 Fig. 4F, Maximal branch order Unpaired t test t(10) = 0.77, p = 0.458 Fig. 4F, Total dendritic length Unpaired t test t(10) = 3.81, p = 0.004 Fig. 6D, Drd1aCre 2-way RM ANOVA genotype: F(1, 10) = 0.24, p = 0.636
treatment: F(1,10) = 0.2, p = 0.663
genotype x treatment: F(1,10) = 0.09, p = 0.765
Fig. 6D, Drd2Cre 2-way RM ANOVA genotype: F(1,12) = 0.01, p = 0.928
treatment: F(1,12) = 12.77, p = 0.004
genotype x treatment: F(1,12) = 1.72, p = 0.214
Fig. 6E, Drd1aCre No-grab 2-way RM ANOVA genotype: F(1,10) = 0.1, p = 0.763
treatment: F(1,10) = 0.01, p = 0.93
genotype x treatment: F(1,10) = 0.03, p = 0.859
Fig. 6E, Drd1aCre Miss 2-way RM ANOVA genotype: F(1,10) = 0.21, p = 0.66
treatment: F(1,10) = 0.27, p = 0.614
genotype x treatment: F(1,10) = 2.23, p = 0.166
Fig. 6E, Drd1aCre Drop 2-way RM ANOVA genotype: F(1,10) = 0.49, p = 0.499
treatment: F(1,10) = 0.57, p = 0.469
genotype x treatment: F(1,10) = 1.05, p= 0.33
Fig. 6E, Drd2Cre No-grab 2-way RM ANOVA genotype: F(1,12) = 0.29, p = 0.599
treatment: F(1,12) = 15.34, p = 0.002
genotype x treatment: F(1,12) = 1.16, p = 0.303
Fig. 6E, Drd2Cre Miss 2-way RM ANOVA genotype: F(1,12) = 1.06, p = 0.323
treatment: F(1,12) = 2.77, p = 0.122
genotype x treatment: F(1,12) = 0.02, p = 0.9
Fig. 6E, Drd2Cre Drop 2-way RM ANOVA genotype: F(1,12) = 0.02, p = 0.887
treatment: F(1,12) = 0.28, p = 0.609
genotype x treatment: F(1,12) = 2.06, p = 0.177
Fig. 6F, Drd1aCre Distance 2-way RM ANOVA genotype: F(1,10) = 0.01, p = 0.916
treatment: F(1,10) = 4, p = 0.074
genotype x treatment: F(1,10) = 1.06, p = 0.328
Fig. 6F, Drd1aCre Velocity 2-way RM ANOVA genotype: F(1,10) = 4.95, p = 0.05
treatment: F(1,10) = 0.21, p = 0.658
genotype x treatment: F(1,10) = 0.1, p = 0.762
Fig. 6F, Drd2Cre Distance 2-way RM ANOVA genotype: F(1,12) = 0.12, p = 0.739
treatment: F(1,12) = 3.87, p = 0.073
genotype x treatment: F(1,12) = 1.31, p = 0.274
Fig. 6F, Drd2Cre Velocity 2-way RM ANOVA genotype: F(1,12) = 0.57, p = 0.464
treatment: F(1,12) = 0.55, p = 0.472
genotype x treatment: F(1,12) = 0.07, p = 0.795
Fig. 6G, Drd1aCre Distance Unpaired t test t(10) = 0.82, p = 0.43 Fig. 6G, Drd1aCre Velocity Unpaired t test t(10) = 0.83, p = 0.429 Fig. 6G, Drd2Cre Distance Unpaired t test t(12) = 0.62, p = 0.547 Fig. 6G, Drd2Cre Velocity Unpaired t test t(12) = 0.61, p = 0.551 - Table 2.
Combinations of mRNA species detected in M1 slices obtained from Drd1a-tdTomato and Drd2Cre::Ai14 mice
mRNA Combinationa Drd1a-tdTomato Drd2Cre::Ai14 Layer II-III [Mean ± SEM (%)] Layers V-VI [Mean ± SEM (%)] Layer II-III [Mean ± SEM (%)] Layers V-VI [Mean ± SEM (%)] All vGlut1 62 ± 10 (100) 52 ± 5 (100) 67 ± 6 (100) 48 ± 4 (100) vGlut1 only 53 ± 9 (85) 12 ± 2 (23) 42 ± 5 (63) 44 ± 3 (92) vGlut1/tdTomatob 9 ± 5 (15) 40 ± 6 (77) 25 ± 3 (37) 4 ± 1 (8) All vGAT1 10 ± 1 (100) 8 ± 2 (100) 12 ± 2 (100) 9 ± 1 (100) vGAT1 only 3 ± 1 (30) 2 ± 1 (25) 8 ± 2 (67) 5 ± 1 (56) vGAT1/tdTomatob 7 ± 1 (70) 6 ± 1 (75) 4 ± 1 (33) 4 ± 1 (44) All vGlut1/vGAT1 1 ± 1 (100) 0 (0) 2 ± 1 (100) 1 ± 0.3 (100) vGlut1/vGAT1 only 0 (0) 0 (0) 1 ± 0.3 (50) 1 ± 0.2 (100) vGlut1/vGAT1/tdTomatob 1 ± 1 (100) 0 (0) 1 ± 1 (50) 0 (0) All tdTomatob 18 ± 5 (100) 46 ± 5 (100) 30 ± 2 (100) 9 ± 1 (100) tdTomato onlyb 15 ± 5 (83) 44 ± 5 (95) 29 ± 3 (96) 3 ± 1 (39) tdTomato/D2b,c 3 ± 1 (17) 2 ± 1 (5) – – tdTomato/D1b,c – – 1 ± 1 (4) 6 ± 1 (61) ↵a The data presented in the table correspond to the Euler diagrams shown in Fig. 2A.
↵b tdTomato signifies D1+ neurons in Drd1a-tdTomato mice or D2+ neurons in Drd2Cre::Ai14 mice.
↵c Counting of Drd1a expressing cells in Drd1a-tdTomato mice and Drd2 expressing in Dr2Cre::Ai14 mice was not performed due to the confounding effects of partial receptor sequences included in the transgenes.
Figure 6-1
Validation of chemogenetic inhibition of neuronal activity in Drd1aCre and Drd2Cre mice using striatal neurons as an example. (A) Coronal section obtained from a wild-type (left) and Drd1aCre mouse (right) showing slice placement upon the multi-electrode array (black dots indicate single recording electrodes) and hM4Di-mCherry expression in the striatum after recording. Scale bars, 200 μm. (B) Table summarizing recorded neurons and their responses to CNO application. (C) Temporal heatmaps encoding single-unit activity (SUA) of all recorded neurons and their response to CNO application (10 μM, 10 ml; indicated by a black line). Each row indicates a single unit. White dotted horizontal lines classify units to either Drd1aCre mice (n = 46 units), Drd2Cre mice (n = 34), or wild-type mice (n = 35). The cell activity was normalized from 0 to 1 and was sorted by the defined window corresponding to the CNO response. Bin, 30 s. (D) Examples of neurons recorded during CNO administration in Drd1aCre, Drd2Cre and wild-type mice. Top panels show separated spikes of a single neuron, bottom panels show a corresponding frequency histogram. The orange-shaded rectangle indicates the duration of CNO action. Bin, 60 s. (B-D) Slices were obtained from n = 1 hM4Di-mCherry injected animal for each group, n = 4 slices per animal. Download Figure 6-1, TIF file.
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