Article Figures & Data
Figures
- Figure 1.
Whole-cell recordings of excitatory neurons in forelimb M1 were localized to L2/3 and L5. A, Schematic showing the anterior–posterior span of recorded slices, restricted to the forelimb area of M1. B, Recorded neurons were visualized with streptavidin labeling of biocytin and confirmed as excitatory by negative immunoreactivity for GAD67. GAD67 and merged images are shown at one z-plane depth; biocytin images are shown as a collapsed stack spanning the entire neuron. Open arrows, GAD67– biocytin-filled neurons; closed arrows, neighboring GAD67+ interneurons (not recorded) at the same depth. Scale bar, 50 μm. C, Histologic staining of cytoarchitecture used to define cortical layers. Hoechst 33342 stain is a nuclear counterstain of all cells in the region, Neurotrace was used as a neuron-specific stain for somata, SMI-32 labels a subset of pyramidal neurons in layer 3 and layer 5. Scale bar, 200 μm. Right-most panel, Two example neurons localized to L2/3 and L5; neurons localized within 8–35% of the total cortical depth were defined as L2/3; neurons within 35–76% of cortical depth were defined as L5.
- Figure 2.
Acute D1R blockade shifts excitability of M1 neurons. A, Superimposed responses to hyperpolarizing and depolarizing current steps in individual L2/3 and L5 neurons before and after bath application of D1R antagonist SCH23390 (D1ant, 10 μm). Scale bar: Top, 20 mV, 100 ms; bottom: 10 mV, 100 ms. B–F, Summary excitability plots for excitatory neurons in L2/3 (green) and L5 (blue) before and after D1ant application, in baseline (ACSF) or synaptic blocker (BLK: 20 μm picrotoxin, 20 μm DNQX, 50 μm AP5) conditions. Modified Cumming plots show raw data of individual neurons as lines, overlayed with the mean ± SEM. To the right of each group of raw data are the effect size (black circle), corresponding 95% CIs (black vertical bars), and the underlying bootstrap sampling distribution. B, Dynamic input resistance across hyperpolarizing current steps. C, Voltage dependence of Ih-mediated voltage sag elicited by hyperpolarizing current. D, Action potential threshold at rheobase. E, Action potential half-width at rheobase. F, Action potential frequency during suprathreshold current injections (ACSF L2/3 neurons: N = 6, n = 12; ACSF L5 neurons: N = 6, n = 11; BLK L2/3 neurons: N = 5, n = 10; BLK L5 neurons: N = 6, n = 10. Data are shown as the mean ± SEM. *p ≤ 0.05.
- Figure 3.
Acute D2R blockade shifts excitability of M1 neurons. A, Superimposed responses to hyperpolarizing and depolarizing current steps in individual L2/3 (green) and L5 (blue) neurons before and after bath application of D2R antagonist sulpiride (D2ant, 10 μm) Scale bar: Top, 20 mV, 100 ms; bottom, 10 mV, 100 ms. B–F, Summary excitability plots for excitatory neurons in L2/3 and L5 before and after D2ant application, in baseline (ACSF) or synaptic blocker (BLK: 20 μm picrotoxin, 20 μm DNQX, 50 μm AP5) conditions. Modified Cumming plots show raw data of individual neurons as lines, overlayed with the mean ± SEM. To the right of each group of raw data are the effect size (black circle), corresponding 95% CIs (black vertical bars), and the underlying bootstrap sampling distribution. B, Dynamic input resistance across hyperpolarizing current steps. C, Voltage dependence of Ih-mediated voltage sag elicited by hyperpolarizing current. D, Action potential threshold at rheobase. E, Action potential half-width at rheobase. F, Action potential frequency during suprathreshold current injections. ACSF L2/3 neurons: N = 5, n = 11; ACSF L5 neurons: N = 5, n = 10; Blk L2/3 neurons: N = 4, n = 8; Blk L5 neurons: N = 4, n = 8. Data are shown as mean ± SEM. *p ≤ 0.05.
- Figure 4.
Combined D1R+D2R blockade in dopamine-primed slices of M1 recapitulates the results of individual antagonist experiments. A, Superimposed responses to hyperpolarizing and depolarizing current steps in individual L2/3 (green) and L5 (blue) neurons before and after bath application of SCH23390 (D1ant) and Sulpiride (D2ant). Scale bar: Top, 20 mV, 100 ms; bottom, 10 mV, 100 ms. B–F, Summary excitability plots for excitatory neurons in L2/3 and L5 before and after D1ant+D2ant application, in dopamine (10 μm) ACSF. Modified Cumming plots show raw data of individual neurons as lines, overlayed with the mean ± SEM. To the right of each group of raw data are the effect size (black circle), corresponding 95% CIs (black vertical bars), and the underlying bootstrap sampling distribution. B, Dynamic input resistance across hyperpolarizing current steps. C, Action potential threshold at rheobase. D, Action potential half-width at rheobase. E, Voltage dependence of Ih-mediated voltage sag elicited by hyperpolarizing current. F, Action potential frequency during suprathreshold current injections. DOPA L2/3 neurons: N = 7, n = 11; DOPA L5 neurons: N = 5, n = 10. Data are shown as the mean ± SEM. *p ≤ 0.05.
- Figure 5.
Validation of the 6OHDA model of Parkinson’s disease. A, Unilateral injection of 6OHDA or vehicle, centered on the SNc. B, Schematic of the cylinder motor assessment. C, Quantification of weight-bearing wall touches measured as a ratio of forelimb use contralateral versus ipsilateral to the injected hemisphere. D, Immunolabeled TH+ dopaminergic neurons visualized with DAB in the SNc and VTA. E, Summary of stereological counts of TH+ neurons in the SNc and VTA of lesioned or vehicle-injected animals. (vehicle animals, N = 18; 6OHDA animals, N = 17; data are shown as the mean ± SEM. *p < 0.0001. F, Summary stereological counts of TH+ boutons in M1 of a subset of animals. Vehicle animals, N = 2; 6OHDA animals, N = 2. Data are shown as the mean ± SEM. *p ≤ 0.05.
- Figure 6.
Nigral 6OHDA lesion shifts M1 neuron excitability, partially recapitulating the effects of D1R antagonist. A, Superimposed responses to hyperpolarizing and depolarizing current steps in individual L2/3 (green) and L5 (blue) neurons of vehicle- and 6OHDA-injected animals. All traces shown are in ACSF conditions. Scale bar: Top, 20 mV, 100 ms; bottom, 10 mV, 100 ms. B–F, Summary excitability plots for excitatory neurons in L2/3 and L5 of vehicle and 6OHDA animals, in baseline (ACSF) or synaptic blockers (BLK; 20 μm picrotoxin, 20 μm DNQX, 50 μm AP5) conditions. Modified Cumming plots show raw data of individual neurons as swarm plots, with the mean ± SEM offset to the right. Further right of each group of raw data are the effect size (black circle), corresponding 95% CIs (black vertical bars), and the underlying bootstrap sampling distribution. B, Dynamic input resistance across hyperpolarizing current steps. C, Voltage dependence of Ih-mediated voltage sag elicited by hyperpolarizing current. D, Action potential threshold at rheobase. E, Action potential half-width at rheobase. F, Action potential frequency during suprathreshold current injections. ACSF L2/3 vehicle neurons: N = 11, n = 16; ACSF L2/3 6OHDA neurons: N = 13, n = 21; BLK L2/3 vehicle neurons: N = 3, n = 7; BLK L2/3 6OHDA neurons: N = 4, n = 8; ACSF L5 vehicle neurons: N = 11, n = 29; ACSF L5 6OHDA neurons: N = 11, n = 29; BLK L5 vehicle neurons: N = 5, n = 14; BLK L5 6OHDA neurons: N = 6, n = 12. Data are shown as the mean ± SEM. *p ≤ 0.05.
- Figure 7.
Chronic M1 dopamine depletion impacts L2/3 intrinsic excitability. A, Unilateral injection of 6OHDA or vehicle into forelimb region of M1. B, Quantification of weight-bearing wall touches in cylinder motor assessment. C, TH+ axons and boutons labeled in L2/3 of M1 ipsilateral and contralateral to the injection site. Magnification, 40×. Scale bar, 50 μm. D, Stereological counts of TH+ boutons in M1 in lesioned (N = 4) or vehicle-injected (N = 5) animals. E, Stereological counts of TH+ neurons in the SNc and VTA of a subset of animals (vehicle, N = 4; 6OHDA, N = 4). F–K, Summary excitability plots for excitatory neurons in L2/3 (green) and L5 (blue) of vehicle or 6OHDA-injected animals, performed in synaptic blockers (BLK: 20 μm picrotoxin, 20 μm DNQX, 50 μm AP5). Modified Cumming plots show raw data of individual neurons as swarm plots, with the mean ± SEM offset to the right. Further right of each group of raw data are the effect size (black circle), corresponding 95% CIs (black vertical bars), and the underlying bootstrap sampling distribution. F, Superimposed responses to hyperpolarizing and depolarizing current steps in individual L2/3 and L5 neurons in vehicle- or 6OHDA-injected animals. Scale bar: Top, 20 mV, 100 ms; bottom, 10 mV, 100 ms. G, Dynamic input resistance across hyperpolarizing current steps. H, Action potential threshold at rheobase. I, Action potential half-width at rheobase. J, Voltage dependence of Ih-mediated voltage sag elicited by hyperpolarizing current. K, Action potential frequency during suprathreshold current injections. Vehicle L2/3 neurons: N = 4, n = 12; vehicle L5 neurons: N = 5, n = 16; 6OHDA L2/3 neurons: N = 4, n = 14; 6OHDA L5 neurons: N = 4, n = 16. Data are shown as the mean ± SEM. *p ≤ 0.05.
Tables
Experiment Parameter n Mean
differenceCI lower
limitCI upper
limitp Value Permutation t test Student’s t test ACSF D1ant DIR 12 29.15 13.25 46.09 0.010 0.007 AP threshold 12 –1.86 –2.89 –0.83 0.009 0.007 AP half-width 12 0.11 0.05 0.21 0.011 0.017 BLK D1ant DIR 12 62.99 48.57 95.31 0.001 0.000 AP threshold 10 –1.90 –3.69 –0.58 0.049 0.048 AP half-width 10 0.07 0.02 0.12 0.024 0.024 ACSF D2ant DIR 11 20.22 8.05 35.17 0.016 0.019 AP threshold 11 –1.50 –2.24 –0.61 0.007 0.006 AP half-width 11 –0.01 –0.04 0.03 0.476 0.472 BLK D2ant DIR 8 10.84 1.70 25.40 0.152 0.126 AP threshold 8 –0.89 –2.62 1.16 0.392 0.403 AP half-width 8 –0.03 –0.06 0.00 0.125 0.113 DOPA D1+D2ant DIR 11 26.52 8.57 54.42 0.032 0.046 AP threshold 11 –1.99 –2.69 –1.47 0.000 0.000 AP half-width 11 0.04 0.01 0.07 0.050 0.042 SNc 6OHDA ACSF Vehicle = 16 DIR 6OHDA = 21 –9.64 –46.15 28.89 0.595 0.609 Vehicle = 16 AP threshold 6OHDA = 21 0.68 –1.36 2.49 0.501 0.504 Vehicle = 16 AP half-width 6OHDA = 21 0.07 –0.05 0.20 0.305 0.303 SNc 6OHDA BLK Vehicle = 7 DIR 6OHDA = 8 4.78 –82.51 83.74 0.907 0.917 Vehicle = 7 AP threshold 6OHDA = 7 1.50 –2.33 5.91 0.534 0.527 Vehicle = 7 AP half-width 6OHDA = 7 0.13 –0.07 0.35 0.310 0.302 M1 6OHDA BLK Vehicle = 12 DIR 6OHDA = 14 2.90 –31.25 41.27 0.877 0.884 Vehicle = 12 6OHDA = 12 2.35 –0.12 4.53 0.063 0.067 Vehicle = 12 AP half-width 6OHDA = 12 –0.18 –0.33 –0.01 0.053 0.050 Permutation t test p values are listed alongside Student’s t test p values for comparison. Bold p values indicate a p value ≤ 0.05. AP, Action potential; DIR, dynamic input resistance.
Experiment Parameter n Mean
differenceCI lower
limitCI upper
limitp Value Permutation t test Student’s t test ACSF D1ant DIR 11 16.95 8.42 27.31 0.002 0.007 AP threshold 11 –1.30 –2.10 –0.58 0.007 0.009 AP half-width 11 0.02 0.00 0.04 0.067 0.065 BLK D1ant DIR 12 45.56 30.50 65.71 0.000 0.000 AP threshold 10 –1.66 –3.06 –0.38 0.058** 0.047 AP half-width 10 0.03 0.00 0.06 0.092 0.099 ACSF D2ant DIR 10 9.91 4.09 22.43 0.023** 0.053 AP threshold 10 –2.59 –3.76 –1.40 0.003 0.003 AP half-width 10 –0.04 –0.06 –0.02 0.013 0.018 BLK D2ant DIR 8 16.94 9.55 36.71 0.000 0.027 AP threshold 8 –2.24 –4.83 –0.95 0.024** 0.055 AP half-width 8 –0.06 –0.08 –0.04 0.000 0.000 DOPA D1+D2ant DIR 10 11.08 2.36 20.04 0.042 0.047 AP threshold 10 –1.21 –1.71 –0.69 0.004 0.002 AP half-width 10 0.04 0.01 0.06 0.017 0.017 SNc 6OHDA ACSF Vehicle = 29 DIR 6OHDA = 29 –2.55 –23.05 15.48 0.808 0.803 Vehicle = 29 AP threshold 6OHDA = 28 0.67 –0.81 2.15 0.391 0.392 Vehicle = 29 AP half-width 6OHDA = 28 0.08 –0.03 0.19 0.177 0.173 SNc 6OHDA BLK Vehicle = 14 DIR 6OHDA = 12 –26.11 –71.51 –1.13 0.165 0.168 Vehicle = 14 AP threshold 6OHDA = 11 0.05 –2.25 2.56 0.970 0.971 Vehicle = 14 AP half-width 6OHDA = 11 –0.06 –0.19 0.05 0.391 0.396 M1 6OHDA BLK Vehicle = 16 DIR 6OHDA = 16 –9.21 –37.70 16.81 0.532 0.519 Vehicle = 16 AP threshold 6OHDA = 16 0.64 –1.37 2.70 0.547 0.542 Vehicle = 16 AP half-width 6OHDA = 16 –0.06 –0.20 0.07 0.434 0.416 Permutation t test p values are listed alongside Student’s t test p values for comparison. Bold p values indicate p ≤ 0.05. AP, Action potential; DIR, dynamic input resistance.
**Instances where one statistics test was over/under the 0.05 p value threshold when the other was not.
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