Recovery of the full in vivo firing range in post-lesion surviving DA SN neurons associated with Kv4.3-mediated pacemaker plasticity
- Institute for Neurophysiology, Neuroscience Center, Goethe University Frankfurt, Germany
- Department of Neurology, University Medical Centre of the Johannes Gutenberg University Mainz, Germany
- Neuroscience Center at Fundación de Investigación HM Hospitales, CEU San Pablo University, Spain
- Eye Center, Medical Center-University of Freiburg, Germany
- Department of Psychology, University of Alabama, United States
- School of Biology, Indian Institute of Science Education and Research (IISER), India
Figures
Figure 1 with 5 supplements
Unilateral striatal 6-hydroxy-dopamine (6-OHDA) mouse model with stable loss of substantia nigra (SN) dopamine (DA) neurons resulted in delayed partial behavioral recovery.
(A) Experimental design, illustrating timeline of behavioral assays with termination points – groups ended either on the 21st, or later than the 64th post-infusion day. (B) Left panel: TH-DAB staining of the SN, ×10 magnification and ×60 magnification for vehicle and 6-OHDA-infused mouse. Scale bar left 200 µm, right 25 µm. Right panel: ratio of ipsilateral (infusion side) to contralateral side of surviving TH-positive neurons in SN at 21st post-infusion day. (C) Left: spontaneous locomotion of mice in open field arena for a 10 min session. Left: examples of an ACSF-infused mouse (vehicle), right example of a 6-OHDA-infused mouse at baseline (upper panels), 4th post-infusion day (middle panels), and 68th post-infusion day (lower panels). Right: ratio of ipsilateral to contralateral turning behavior for all experimental groups plotted against session days. Note recovery in the 6-OHDA-treated mice from day 4 to day 68 after initial strong turning bias (>90%). (D) Cylinder test quantified by ratio of contra- to ipsilateral forepaw use. Note significant loss of contralateral forepaw involvement, both at 21 and 64 days. All data are presented as mean ± standard error of mean (SEM).
Figure 1—figure supplement 1
Characterization of the unilateral partial striatal 6-hydroxy-dopamine (6-OHDA) model in male mice.
(A, B) Comparison of ipsilateral, infusion side, to contralateral side as percentage of relative TH immunohistochemistry signal in the dorsal striatum (dSTR) and ventral striatum (vSTR), at the corresponding early (A) and late phase (B). Note the stability of TH density loss in the dSTR through time. (A) dSTR, 21st post-infusion day: vehicle 98.4 ± 2.2%, 6-OHDA 42.5 ± 3%, p<0.0001, Mann–Whitney test; vSTR, 21st post- infusion day: vehicle 94.6 ± 1.9%, 6-OHDA 81.4 ± 3.5%, p=0.0082, Mann–Whitney test. (B) dSTR, >64th post-infusion day: vehicle 94.4 ± 2.6%, 6-OHDA 48.3 ± 3.6%, p<0.0001, Mann–Whitney test; vSTR, >64th post-infusion day: vehicle 97.8 ± 1.8%, 6-OHDA 75.2 ± 5%, p=0.0001, Mann–Whitney test. (C) Ratio of ipsilateral (infusion side) to contralateral side of surviving TH-positive neurons in SN in the late phase (vehicle 102.6 ± 7.8%, 6-OHDA 39.9 ± 3.6%, p=0.0262, Mann–Whitney test). (D) Mean track length from all mice for each open field session. Note the post-infusion drop in performed track in the 6-OHDA groups, which gradually recovers. (Infusion day marked as a thin gray line.) Two-way ANOVA, p-value across time p<0.0001, p-value across groups p=0.0001, significant difference between vehicle and 6-OHDA group 4th–52nd day and on 60th, Šídák’s multiple comparisons test. (E) Ratio of TH+ cell loss in SN (ipsilateral [infusion side] to contralateral side)>21st post-infusion day from different infused 6-OHDA doses.
Figure 1—figure supplement 2
Characterization of the unilateral partial striatal 6-hydroxy-dopamine (6-OHDA) model in female mice.
(A) Ratio of ipsilateral to contralateral turning behavior for female ACSF and 6-OHDA-infused groups plotted against session days and with underlaid the corresponding ratio from the male ACSF and 6-OHDA groups as a mean line (two-way ANOVA, p-value across time p<0.0001, p-value across groups p=0.0002, significant difference between vehicle and 6-OHDA group 4th–28th day, Šídák’s multiple comparisons test). (B) Ratio of ipsilateral (infusion side) to contralateral side of surviving TH-positive neurons in SN at >64st post-infusion day from female ACSF and 6-OHDA-infused mice (vehicle 100 ± 4.4%, 6-OHDA 41.1 ± 5.6%, p=0.0002, Mann–Whitney test). (C) Comparison of ipsilateral, infusion side, to contralateral side as percentage of relative TH immunohistochemistry signal in the dorsal striatum (dSTR) at >64st post-infusion day from female ACSF and 6-OHDA-infused mice (vehicle 98.7 ± 2%, 6-OHDA 51.3 ± 4.9%, p=0.0001, Mann–Whitney test).
Figure 1—figure supplement 3
Turning analysis during open field locomotion in the unilateral partial striatal 6-hydroxy-dopamine (6-OHDA) model.
For two representative vehicle- and 6-OHDA-treated mice, schematic representation of turning sequences. Left turn sequences are plotted as negative values, whereas right turn sequences are plotted as positive values. The count represents the number of same direction turns within a sequence. On the left panels are turning sequences for the control animal and on the right panels for the 6-OHDA-infused animal, correspondingly in a baseline session (A), 4th postoperative day (B), 20th postoperative day (C), and 64th postoperative day (D).
Figure 1—figure supplement 4
Ipsi- and contralateral turning features during open field locomotion in the unilateral partial striatal 6-hydroxy-dopamine (6-OHDA) model.
Correlation pair plots comparing turning features, such as velocity (cm/s), diameter (cm), and duration for all contralateral turns (left panels), and all ipsilateral turns performed from all vehicle-infused mice (in orange) and all 6-OHDA-treated mice (in blue) across different time points (baseline, 4th, 20th, and 64th post-infusion day).
Figure 1—figure supplement 5
Analysis of α-synuclein aggreates in the unilateral partial striatal 6-hydroxy-dopamine (6-OHDA) model.
(A, B) Multilabeling immunohistochemistry for α-synuclein-expressing AAV injections in mice as a positive control group for α-synuclein-proteinopathy (i.e., aggregates and phospho-α-synuclein). Top: ×4 magnification of midbrain. Bottom: corresponding ×60 magnification within the SN from 4× image (for A: green, TH; red, α-synuclein aggregate; for B: green, TH; red, phospho-α-synuclein). Scale bars: top, 200 μm; bottom, 20 μm. (C–F) Multilabeling immunohistochemistry for α-synucleinopathy at 21st day (C, D) and after >64 days (E, F) in the 6-OHDA-infused mice (for C and E: green, TH; red, α-synuclein aggregate; for D and F: green, TH; red, phospho-α-synuclein). Notice the absence of α-synucleinopathy in the 6-OHDA-infused mice both at the early and late time points.
Figure 2 with 2 supplements
Surviving substantia nigra (SN) dopamine (DA) neurons at early post-6-hydroxy-dopamine (6-OHDA) phase exhibited a compressed dynamic range with a 10-fold decrease in in vivo bursting and a 5-fold decrease in in vitro pacemaker regularity.
(A, I) Experimental design, illustrating timeline of behavioral assays, followed by terminal in vivo juxtacellular recordings (A) or by terminal in vitro whole-cell recordings (I) at the 21st post-infusion day. (B), (C) Top: 10 s original recording trace of spontaneous in vivo single-unit activity from SN DA neurons in vehicle (B) and 6-OHDA-infused mouse (C). Scale bars: 1 s, 0.2 mV. Below, left: corresponding interspike interval (ISI) histograms. Below, right: corresponding confocal images of juxtacellularly labeled and immunohistochemically identified DA neuron. Note the sparse bursting of the surviving SN DA neuron from the 6-OHDA-infused mouse at early phase. (D, L) Anatomical mapping of all extracellularly recorded and juxtacellularly labeled DA neurons (D), and of all in vitro recorded and filled DA neurons (L), projected to bregma –3.08 mm. Location of example SN DA neurons in (B, C, J, K) is highlighted. Smaller symbols represent DA neurons that have been recorded and identified, but not included in the group data analysis in (E–H). (E–H) Scatter dot-plots, showing significant decrease of in vivo mean firing rate (E), percentage of spikes fired in bursts (SFB) (G) and burst rate (H) and no significant differences in coefficient of variance (CV) (F) between the vehicle and 6-OHDA-infused mice. Note the 10-fold decrease in SFB for 6-OHDA-infused mice. (J, K) Top: 10 s original recording trace of in vitro whole-cell recording of spontaneous SN DA neuron activity in a vehicle (J) and 6-OHDA-infused (K) mouse. Scale bars: 1 s, 20 mV. Note that the 6-OHDA DA neuron has a highly irregular pacemaking. Below, left: corresponding to hyperpolarizing current injection. Below, right: confocal images of NB-filled and immunohistochemically identified DA neuron. (M–P) Scatter dot-plots, showing no difference in in vitro mean firing rate (M), rebound delay (O), or sag-component (P) and a fivefold increase in CV (N). Immunohistochemical imaging for all four DA neurons is displayed in ×10 and ×60 magnifications (green, TH; red, NB), scale bars: 200 µm, 20 µm. All data are presented as mean ± standard error of mean (SEM).
Figure 2—figure supplement 1
Analysis of in vivo electrophysiological parameters in surviving dopamine (DA) substantia nigra (SN) neuron in the early phase.
Scatter dot-plots, showing no significant difference of in vivo mean intra-burst (IB) frequency (A), mean maximum (max) intra-burst (IB) frequency (B), burst duration (C), number of spikes per burst (D), single spike frequency (SSF) (E), single-spike coefficient of variance (CV) (F), and action potential (AP) width (G) between the vehicle and 6-hydroxy-dopamine (6-OHDA)-infused mice in the early phase (A: vehicle 12.54 ± 1.9, 6-OHDA 20.31 ± 3.6, p=0.0673, Mann–Whitney test; B: vehicle 15.84 ± 2.8, 6-OHDA 21.18 ± 3.3, p=0.3723, Mann–Whitney test; C: vehicle 0.359 ± 0.12, 6-OHDA 0.12 ± 0.05, p=0.1042, Mann–Whitney test; D: vehicle 4.65 ± 1.2, 6-OHDA 2.47 ± 0.35, p=0.0811, Mann–Whitney test; E: vehicle 5.74 ± 0.48, 6-OHDA 4.61 ± 0.6, p=0.0955, Mann–Whitney test; F: vehicle 47.3 ± 5.8, 6-OHDA 35.4 ± 3.6, p=0.1, Mann–Whitney test; G: vehicle 1.896 ± 0.1, 6-OHDA 1.99 ± 0.1, p=0.5173, Mann–Whitney test). (H) Normalized stacked bar plots of different in vivo firing patterns based on ACH (H: chi-square test, p<0.0001). (I) ISI distributions from all in vivo recorded and labeled mSN DA neurons from vehicle- and 6-OHDA-treated mice in the early phase. Inset, cumulative representation of the same distributions (I: two-sample Kolmogorov–Smirnov test with a nonparametric hypothesis, p<0.0001, D=0.285).
Figure 2—figure supplement 2
Analysis of in vitro electrophysiological parameters in surviving dopamine (DA) substantia nigra (SN) neuron in the early phase.
Scatter dot-plots, showing no significant difference of in vitro afterhyperpolarization (AHP) minimum (A), threshold (B), spike amplitude (C), action potential (AP) width (D), and input resistance (E) between the vehicle and 6-hydroxy-dopamine (6-OHDA)-infused mice in the early phase (A: vehicle –54.6 ± 0.9, 6-OHDA –52.99 ± 0.7, p=0.3033, Mann–Whitney test; B: vehicle –27.9 ± 0.9, 6-OHDA –29.01 ± 0.7, p=0.3219, Mann–Whitney test; C: vehicle 26 ± 1.2, 6-OHDA 29.1 ± 1.4, p=0.1293, Mann–Whitney test; D: vehicle 4.1 ± 0.2, 6-OHDA 4.03 ± 0.13, p=0.6661, Mann–Whitney test; E: vehicle 249.9 ± 20.1, 6-OHDA 278.5 ± 33.9, p=0.5978, Mann–Whitney test). (F, G) Paired scatter dot-plots of firing frequency (F) and coefficient of variance (G) during on-cell recording and whole-cell recording in the early phase (F: Wilcoxon matched-pairs signed rank test, vehicle p=0.3894, 6- OHDA p=0.7316; G: Wilcoxon matched-pairs signed rank test, vehicle p=0.5614, 6-OHDA Pp=0.5016). (H) ISI distributions from all in vitro whole-cell recorded and labeled mSN DA neurons from vehicle- and 6-OHDA-treated mice in the early phase. Inset, cumulative representation of the same distributions (H: two-sample Kolmogorov–Smirnov test with a nonparametric hypothesis, p<0.0001, D=0.498) (I) Paired scatter dot-plot indicating no significant difference between the mean voltage during spike-pauses and the rest of the firing activity (paired t-test, p=0.2604).
Figure 3 with 2 supplements
Surviving substantia nigra (SN) dopamine (DA) neurons at late 6-hydroxy-dopamine (6-OHDA) phase recovered in vivo burst firing and doubled their intrinsic pacemaker frequency in vitro.
(A, I) Experimental design, illustrating timeline of behavioral assays, followed by terminal in vivo juxtacellular recordings (A) or by terminal in vitro whole-cell recordings (I) after >64 post-infusion days. (B, C) Top: 10 s original recording trace of spontaneous in vivo single-unit activity from SN DA neurons in vehicle (B) and 6-OHDA-infused mouse (C). Scale bars: 1 s, 0.2 mV. Below, left: corresponding ISI histograms. Below, right: corresponding confocal images of juxtacellularly labeled and immunohistochemically identified DA neuron. (D, L) Anatomical mapping of all extracellularly recorded and juxtacellularly labeled DA neurons (D), and of all in vitro recorded and filled DA neurons (L), projected to bregma –3.40 mm (D) and –3.08 mm (L). Location of example SN DA neurons in (B, C, J, K) is highlighted. Smaller symbols represent DA neurons that have been recorded and identified but not included in the group data analysis in (E–H). (E–H) Scatter dot-plots, showing no significant difference in mean firing rate (E), coefficient of variance (CV) (F), spikes fired in bursts (SFB) (G), and burst rate (H). (J, K) Top: 10 s original recording trace of in vitro whole-cell recording of spontaneous activity from SN DA neurons in a vehicle (J) and 6-OHDA-infused (K) mouse. Scale bars: 1 s, 20 mV. Note that the 6-OHDA DA neuron has enhanced, but regular pacemaking. Below, left, corresponding to hyperpolarizing current injection. Below, right, confocal images of NB-filled and immunohistochemically identified DA neuron. (M–P) Scatter dot-plots, showing doubling of the firing rate (M), no difference in CV (N), decrease in rebound delay (O), and increase of sag-component (P) for the 6-OHDA-treated mice in comparison to vehicle group. Immunohistochemical imaging for all four DA neurons is displayed in ×10 and ×60 magnifications (green, TH; red, NB), scale bars: 200 µm, 20 µm. All data are presented as mean ± standard error of mean (SEM).
Figure 3—figure supplement 1
Analysis of in vivo electrophysiological parameters in surviving dopamine (DA) substantia nigra (SN) neuron in the late phase.
Scatter dot-plots, showing no significant difference of in vivo mean intra-burst (IB) frequency (A), mean maximum (max) intra-burst (IB) frequency (B), burst duration (C), number of spikes per burst (D), single spike frequency (SSF) (E), single spike coefficient of variance (CV) (F), and action potential (AP) width (G) between the vehicle and 6-hydroxy-dopamine (6-OHDA)-infused mice in the late phase (A: vehicle 15.2±1.1, 6-OHDA 12.78±2.1, p=0.1128, Mann–Whitney test; B: vehicle 17.82±1.95, 6-OHDA 15.59±2.7, p=0.447, Mann–Whitney test; C: vehicle 0.25±0.12, 6-OHDA 0.25±0.05, p=0.1333, Mann–Whitney test; D: vehicle 3.92±1.3, 6-OHDA 3.57±0.5, p=0.1128, Mann–Whitney test; E: vehicle 5.57±0.49, 6-OHDA 5.94±0.58, p=0.6346, Mann–Whitney test; F: vehicle 48.9±9.9, 6-OHDA 46.7±10.2, p=0.8804, Mann–Whitney test; G: vehicle 1.89±0.07, 6-OHDA 1.83±0.06, p=0.4982, Mann–Whitney test). (H) Normalized stacked bar plots of different in vivo firing patterns based on ACH (H: chi-square test, p=0.0016). (I) ISI distributions from all in vivo recorded and labeled mSN DA neurons from vehicle- and 6-OHDA-treated mice in the late phase. Inset, cumulative representation of the same distributions (I: two-sample Kolmogorov–Smirnov test with a nonparametric hypothesis, p<0.0001, D=0.0928).
Figure 3—figure supplement 2
Analysis of in vitro electrophysiological parameters in surviving dopamine (DA) substantia nigra (SN) neurons in the late phase.
Scatter dot-plots, showing no significant difference of in vitro afterhyperpolarization (AHP) minimum (A), threshold (B), spike amplitude (C), action potential (AP) width (D), and input resistance (E) between the vehicle and 6-hydroxy-dopamine (6-OHDA)-infused mice in the late phase (A: vehicle –55.1±0.8, 6-OHDA –54.2±0.7, p=0.5535, Mann–Whitney test; B: vehicle –26.6±1.1, 6-OHDA –25.84±1.1, p=0.5973, Mann–Whitney test; C: vehicle 23.2±1.5, 6-OHDA 23.9±1.1, p=0.6198, Mann–Whitney test; D: vehicle 3.7±0.11, 6-OHDA 3.9±0.11, p=0.6198, Mann–Whitney test; E: vehicle 298.6±28.1, 6-OHDA 287.2±19.4, p=0.73, Mann–Whitney test). (F, G) Paired scatter dot-plots of firing frequency (F) and coefficient of variance (G) during on-cell recording and whole-cell recording in the late phase (F: Wilcoxon matched-pairs signed rank test, vehicle p=0.3804, 6-OHDA p=0.0004; G: Wilcoxon matched-pairs signed rank test, vehicle p=0.0522, 6-OHDA p=0.0173). (H) ISI distributions from all in vitro whole-cell recorded and labeled mSN DA neurons from vehicle- and 6-OHDA-treated mice in the late phase. Inset, cumulative representation of the same distributions (H: two-sample Kolmogorov–Smirnov test with a nonparametric hypothesis, p<0.0001, D=0.653).
Figure 4 with 3 supplements
Enhanced pacemaker frequency of substantia nigra (SN) dopamine (DA) neurons at the late post-6-hydroxy-dopamine (6-OHDA) phase was mediated by Kv4.3 channel downregulation.
(A, I) Experimental design, illustrating timeline of behavioral assays, followed by terminal in vitro whole-cell recordings under Kv4.3 channel blocker AmmTx3 (A) or in vitro voltage-clamp whole-cell recordings (I) after >64 post-infusion days. (B, C) Top: 10 s original recording traces of in vitro whole-cell recording of spontaneous activity from DA SN neurons in AmmTx3-preincubated slices for a vehicle (B) and 6-OHDA-infused (C) mouse. Below, left: corresponding hyperpolarizing current injection. Scale bars: 1 s, 20 mV. Below, right: confocal images of NB-filled and immunohistochemically identified DA neuron. (D, L) Anatomical mapping of all in vitro recorded and filled DA neurons, projected to bregma –2.92 mm (D) and –3.16 mm (L). (E–H) Scatter dot-plots, showing no differences in mean firing rate (E), coefficient of variation (F), rebound delay duration (G), and sag-amplitude (H). (J, K) Original recording trace of in vitro voltage-clamp recordings from DA SN neurons in a vehicle (J) and 6-OHDA-infused (K) mouse. Top, right: zoom-in from a Kv4.3 channel activation. Top, left: zoom-in from a Kv4.3 channel inactivation. Below, right: zoom-in from HCN-channel activation. Below, right, confocal images of NB-filled and immunohistochemically identified DA neuron. Scale bars first row: 3 nA, 100 ms. Scale bars below, left: 5 nA, 500 ms. Note the small Kv4.3 channel in/activation peak in a surviving DA neuron from a 6-OHDA mouse in comparison to the one from an ACSF-treated mouse. Immunohistochemical imaging for all four DA neurons is displayed in ×10 and ×60 magnifications (green, TH; red, NB), scale bars: 200 µm, 20 µm. All data are presented as mean ± standard error of mean (SEM). (M) Scatter dot-plots, showing a significant, half of maximum Kv4.3 channel conductance in surviving DA neurons. (N) Scatter dot-plots, showing no difference in HCN channel delta current (Ipeak – Isteady) between groups. (O) Normalized (Norm.) Kv4.3 conductance at different voltage steps, resulting in inactivation and activation curve for both groups. All data are presented as mean ± standard error of mean (SEM).
Figure 4—figure supplement 1
Analysis of in vitro electrophysiological parameters in surviving dopamine (DA) substantia nigra (SN) neuron in the late phase during inhibition of Kv4 channels.
Scatter dot-plots, showing minor significant difference of in vitro afterhyperpolarization (AHP) minimum (A), and no difference in threshold (B), spike amplitude (C), action potential (AP) width (D), and input resistance (E) between the vehicle and 6-hydroxy-dopamine (6-OHDA)-infused mice in the late phase under 1 μM AmmTx3 (A: vehicle –50.6±1.3, 6-OHDA –54.2±1.2, p=0.0411, Mann–Whitney test; B: vehicle –27.65±1, 6-OHDA –27.78±1.0, p=0.8805, Mann–Whitney test; C: vehicle 19.9±1.4, 6-OHDA 23.2±1.3, p=0.1213, Mann–Whitney test; D: vehicle 4.3±0.2, 6-OHDA 4.46±0.16, p=0.5854, Mann–Whitney test; E: vehicle 245±23.1, 6-OHDA 241±15, p=0.7148, Mann–Whitney test). (F, G) Paired scatter dot-plots of firing frequency (F) and coefficient of variance (G) during on-cell recording and whole-cell recording in the late phase under 1 μM AmmTx3 (F: Wilcoxon matched-pairs signed rank test, vehicle p<0.0001, 6-OHDA p=0.0676; G: Wilcoxon matched-pairs signed rank test, vehicle p=0.5995, 6-OHDA p=0.1040). (H) ISI distributions from all in vitro whole-cell recorded and labeled mSN DA neurons from vehicle- and 6-OHDA-treated mice in the late phase under 1 μM AmmTx3. Inset, cumulative representation of the same distributions (H: two-sample Kolmogorov–Smirnov test with a nonparametric hypothesis, p<0.0001, D=0.293).
Figure 4—figure supplement 2
Analysis of Kv4 whole-cell currents in surviving dopamine (DA) substantia nigra (SN) neurons in the late phase during inhibition of Kv4 channels.
(A–D) Measurements of transient outward (A-type) potassium biophysical parameters in whole-cell voltage-clamp recordings of Kv4.3 channel activation and inactivation in vehicle relative to 6-hydroxy-dopamine (6-OHDA)-infused mice. (E, F) No difference in series resistance (Rs) or in slow capacitance between groups (A: vehicle 35±2.2, 6-OHDA 46.1±7.8, p=0.1770, Mann–Whitney test; B: vehicle 95.6±12.9, 6-OHDA 46.2±10.2, p=0.0018, Mann–Whitney test; C: vehicle –32.3±1.3, 6-OHDA –33.7±2.9, p=0.8245, Mann–Whitney test; D: vehicle 6.25±0.3, 6-OHDA 10.58±0.7, p<0.0001, Mann–Whitney test; E: vehicle 5.48±0.5, 6-OHDA 5.45±0.4, p=0.8136, Mann–Whitney test; F: vehicle 29.3±1.7, 6-OHDA 31.8±3.2, p=0.8218, Mann–Whitney test).
Figure 4—figure supplement 3
Analysis of HCN whole-cell currents in surviving dopamine (DA) substantia nigra (SN) neurons in the late phase.
(A, B) HCN current amplitudes (A) and activation kinetics (B) in whole-cell voltage-clamp recordings of medial DA SN neurons from vehicle-infused compared to 6-hydroxy-dopamine (6-OHDA)-infused mice. For (A): two-way ANOVA, p-value across voltage p<0.0001, p-value across groups p=0.98, no significant difference between vehicle and 6-OHDA, Šídák’s multiple comparisons test. For (B): two-way ANOVA, p-value across voltage p<0.0001, p-value across groups p=0.27, no significant difference between vehicle and 6-OHDA, Šídák’s multiple comparisons test.
Figure 5 with 1 supplement
Surviving dopamine (DA) substantia nigra (SN) neurons at the late post-6-hydroxy-dopamine (6-OHDA) phase showed lower Kv4.3 channel immunohistochemical signal.
(A) Experimental design, illustrating timeline of behavioral assays, followed by multilabeling immunohistochemistry for exploring Kv4.3-channel expression differences after >64 post-infusion days. (B, C) Top: ×4 magnification of midbrain of a 6-OHDA-infused mouse, >64 days post-lesion – contralateral side (B), and corresponding ipsilateral side (C). Middle: ×60 magnification in the highlighted area from 4× image (green, TH; red, Kv4.3). Bottom, left: zoom-in on an example ROI (highlighted in 60× image). Bottom, right: color-coded Kv4.3-channel immunohistochemical signal intensity in the example ROI. Note Kv4.3-channel signal decrease in surviving DA neurons on the ipsilateral to the injection side. Scale bars: 200 µm, 20 µm. (D) Histogram showing intensity of Kv4.3 immunosignals for all TH-positive ROIs, from ipsilateral, lesioned, side (in red) and from contralateral side (in gray). Inset, same data shown as a cumulative distribution. Note a clear right shift to lower intensities for the ipsilateral side. (E) Comparison of mean TH-colocalized Kv4.3 immunosignals from medial SN from ipsilateral, lesioned, side, and contralateral, as control side. Inset, cumulative distribution of the same data, demonstrating a rightward shift towards lower Kv4.3 immunosignal intensities (Norm.=normalized). All data are presented as mean ± standard error of mean (SEM).
Figure 5—figure supplement 1
Analysis of Kv4.3 immunoreactivity in surviving dopamine (DA) substantia nigra (SN) neuron in the late phase.
(A) Example illustration of an image from neuronal TH-immunosignal, transformed as a TH mask and overlayed on top of Kv4.3 immunosignal. Further segregation of the membrane and cytoplasm compartment with the corresponding Kv4.3 immunosignal intensity. (B) Distributions of all ROIs area sizes based on TH-masks from vehicle- (gray) and 6-hydroxy-dopamine (6-OHDA)-treated mice (red) in the late phase. Inset, cumulative representation of the same distributions (two-sample Kolmogorov–Smirnov test with a nonparametric hypothesis, p- and D-values noted in the lower right corner in the graph). (C) Scatter dot-plots, illustrating the medio-lateral gradient of Kv4.3 immunosignal on the contralateral and ipsilateral side from 6-OHDA-treated mice in the late phase (ordinary one-way ANOVA, p<0.0001, multiple comparisons statistics: upper row p=0.0028, middle row p<0.0001, lower row left to right: p<0.0001, p=0.0005, p<0.0001). (D) Scatter dot-plots, showing significant decrease in Kv4.3 immunosignal in both membrane and cytoplasm compartments in the ipsilateral (6-OHDA-infused) side compared to the contralateral (control) side. Membrane: p=0.0003, contralateral side 67.2±0.9, ipsilateral side 56.7±1.1 (about 16% reduction); Cytoplasm: p<0.0001, contralateral side 55.8±0.9, ipsilateral side 45.9±1.1 (about 18% reduction) (E) Top: ×4 magnification of midbrain of a vehicle-infused mouse,>64 days post-lesion – contralateral side (left panel), and corresponding ipsilateral side (right panel). Middle: ×60 magnification in the highlighted area from 4× image (green, TH; red, Kv4.3). Bottom, left: zoom-in on an example ROI (highlighted in 60× image). Bottom, right: color-coded Kv4.3-channel immunohistochemical signal intensity in the example ROI. (F) Histogram showing intensity of Kv4.3 immunosignals for all TH-positive ROIs, from ipsilateral, vehicle-infused, side (in orange) and from contralateral side (in green). Inset, same data shown as a cumulative distribution (two-sample Kolmogorov–Smirnov test with a nonparametric hypothesis, p=0.0005, D=0.0287). (G) Scatterplots of Kv4.3 immunosignal per area with a medial-lateral distribution and shown across animals. The lines represent the mean intensity for each region.
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Recovery of the full in vivo firing range in post-lesion surviving DA SN neurons associated with Kv4.3-mediated pacemaker plasticity
eLife 14:e104037.
https://doi.org/10.7554/eLife.104037
