Refining the Identity and Role of Kv4 Channels in Mouse Substantia Nigra Dopaminergic Neurons

Spontaneous activity and postinhibitory rebound are profoundly altered in Kv4.3−/− SNc DA neurons. A, Representative cell-attached recordings showing the spontaneous pattern of activity in WT (black trace, left) and Kv4.3−/− SNc DA neurons (red trace, right). B, Box and whisker plots showing the distribution of values for extracellularly recorded spontaneous ISI (extracellular ISI; left) and ISI CV (CV_{extra. ISI}; right) in WT and Kv4.3−/− SNc DA neurons. C, Representative current-clamp recordings showing the voltage response of SNc DA neurons to a current step (gray trace) hyperpolarizing membrane voltage to ∼–120 mV in WT (black trace, left) and Kv4.3−/− mice (red trace, right). The recordings come from the same neurons as the cell-attached recordings presented in A. D, Box and whisker plots showing the distribution of values for voltage sag amplitude (left) and postinhibitory rebound delay (right) in WT and Kv4.3−/− SNc DA neurons; ***p < 0.001. Dotted lines in the box and whisker plots indicate the distribution of data (violin plots). Scale bars: 1 s (A, horizontal), 500 ms (C, top, horizontal), 40 mV (C, top, vertical), 100 pA (C, bottom, vertical), and –60 mV (C, horizontal dotted lines).

AP and excitability properties in Kv4.3−/− SNc DA neurons. A, left, Current-clamp recordings showing the shape of the AP in the WT (black traces) and Kv4.3−/− mice (red traces) on a slow (top) and fast time-scale (bottom). Right, Box and whisker plots showing the distribution of values for AP threshold (top left), amplitude (top right), half-width (AP half-width, bottom left), and AHP trough (bottom right) in WT and Kv4.3−/− SNc DA neurons. B, left, Current-clamp recordings showing the voltage response of SNc DA neurons to 100- and 200-pA depolarizing current steps (gray traces) in WT (top, black traces) and Kv4.3−/− mice (bottom, red traces). Gray dotted rectangles indicate the ISI used to calculate the gain start and gain end on each train of APs. Right, Box and whisker plots showing the distribution of values for gain start (top left), gain end (top right), SFA index (bottom left), and start frequency at 100 pA (bottom right) in WT and Kv4.3−/− SNc DA neurons; *p < 0.05, **p < 0.01, ***p < 0.001. Dotted lines in the box and whisker plots indicate the distribution of data (violin plots). Scale bars: 50 ms (A, top, horizontal), 20 mV (A, top, vertical), 2 ms (A, bottom, horizontal), 20 mV (A, bottom, vertical), –40 mV (A, horizontal dotted lines), 500 ms (B, voltage, horizontal), 40 mV (B, voltage, vertical), and 100 pA (B, current, vertical).

Voltage-clamp analysis of I_A in WT and Kv4.3−/− SNc DA neurons. A, Voltage-clamp traces showing representative I_A recordings obtained from a WT (black trace) and a Kv4.3−/− SNc DA neuron (red trace) in response to a voltage step to –40 mV (gray trace). The small residual current present in the Kv4.3−/− mice is blocked by AmmTX3 (inset, orange trace). B, Box and whisker plot showing the distribution of values for I_A amplitude in WT and Kv4.3−/− SNc DA neurons. C, Box and whisker plot showing the distribution of values for I_A time constant of inactivation (I_A tau) in WT and Kv4.3−/− SNc DA neurons. The green dotted rectangle highlights five Kv4.3−/− outliers displaying unusually large values for I_A tau. D, Box and whisker plot showing the distribution of values for I_A charge in WT and Kv4.3−/− SNc DA neurons. The green dotted rectangle highlights five Kv4.3−/− outliers displaying unusually large values for I_A charge (same cells as in C). E, left, Scatter plot showing the relationship between I_A tau and charge in WT (gray dots) and Kv4.3−/− SNc DA neurons (red dots). Please note that 5 of the Kv4.3−/− measurements lie in the WT region of space (green dotted ellipse). Right, Voltage-clamp traces showing one example of the atypical I_A recording (green trace, corresponding to the large green circle in the scatter plot) encountered in one of the 5 Kv4.3−/− outliers highlighted in panels C, D, compared with the typical recording obtained in Kv4.3−/− neurons (red trace, same as in panel A, corresponding to the large red circle in the scatter plot); ***p < 0.001. Dotted lines in the box and whisker plots indicate the distribution of data (violin plots). Scale bars: 200 ms (A, E, horizontal) and 1 nA (A, E, vertical).

Kv4.2 channels are expressed by a minority of WT and Kv4.3−/− SNc DA neurons. A, top, Low-magnification pictures of the SNc showing (from left to right) TH (red), Kv4.3 (green), and DAPI (blue) stainings in WT (top row) and Kv4.3−/− mice (bottom row). Bottom, High-magnification pictures of SNc DA neurons showing (from left to right) TH (red), Kv4.3 (green), and DAPI (blue) stainings in WT (top row) and Kv4.3−/− mice (bottom row). B, Effect of the AR procedure on Kv4.2 detection in the SNc. Top, Low-magnification pictures showing (from left to right) TH (red), Kv4.2 (green), and DAPI staining (blue) without (top row) or with AR (bottom row). Bottom, left, High-magnification pictures of the insets depicted in the merged low-magnification picture and corresponding to a Kv4.2-negative (left) and a Kv4.2-positive SNc DA neuron (middle), for which inset pictures (right) illustrate the region selected to characterize the immunofluorescence profile of Kv4.2 and TH stainings (3 μm yellow bar). Bottom, right, Immunofluorescence profiles of Kv4.2 (green, top graph), Kv4.3 (green, bottom graph), and TH staining (red, both graphs) showing that both Kv4.2 and Kv4.3 profiles are very similar and strongly suggestive of specific plasma membrane expression. The average profiles (n = 5) shown here were defined over 3-μm selected regions of interest (yellow bar above the graph), such as the one shown on the inset pictures on the left. C, left, High-magnification pictures showing (from left to right) TH (red), Kv4.2 (green), and DAPI staining (blue) after AR in WT (top row) and Kv4.3−/− mice (bottom row). Right, Expanded view of the Kv4.2-positive cells (1, 2) highlighted in the merged pictures on the left. To overcome differences regarding levels of TH expression in different DA neurons, different minimum and maximum display settings were applied for the TH channel. D, Bar plot showing the counts of Kv4.2-negative (dark colors) and Kv4.2-positive (light colors) cells observed in the SNc of WT (black bar) and Kv4.3−/− mice (red bar). n.s., non-significant. Scale bars: 100 μm (A, top row), 10 μm (A, bottom row), 10 μm (B, top row), 10 μm (B, bottom row), 2 μm (B, inset), and 10 μm (C).

Comparing the alterations in electrophysiological phenotype after acute blockade of Kv4 channels with the Kv4.3−/− mouse model. A, Current-clamp recordings showing the spontaneous pattern of activity of a WT SNc DA neuron in control condition (black trace, left) and after AmmTX3 application (red trace, right). B, left, Line and scatter plot showing the change in spontaneous firing frequency induced by AmmTX3 application in individual WT SNc DA neurons. Right, Bar plot comparing the average change in spontaneous firing frequency after AmmTX3 application (left, light colors) or Kv4.3 channel deletion (right, dark colors). C, Current-clamp recordings showing the voltage response of a WT SNc DA neuron to a hyperpolarizing current step (bottom gray traces) in control condition (left, black trace) and after AmmTX3 application (right, red trace). D, left, Line and scatter plot showing the change in rebound delay induced by AmmTX3 application in individual WT SNc DA neurons. Right, Bar plot showing the average change in rebound delay after AmmTX3 application (left, light colors) or Kv4.3 channel deletion (right, dark colors); **p < 0.01, ***p < 0.001. Scale bars: 1 s (A, horizontal), 20 mV (A, vertical), –60 mV (A, horizontal gray dotted lines), 500 ms (C, horizontal), 20 mV (C, vertical).

Absence of compensatory changes in delayed rectifier and I_H currents in the Kv4.3−/− SNc DA neurons. A, Properties of the delayed rectifier potassium current (I_KDR) in SNc DA neurons in WT and Kv4.3−/− mice. Left, Voltage-clamp recordings of I_KDR obtained in a WT (black trace) and a Kv4.3−/− mouse (red trace) in response to a voltage step to +20 mV (gray trace). The peak and steady-state components of I_KDR are indicated by arrowheads. Right, Line and scatter plots representing the average current–voltage relationships of the peak (left) and steady-state I_KDR (right) obtained from WT (gray dots) and Kv4.3−/− SNc DA neurons (red dots). B, Properties of I_H in SNc DA neurons in WT and Kv4.3−/− mice. Left, Voltage-clamp recordings of I_H obtained in a WT (black traces) and a Kv4.3−/− mouse (red traces) in response to increasingly hyperpolarized voltage steps. Right, Box and whisker plot showing the distribution of values for I_H amplitude (left) and voltage sensitivity (right) in WT and Kv4.3−/− SNc DA neurons; ***p < 0.001. Dotted lines in the box and whisker plots indicate the distribution of data (violin plots). Scale bars: 200 ms (A, horizontal), 1 nA (A, vertical), 2 s (B, horizontal), and 500 pA (B, vertical).

Linking biophysical changes in I_A to changes in electrophysiological phenotype in WT and Kv4.3−/− SNc DA neurons. A, Recordings representative of the variation in spontaneous activity, postinhibitory rebound and I_A in WT and Kv4.3−/− SNc DA neurons. Left, Cell-attached voltage-clamp recordings of spontaneous pacemaking activity in two WT (black traces) and two Kv4.3−/− neurons (red traces). The value of the average ISI is indicated above the trace. Middle, Current-clamp recordings of the postinhibitory rebound obtained in the same neurons. The value of rebound delay is indicated above the trace. Right, I_A voltage-clamp recordings obtained at –40 mV in the same neurons. The values of I_A amplitude and tau are indicated above the trace. B, Scatter plot showing the significant positive correlation between extracellularly recorded ISI and rebound delay observed in WT (gray dots) and Kv4.3−/− SNc DA neurons (red dots). The plain black and red lines correspond to the linear regression of the data (r, n, and p values are shown on the graph), while the gray and pink dotted lines indicate the regression confidence intervals. The diamond and square symbols correspond to the recordings presented in panel A. C, Scatter plots showing the relationships between biophysical variables and neuronal output. Correlations between I_A tau, I_H amplitude, I_A amplitude or I_A V₅₀ (from left to right) and rebound delay or extracellular ISI (from top to bottom) were tested in both WT (gray dots) and Kv4.3−/− neurons (red dots). Please note that only I_A tau and I_H amplitude were significantly correlated with both ISI and rebound delay in WT and Kv4.3−/− neurons. The plain black and red lines correspond to the linear regression of the data (r/ρ, n, and p values are shown on the graph), while the gray and pink dotted lines indicate the regression confidence intervals. Dashed black and red lines indicate non-significant correlations. The diamond and square symbols correspond to the recordings presented in panel A. D, Scatter plots showing the significant correlations between I_A and I_H properties. Left, Scatter plot showing the positive correlation between I_A inactivation V₅₀ and I_H activation V₅₀ in WT neurons. Right, Scatter plot showing the negative correlation between I_A tau and I_H amplitude observed in both WT (gray dots) and Kv4.3−/− neurons (red dots). The plain black and red lines correspond to the linear regression of the data (r/ρ, n, and p values are shown on the graph), while the gray and pink dotted lines indicate the regression confidence intervals. E, left, Scatter plot showing the multiple linear regression of extracellular ISI versus I_A tau and I_H amplitude (predicted ISI) in WT neurons. Right, Scatter plot showing the multiple linear regression of rebound delay versus I_A tau, I_A inactivation V₅₀, and I_H amplitude (predicted rebound delay) in WT neurons. The corresponding equations are indicated below the x-axis of each graph. The plain black lines correspond to the linear regression of the data (r, n, and p values are shown on the graph), while the gray dotted lines indicate the regression confidence intervals. Scale bars: 1 s (A, left horizontal), 500 ms (A, middle horizontal), 50 mV (A, vertical), 250 ms (A, right horizontal), and 2 nA (A, right vertical).