Figure 8. AIS-positive DA neurons have greater intrinsic excitability.

(A) Example image of a fixed, 50 µm OB slice from a P28 DAT-tdT (red) mouse, immunostained with an anti-TH antibody (blue). While most TH-positive neurons exhibit red tdT fluorescence, some are tdT-negative (arrows). (B) Soma size distributions of all DAT-tdT-positive cells (red), and of DA neurons that are DAT-tdT-negative but TH-positive (blue). Inset: percentages of AIS-positive/TH-positive DA cells that are either tdT-positive or –negative (n = 50, N = 5). (C) Example image of a DAT-tdT-labelled DA cell (red) stained with the axonal marker TRIM-46 (green) and the AIS marker AnkG (greyscale). Asterisks indicate the soma; line indicates axon start; arrows indicate start and end position of the TRIM (green) and AnkG (white) label. (D) Schematic representation of the experimental strategy for whole-cell recordings: acute 300 µm OB slices were obtained from P21-35 DAT-tdT mice, and tdT-positive DA cells of either subtype were targeted for whole-cell patch-clamp recording. (E) Example current-clamp traces of single APs fired by monophasic (AIS-negative, black, n = 15) and biphasic (AIS-positive, magenta, n = 11) DAT-tdTomato neurons. Left: action potentials fired to threshold 10 ms somatic current injection. Right: phase plane plots of the spikes shown on the left. Arrow points to the AIS-dependent first action potential phase. (F) Quantification of soma area (t-test; ***p=0.0006), current threshold (Welch-corrected t-test; *p=0.017), and onset rapidness (Welch-corrected t-test; *p=0.035) in monophasic and biphasic cells. Empty circles show values from individual cells, filled circles show mean ± SEM. (G) Top: Example current-clamp traces of multiple APs fired in response to a 300pA/500 ms somatic current injection in monophasic and biphasic cells. Bottom: input-output curve of injected current density versus mean ± SEM spike number for each group. (H). Quantification of input-output slope (t-test; *p=0.044), and of the maximum number of action potentials fired by each cell over the whole range of injected current intensities (t-test; **p=0.0092). Empty circles show values from individual cells; filled circles show mean ± SEM. (I) Classification of DAT-tdT neurons based on values obtained from whole-cell recordings. Each circle shows one cell, plotted according to its primary and secondary PCA component scores (these components accounted for 92% and 7% of the variance in the data, respectively). Filled circles show cells correctly classified by k-means analysis; open circles show the few cells (3/26 overall) that were incorrectly classified.

Figure 8—figure supplement 1. AIS-positive DA neurons also have greater intrinsic excitability with strict, objective phase plane plot classification.

(A) Example current-clamp traces of phase plane plots of single APs fired by monophasic (AIS-negative, black, top) and biphasic (AIS-positive, magenta, bottom) DAT-tdTomato neurons. Dashed box indicates the AIS-dependent first action potential phase, which is shown at higher detail on the right and fitted with two methods. The ratio between the errors of the linear fit (green) and the exponential fit (red) is indicated near the trace. (B) Quantification of soma area (t-test; p=0.04), voltage threshold (t-test; p=0.046), onset rapidness (Mann-Withney test; p=0.003), rheobase (t-test; p=0.039) and first spike delay (t-test; p=0.0431) in monophasic and biphasic cells. Empty circles show values from individual cells, filled circles show mean ± SEM; *p<0.05; **p<0.01. (C) Input-output curve of injected current density versus mean ±SEM spike number for each group.