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. 2019 Jul 10:13:317.
doi: 10.3389/fncel.2019.00317. eCollection 2019.

Biophysical Properties of Somatic and Axonal Voltage-Gated Sodium Channels in Midbrain Dopaminergic Neurons

Jun Yang  1 , Yujie Xiao  1 , Liang Li  1 , Quansheng He  1 , Min Li  1 , Yousheng Shu  1
Affiliations

Biophysical Properties of Somatic and Axonal Voltage-Gated Sodium Channels in Midbrain Dopaminergic Neurons

Jun Yang et al. Front Cell Neurosci. .

Abstract

Spiking activities of midbrain dopaminergic neurons are critical for key brain functions including motor control and affective behaviors. Voltage-gated Na+ channels determine neuronal excitability and action potential (AP) generation. Previous studies on dopaminergic neuron excitability mainly focused on Na+ channels at the somatodendritic compartments. Properties of axonal Na+ channels, however, remain largely unknown. Using patch-clamp recording from somatic nucleated patches and isolated axonal blebs from the axon initial segment (AIS) of dopaminergic neurons in mouse midbrain slices, we found that AIS channel density is approximately 4-9 fold higher than that at the soma. Similar voltage dependence of channel activation and inactivation was observed between somatic and axonal channels in both SNc and VTA cells, except that SNc somatic channels inactivate at more hyperpolarized membrane potentials (V m). In both SNc and VTA, axonal channels take longer time to inactivate at a subthreshold depolarization V m level, but are faster to recover from inactivation than somatic channels. Moreover, we found that immunosignals of Nav1.2 accumulate at the AIS of dopaminergic neurons. In contrast, Nav1.1 and Nav1.6 immunosignals are not detectible. Together, our results reveal a high density of Na+ channels at the AIS and their molecular identity. In general, somatic and axonal channels of both SNc and VTA dopaminergic neurons share similar biophysical properties. The relatively delayed inactivation onset and faster recovery from inactivation of axonal Na+ channels may ensure AP initiation at high frequencies and faithful signal conduction along the axon.

Keywords: action potential; axon; dopaminergic neuron; sodium channel subtype; voltage-gated sodium (Nav) channels.

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Figures

FIGURE 1
FIGURE 1
Action potentials (APs) are initiated at the AIS of SNc TH-positive neurons. (A) An example recorded SNc TH-GFP neuron. (B) Dual whole-cell recording from the soma (blue) and the axon (red, 10 μm away from the soma) showing pace making activity at both recording sites. (C) Negative DC current injection to the soma prevents spontaneous firing. Positive current pulses (500 ms in duration) at the AIS cause repetitive firing at the axon and the soma. Note the absence and presence of the initial AP (arrows) in response to weak and strong current pulses (100 vs. 150 pA). (D) Injection of current pulses to the soma evoke repetitive firing at both recording sites. Also note the absence of presence of the initial AP. (E) Top, expansion of the APs indicated in B–D. Axonal APs precede the somatic APs in all cases. Bottom, the first derivative (dV/dt) of the corresponding APs. The two components at the rising phase of the somatic dV/dt trajectory (blue) correspond to the AIS and SD potentials, respectively. Note the difference of the rising phase between somatic and axonal dV/dt traces.
FIGURE 2
FIGURE 2
Na+ channel density at the soma and the AIS of midbrain TH neurons. (A) Fluorescence and DIC images of the recorded somatic nucleated patch and isolated AIS bleb. The recording pipettes contained Alexa Fluo-594, which could label the recorded nucleated patch and axonal bleb. (B,C) Example Na+ currents obtained from SNc (B) and VTA cells (C) by stepping the holding voltage from −120 mV (35 ms) to 0 mV (25 ms). (D) Averaged peak amplitudes of Na+ currents. (E) Averaged channel density at the soma and the AIS of SNc and VTA cells. *p < 0.05, comparison between SNc and VTA axonal channel density.
FIGURE 3
FIGURE 3
Activation of transient Na+ currents in somatic nucleated patches and isolated AIS blebs. (A) Top, Na+ currents evoked by activation voltage commands (inset) in a somatic nucleated patch and an isolated axonal bleb in SNc. Bottom, activation curves from somatic and axonal Na+ currents of SNc cells. The plots are fitted with Boltzmann functions. (B) Similar as in A but from VTA cells. (C) Group data showing the half-activation voltage (V1/2) and the slope of Na+ channel activation curves. ∗∗p < 0.01.
FIGURE 4
FIGURE 4
Kinetics of activation and inactivation of Na+ currents. (A,B) Example Na+ currents of SNc (A) and VTA cells (B) evoked by stepping the voltage from a 35-ms prepulse of –120 to a test pulse of –20 mV. The rising phase of the currents was fitted by an exponential function with a delayed onset. The decay phase was fitted with a single exponential function. (C,D) Comparison of the rise and decay time between somatic and axonal Na+ currents. ∗∗∗p < 0.001.
FIGURE 5
FIGURE 5
Time course of Na+ channel deactivation in TH neurons. (A,B) Families of Na+ currents in SNc (A) and VTA cells (B) evoked by the channel deactivation voltage protocol (inset). Immediately after the test pulse (from a 30-ms –120 mV prepulse to 0 mV, 0.2 ms), the Vm was held at different levels (from –100 to –30 mV with increment of 10 mV). (C) Fitting the decay of the currents with single exponential functions. (D) The deactivation time constants at different voltage levels in SNc (top) and VTA cells (bottom). *p < 0.05; ∗∗p < 0.01.
FIGURE 6
FIGURE 6
Steady-state inactivation of transient Na+ currents in midbrain TH neurons. (A) Example families of Na+ currents (top) evoked by the voltage commands (inset) for steady-state channel inactivation in a SNc somatic nucleated patch and an isolated AIS bleb. Bottom, inactivation curves of SNc Na+ currents (n = 7 somatic nucleated patches and 7 isolated AIS blebs). (B) Similar as in A but from VTA cells. (C,D) Group data comparing the half-inactivation voltages and the inactivation curve slopes. *p < 0.05; ∗∗p < 0.01.
FIGURE 7
FIGURE 7
Time course of Na+ channel inactivation onset in TH neurons. (A) Example currents evoked by the voltage commands (inset) in SNc neurons. The Vm was stepped from a prepulse of –120 mV (20 ms) to –55 mV with varying intervals followed by a test pulse to 0 mV (5 ms). (B) Similar as in A but from VTA neurons. (C,D) Plots of the currents as a function of intervals. Currents were normalized to the Na+ currents without time interval. The plots were fitted with single exponential functions. Inset, expanded plots showing differences between the two groups. (E) Time constants for the development of Na+ channel inactivation at –55 mV. *p < 0.05; ∗∗∗p < 0.001.
FIGURE 8
FIGURE 8
Recovery from inactivation of Na+ channels in TH neurons. (A,B) Representative currents evoked by two paired pulses (from –120 to 0 mV) with varying intervals (inset) in SNc (A) and VTA neurons (B). (C,D) Plots of the currents in SNc (C) and VTA cells (D) as a function of intervals between the two pulses. Currents were normalized to the peak amplitude of the current evoked by the first pulse. (E) Comparison of the recovery time constants from inactivation between Na+ currents in different groups. ∗∗∗p < 0.001.
FIGURE 9
FIGURE 9
The AIS of midbrain TH-positive neurons express Nav1.2. (A) Double staining of AnkG (Blue) and Nav1.2 (Red) in neocortex. (B) Triple staining of TH (green), AnkG (Blue) and Nav1.2 (Red) in two example SNc cells. White arrowheads indicate neurite segments positive to all three antibodies. (C) Similar as in B but for cells in VTA. (D) Percentages of TH/AnkG-positive neurites in SNc (top) and VTA (bottom) that are positive (blue) or negative (yellow) to Nav1.2 staining.
FIGURE 10
FIGURE 10
Absence of Nav1.1 and Nav1.6 from the AIS of midbrain TH-positive neurons. (A) Double staining of AnkG (Blue) and Nav1.1 (Red) in neocortex. (B) Triple staining of TH (green), AnkG (Blue) and Nav1.1 (Red) in SNc. White arrowheads indicate neurites positive to AnkG and Nav1.1 but negative to TH. (C) Similar as in B but for VTA. (D–F) Similar to A–C but for Nav1.6 staining in the neocortex (D), SNc (E) and VTA (F). Note the absence of Nav1.6 immunosignals from the TH-positive neurites (arrowheads). Arrows indicate neurites positive to AnkG and Nav1.6 but negative to TH.
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