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. 2018 Jun:135:343-354.
doi: 10.1016/j.neuropharm.2018.03.028. Epub 2018 Mar 23.

CaV3.1 isoform of T-type calcium channels supports excitability of rat and mouse ventral tegmental area neurons

Matthew E Tracy  1 , Vesna Tesic  1 , Tamara Timic Stamenic  1 , Srdjan M Joksimovic  1 , Nicolas Busquet  2 , Vesna Jevtovic-Todorovic  1 , Slobodan M Todorovic  3
Affiliations

CaV3.1 isoform of T-type calcium channels supports excitability of rat and mouse ventral tegmental area neurons

Matthew E Tracy et al. Neuropharmacology. 2018 Jun.

Abstract

Recent data have implicated voltage-gated calcium channels in the regulation of the excitability of neurons within the mesolimbic reward system. While the attention of most research has centered on high voltage L-type calcium channel activity, the presence and role of the low voltage-gated T-type calcium channel (T-channels) has not been well explored. Hence, we investigated T-channel properties in the neurons of the ventral tegmental area (VTA) utilizing wild-type (WT) rats and mice, CaV3.1 knock-out (KO) mice, and TH-eGFP knock-in (KI) rats in acute horizontal brain slices of adolescent animals. In voltage-clamp experiments, we first assessed T-channel activity in WT rats with characteristic properties of voltage-dependent activation and inactivation, as well as characteristic crisscrossing patterns of macroscopic current kinetics. T-current kinetics were similar in WT mice and WT rats but T-currents were abolished in CaV3.1 KO mice. In ensuing current-clamp experiments, we observed the presence of hyperpolarization-induced rebound burst firing in a subset of neurons in WT rats, as well as dopaminergic and non-dopaminergic neurons in TH-eGFP KI rats. Following the application of a pan-selective T-channel blocker TTA-P2, rebound bursting was significantly inhibited in all tested cells. In a behavioral assessment, the acute locomotor increase induced by a MK-801 (Dizocilpine) injection in WT mice was abolished in CaV3.1 KO mice, suggesting a tangible role for 3.1 T-type channels in drug response. We conclude that pharmacological targeting of CaV3.1 isoform of T-channels may be a novel approach for the treatment of disorders of mesolimbic reward system.

Keywords: Burst firing; Dopamine; Low-voltage-activated; Rebound spiking; T-type calcium channel; TTA-P2; Ventral tegmental area.

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Conflict of interest statement

Conflict of interest

The authors received no compensation, nor do they have any conflicting financial interests in regards to the work described in this manuscript.

Figures

Figure 1
Figure 1. Rebound burst firing in a subpopulation of VTA neurons in WT rats
(A) Depiction of two current injection traces of 50 pA (red trace) and 100 pA (black trace) from the dual step protocol utilized to elicit depolarization-induced firing mode and after-hyperpolarization-induced rebound spiking. The procedure begins with an injection of positive current at successive steps for 200 ms, followed by a current injection of negative current for 500 ms to attempt to elicit a series of rebound spiking. (B) Representative traces from the dual step protocol of a neuron in the VTA which produces depolarization-induced firing and hyperpolarization-induced rebound spiking. (C) Representative trace from another neuron that fired only after injections of depolarizing currents, but did not produce hyperpolarization-induced rebound spiking. Recorded neurons in B and C were labeled with biocytin (b and c, respectively), which was included in the recording pipette (scale bar = 15 microns).
Figure 2
Figure 2. Selective inhibition of T-channels abolished rebound bursting in rat VTA neurons
(A) The stimulus waveform for the dual step protocol (top panel) overlapped with baseline control traces (middle panel) and ten minutes after application of 5 μM of TTA-P2 in the same neuron (bottom panel). A barrage of depolarization-induced APs and prominent hyperpolarization-induced rebound bursting can be seen. Very little interaction in depolarization-induced firing of APs is evident 10 min after exposure to the specific T-type channel blocker, however the rebound spikes are completely ablated. (B–D) Aggregation of eleven observations following exposure to TTA-P2 from seven WT rats were averaged and compared to baseline control for statistical significance. (B) TTA-P2 (red filled square symbols) produced a significant interaction in hyperpolarization-induced rebound spiking [F(10, 100) = 8.875, ***p=0.001 (Two-way mixed-design ANOVA)] resulting in decreased spiking from 30-100% of maximal current intensity when compared to baseline controls (open black circle symbols). (C) TTA-P2 failed to produce a significant change in the depolarization-induced firing frequency in the same neurons. (D) Application of TTA-P2 produced a significant interaction when compared to baseline control (open circles) [F(20, 180) = 3.008, ***p=0.001] resulting in an increase in input resistance at 10–100% of maximal current at the both 5 min (red squares) and 10 min (black triangles) time points.
Figure 3
Figure 3. Characterization of voltage-dependent activation kinetics of T-currents in rat VTA neurons
(A) Voltage-clamp stimulus waveform and representative current traces (Vh −90 mV, Vt −80 through −20 mV) for the current-voltage activation protocol in WT rats. (B) Analysis of peak current for each trace across subjects was aggregated and normalized to determine an estimated V50 of −59.6 ± 0.4 mV and slope factor of 4.3 ± 0.3 mV for the voltage-dependent activation of well isolated T-currents. Individual data points are in 2.5 mV incremental steps and are averages from multiple determinations. Red solid line on the graph is best fit of data points using Boltzmann equation. (C) The same data points from graph B plotted as current density (peak current amplitude divided by cell’s capacitance) against test potential for the current-voltage activation protocol. (D) Inactivation time constants at different points ranging from −55 to −20 mV plotted against test potential from the current-voltage relationship from the same cells presented on panel B of this figure. Note that inactivation tau becomes progressively faster with stronger depolarizing steps and becomes largely voltage-independent at more positive potentials.
Figure 4
Figure 4. Characterization of voltage-dependent inactivation kinetics of T-currents in rat VTA neurons
(A) Voltage-clamp stimulus waveform and representative current traces for the voltage-dependent inactivation protocol. (B) Analysis of peak current for each trace across subjects was aggregated and normalized to determine a V50 of −94.4 ± 0.6 mV and a slope factor of 5.8 ± 0.6 mV for the voltage-dependent steady-state inactivation in rat VTA neurons. Individual data points are in 5 mV incremental steps and are averages from multiple determinations. Red solid line on the graph is best fit of data points using Boltzmann equation. (C) Normalized current and conductance plotted against test potential for both the inactivation (black) and activation (red) protocol, respectively show the overlapping region, termed T-type “window” current.
Figure 5
Figure 5. Diminished T-current amplitudes in CaV3.1 knock-out (KO) mice when compared to wild-type mice
(A) Representation of the voltage-clamp stimulus waveform for the single step activation protocol. (B) Representative sweeps from traces of neurons in the VTA for a wild-type (black trace) and CaV3.1 KO mouse (red trace). (C) Aggregation of T-current peaks across 10 observations of wild-type mice from 4 animals and 10 observations of CaV3.1 KO mice from 3 animals were averaged and compared for statistical significance; t-test, two-tailed. Current amplitudes were greatly diminished in KO mice (3.7 ± 1.7 pA) when compared to WT mice (91.3 ± 15.6 pA) (***, p < 0.001).
Figure 6
Figure 6. Immunohistochemistry of VTA neurons from TH-eGFP transgenic rats
A) Double immunofluorescence image of a brain slice showing co-localization of tyrosine-hydroxylase-eGFP positive neurons (green) and NeuN positive neurons (red) in the VTA. (B) Magnified images of the white square in (A). Upper panel shows TH-eGFP positive neurons (green), bottom panel with NeuN positive neurons (red) and the middle panel shows merged image. Red nuclei lacking a green signal demonstrates not all of the neurons in VTA are dopaminergic. Scale bar = 150 μm.
Figure 7
Figure 7. Comparison of depolarization-induced and rebound burst firing properties in TH-positive and TH-negative VTA neurons of transgenic TH-eGFP knock-in rat
(A) Representative sweep of current injection protocol used to study AP firing patterns in VTA neurons (top panel). Family of sweeps from traces from a TH-positive (TH-POS, green) and TH-negative (TH-NEG, red) neuron, which both produced depolarization-induced firing of APs and rebound burst firing, are depicted in the middle and bottom panel, respectively. (B) The input-output curve as a function of the number of rebound APs presented as the percentage of maximal current injection between TH-POS and TH-NEG neurons. TH-NEG neurons fired significantly more APs than TH-POS for the 70%, 90%, and 100% data points (p < 0.05). Two-way ANOVA showed borderline interaction p=0.092 and treatment p=0.057. Hence, we performed multiple comparisons (Benjamini, Krieger and Yekutieli method, Graph-Pad Prism) which showed significant p = 0.015 at 70%, p = 0.001 at 90% and p = 0.019 at 100% normalized current injections. (C) However, for the initial depolarizing phase there was no statistical difference in the maximal firing frequency between TH-POS and TH-NEG neurons. (D) TTA-P2 at 5 μM (gray trace) completely inhibited one rebound AP present at control baseline (green trace) in this TH-POS neuron (the same neuron shown in the middle of panel A). (E) TTA-P2 at 5 μM (gray trace) completely inhibited two rebound APs present at control baseline (red trace) in this TH-NEG neuron (the same neuron shown in the bottom of panel A).
Figure 8
Figure 8. MK-801-induced hyperlocomotion
(A) The effects of CaV3.1 T-channels on hyperlocomotion induced by 0.1 mg/kg MK-801. The baseline locomotor activity and activity after saline injection was not significantly different between WT and CaV3.1 KO mice (two-way mixed-design ANOVA). When monitoring the mice for an hour immediately after MK-801 injection (5 min time-bins), WT but not Cav3.1 KO mice exhibited a dramatic increase in locomotor activity. (B) Bar graphs show a significant decrease in the total moved distance in CaV3.1 KO, as compared to WT mice, following MK-801 challenge. *p<0.05
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