Methamphetamine Regulation of Firing Activity of Dopamine Neurons

Abstract

Methamphetamine (METH) is a substrate for the dopamine transporter that increases extracellular dopamine levels by competing with dopamine uptake and increasing reverse transport of dopamine via the transporter. METH has also been shown to alter the excitability of dopamine neurons. The mechanism of METH regulation of the intrinsic firing behaviors of dopamine neurons is less understood. Here we identified an unexpected and unique property of METH on the regulation of firing activity of mouse dopamine neurons. METH produced a transient augmentation of spontaneous spike activity of midbrain dopamine neurons that was followed by a progressive reduction of spontaneous spike activity. Inspection of action potential morphology revealed that METH increased the half-width and produced larger coefficients of variation of the interspike interval, suggesting that METH exposure affected the activity of voltage-dependent potassium channels in these neurons. Since METH has been shown to affect Ca²⁺ homeostasis, the unexpected findings that METH broadened the action potential and decreased the amplitude of afterhyperpolarization led us to ask whether METH alters the activity of Ca²⁺-activated potassium (BK) channels. First, we identified BK channels in dopamine neurons by their voltage dependence and their response to a BK channel blocker or opener. While METH suppressed the amplitude of BK channel-mediated unitary currents, the BK channel opener NS1619 attenuated the effects of METH on action potential broadening, afterhyperpolarization repression, and spontaneous spike activity reduction. Live-cell total internal reflection fluorescence microscopy, electrophysiology, and biochemical analysis suggest METH exposure decreased the activity of BK channels by decreasing BK-α subunit levels at the plasma membrane.

Significance statement: Methamphetamine (METH) competes with dopamine uptake, increases dopamine efflux via the dopamine transporter, and affects the excitability of dopamine neurons. Here, we identified an unexpected property of METH on dopamine neuron firing activity. METH transiently increased the spontaneous spike activity of dopamine neurons followed by a progressive reduction of the spontaneous spike activity. METH broadened the action potentials, increased coefficients of variation of the interspike interval, and decreased the amplitude of afterhyperpolarization, which are consistent with changes in the activity of Ca²⁺-activated potassium (BK) channels. We found that METH decreased the activity of BK channels by stimulating BK-α subunit trafficking. Thus, METH modulation of dopamine neurotransmission and resulting behavioral responses is, in part, due to METH regulation of BK channel activity.

Keywords: BK channels; Ca-activated K channels; amphetamines; dopamine; dopamine neurons; dopamine transporter.

Figure 1.

METH produces a biphasic effect on the spontaneous firing activity of midbrain dopaminergic neurons. A, Top, Representative trace of a spontaneously active dopamine neuron before (baseline, no drug) and after application of 10 μm METH. Middle, Rate histogram of the trace shown in the top panel, in which each bar represents the total spikes within each 10 s interval. Bottom, Representative image of RFP::TH-labeled (left) and neurobiotin-labeled (middle) neurons. Merged image is shown in the right panel (scale bar, 20 μm). B, C, Data are mean ± SEM; the spontaneous firing rate is decreased after prolonged (>4 min) METH treatment. D, E, Mean ± SEM; METH significantly increased the spike half-width. F, Mean ± SEM; interspike intervals were calculated over 1 min of spontaneous firing activity (baseline, no drug). METH-treated neurons exhibited larger coefficients of variation of the interspike interval compared with the baseline, suggesting METH exposure produced a variable firing pattern. In B, D, and F, left y-axis shows absolute values, right y-axis shows normalized values. *p < 0.05; **p < 0.01 (n = 10 per group).

Figure 2.

Single-spike analyses suggest that METH and paxilline modulate the repolarization and AHP. A, Representative traces of single AP at baseline (blue trace) and following METH treatment (10 μm, red trace). B, Representative traces of single APs at baseline (blue trace) and following blockade of BK channels with paxilline (10 μm, green trace). C, Data are mean ± SEM; while METH treatment decreased the half-amplitude of APs, paxilline increased the half-amplitude of APs. D, Mean ± SEM; compared with the baseline untreated condition, both METH and paxilline exposure broadened the spike half-width. E, Mean ± SEM; METH or paxilline decreased the peak amplitude of AHP. Boxplot whiskers indicate maximum and minimum data points. **p < 0.01 (n = 7 or 8 per group).

Figure 3.

METH or paxilline inhibits Ca²⁺-activated K⁺ (BK) channel-mediated outward currents. A₁, B₁, Families of outward currents were evoked by voltage steps from −90 to +50 mV for 200 ms with 10 mV increments every 5 s from the holding potential of −70 mV. A₂, B₂, Outward currents after METH (10 μm) or paxilline (10 μm) treatment. A₃, B₃, METH-sensitive (A₁ − A₂) and paxilline-sensitive (B₁ − B₂) currents. C, Data are mean ± SEM; from 0 to +50 mV membrane potential, the peak current at baseline was significantly larger than METH-sensitive or paxilline-sensitive currents. From +20 to +50 mV membrane potentials, METH-sensitive currents were larger than paxilline-sensitive currents. D, In cells expressing BK-α subunits, families of outward currents were evoked by voltage steps from −70 to +170 mV for 250 ms with 10 mV increments every 5 s from the holding potential of −70 mV, before and after METH administration. E, Mean ± SEM; shows peak I–V curves before (baseline) and after METH application. *p < 0.05 (n = 5, 8, or 11 per group).

Figure 4.

METH regulates the activity of large-conductance Ca²⁺-activated K⁺ (BK) channels. A, Identification of BK channels in midbrain dopamine neurons. Representative currents recorded from an outside-out patch of dopamine neurons at different holding potentials; unitary upward current deflections are visible at potentials >+20 mV. Second trace from the top depicts paxilline blockade of unitary upward current deflections at +80 mV. B, Representative currents recorded from outside-out patch of dopamine neurons held at +80 mV in the absence of METH (top), the presence of METH (second trace), the presence of METH plus NS1619 (an activator of BK channel; third trace), and the presence of NS1619 alone (fourth trace). C, Data are mean ± SEM; interevent interval (IEI; 1 ms bin) histograms of BK currents obtained from the data recorded at the holding potential of +80 mV for 6 s. The frequency of baseline BK currents as determined from IEIs was significantly decreased after METH treatment, but significantly increased in the presence of METH plus NS1619. D, Mean ± SEM; peak I–V curves were plotted against the membrane potential. The single-channel conductance generated by the linear fit in control condition was 209 pS. METH decreased the peak amplitude of unitary currents compared with control. NS1619 treatment attenuated the effect of METH on the unitary current amplitude. E, Mean ± SEM; the activator of the BK channel, NS1619, alone increased the peak amplitude of unitary currents compared with control, the untreated condition. *p < 0.05 (n = 5, 7, 8, 9, or 10 per group).

Figure 5.

Paxilline decreases the spontaneous firing activity of dopamine neurons. A, Top, Representative trace from a spontaneously active dopamine neuron before and after application of paxilline (10 μm). Bottom, A rate histogram from the above trace. B, C, Data are mean ± SEM; analysis of the frequency of the spontaneous firing activity of dopamine neurons after blockade of BK channels. D, E, Mean ± SEM; blockade of BK channels increases the spike half-width. F, Mean ± SEM; interspike intervals were calculated over 1 min of firing activity. Compared with the baseline drug-free condition, blockade of BK channels exhibited larger coefficients of variation of the interspike interval. In B, D, and F, the left y-axis shows absolute values, and the right y-axis shows normalized values. *p < 0.05; **p < 0.01 (n = 9 per group).

Figure 6.

NS1619 attenuates METH alteration of AP repolarization and AHP. A, B, Representative traces of single APs in dopamine neurons in the absence of drug (blue trace), following METH exposure (10 μm, red trace), and following coadministration of the BK channels activator NS1619 (10 μm, green trace). C, Data are mean ± SEM; METH and METH plus NS1619 truncated the amplitude of APs. D, Mean ± SEM; while METH treatment broadened the spike half-width, concomitant NS1619 treatment attenuated the effect of METH on the spike half-width. E, Mean ± SEM; METH suppressed the AHP amplitude, whereas NS1619 decreased the effect of METH on AHP. Boxplot whiskers indicate maximum and minimum data points. **p < 0.01 (n = 6 per group).

Figure 7.

NS1619 attenuates the effect of METH on the spontaneous firing activity of dopamine neurons. A, Top, Representative trace from a spontaneously active dopamine neuron before and after application of METH followed by NS1619 treatment (10 μm). Bottom, Rate histogram of the trace above. B, C, Data are mean ± SEM; NS1619 diminished the effect of METH on the spontaneous firing frequency. D, E, Mean ± SEM; while METH increased the spike half-width, NS1619 attenuated the effect of METH on the spike half-width. F, Mean ± SEM; the coefficients of variation of the interspike intervals were not significantly different among the three groups: baseline, METH, and NS1619. In B, D, and F, the left y-axis shows absolute values, and the right y-axis shows normalized values. **p < 0.01 (n = 9 per group).

Figure 8.

Simultaneous application of METH and paxilline increased the spontaneous firing activity of dopamine neurons. A, Top, Representative trace from a spontaneously active dopamine neuron before and after application of METH and paxilline. Bottom, Rate histogram from the above trace. B, C, Data are mean ± SEM; analysis of the frequency of the spontaneous firing activity of dopamine neurons with combined treatment of paxilline and METH. D, E, Mean ± SEM; the spike half-width decreased after bath application of combined paxilline and METH. F, Interspike intervals were not significantly different. In B, D, and F, the left y-axis shows absolute values, and the right y-axis shows normalized values. **p < 0.01 (n = 11 per group).

Figure 9.

METH exposure reduces plasma membrane localization of the α subunit in dopamine neurons. A, Representative immunofluorescence labeling of the endogenous α subunits of BK channels and cholera toxin subunit B-labeled (CTxB) GM1-positive microdomains at the plasma membrane of dopamine neurons. B, Data are mean ± SEM; colocalization of α subunits and CTxB in the soma and neurites of dopamine neurons before and after METH treatment. C, Western blot analysis detects the α subunit of BK channels in the midbrain and striatal tissue, two brain regions containing dopamine neurons. **p < 0.01 (n = 18, 20 or 29 per group).

Figure 10.

METH or PMA internalizes α subunits, while BIM-1 inhibition of PKC prevented internalization of the GFP-α subunit. A, Representative TIRF microscopy images of the GFP-α subunit at the plasma membrane of HEK 293 cells following vehicle, 10 μm METH, 1 μm BIM-1 plus METH, or 0.1 μm PMA treatment. B, C, Data are mean ± SEM; analyses of relative fluorescent intensities at the surface membrane following vehicle, METH, METH plus BMI-1 or PMA treatments. N = 5 or 7 per group.