Cocaine inhibition of nicotinic acetylcholine receptors influences dopamine release

Abstract

Nicotinic acetylcholine receptors (nAChRs) potently regulate dopamine (DA) release in the striatum and alter cocaine's ability to reinforce behaviors. Since cocaine is a weak nAChR inhibitor, we hypothesized that cocaine may alter DA release by inhibiting the nAChRs in DA terminals in the striatum and thus contribute to cocaine's reinforcing properties primarily associated with the inhibition of DA transporters. We found that biologically relevant concentrations of cocaine can mildly inhibit nAChR-mediated currents in midbrain DA neurons and consequently alter DA release in the dorsal and ventral striatum. At very high concentrations, cocaine also inhibits voltage-gated Na channels in DA neurons. Furthermore, our results show that partial inhibition of nAChRs by cocaine reduces evoked DA release. This diminution of DA release via nAChR inhibition more strongly influences release evoked at low or tonic stimulation frequencies than at higher (phasic) stimulation frequencies, particularly in the dorsolateral striatum. This cocaine-induced shift favoring phasic DA release may contribute to the enhanced saliency and motivational value of cocaine-associated memories and behaviors.

Keywords: addiction; mesolimbic; nAChRs; substantia nigra; ventral tegmental area; voltammetry.

Figure 1

Arrangement for electrical stimulation and FCV recording in striatal brain slices. The striatum is easily identified by its anatomical location and the distinct fiber bundles in horizontal brain slices. Local electrical stimulation in the striatum was delivered using a bipolar tungsten electrode. The two tips of the stimulating electrode were ~150 μm away from each other and from the carbon fiber microelectrode (CFM) tip. GP, globus pallidus; IC, internal capsule; SP, septum.

Figure 2

Cocaine inhibits nicotinic currents in midbrain DA neurons from W-T mice. (A,A1) The membrane properties of presumed DA neurons from the lateral VTA or SNc were typical of DA neurons with a strong I_h sag (arrow). Injected current was 40 pA/step. Spikes were partially truncated for display. (A,A2) These midbrain DA neurons were backfilled with neurobiotin (green) and labeled for TH (red). The co-incidence of neurobiotin and TH is shown as yellow (Overlay), confirming the DA neuron identification. (B,B1) Three traces show nicotinic currents induced in a DA neuron by pressure application of ACh (1 mM, 100 ms pulse) under control conditions (left), with 10 μM cocaine present (middle), or with 25 nM DHβ E present (right), which was applied after recovering from cocaine inhibition. The recordings were made in the presence of 1 μM atropine at a holding potential of −70 mV. (B,B2) Examples showing DHβ E inhibits nicotinic currents in DA neurons. (B,B3) The dose-response relationships for inhibition of nAChR currents. The curves through the data were produced with IC₅₀ = 19.8 μM and Hill coefficient = 1.3 for cocaine, and IC₅₀ = 82 nM and Hill coefficient = 1.1 for DHβ E with n = 3–9 per data point.

Figure 3

Cocaine reduces DA release in the striatum from W-T mice. (A) Cocaine inhibits DA release from the dorsolateral striatum evoked by single-pulse (1 p) stimulation measured using fast-scan cyclic voltammetry. The recordings show the prolonged DA signal after DAT inhibition with 5 μM GBR12909 (left trace). DA release was substantially reduced by 10 μM cocaine (middle trace). After recovery from cocaine inhibition, 25 nM DHβ E induced a similar inhibition of the DA signal (right trace). 1 μM sulpiride (D₂-like antagonist) and SKF83566 (D₁-like antagonist) were used to block local interactions in the striatum. (B) The dose-response relationships for inhibition of DA release. The curves through the data were produced with IC₅₀ = 4.3 μM for cocaine and 22 nM for DHβ E both with a Hill coefficient estimated to be 3 (n = 4–8 per data point). Data points with cocaine concentrations higher than 15 μM were not included in the fitting because a component arising from a local anesthetic effect was also present. (C) Likewise, cocaine inhibits DA release evoked by 1 p stimulation from the NAc shell measured using fast-scan cyclic voltammetry in the presence of 5 μM GBR12909, 1 μM sulpiride, and 1 μM SKF83566. (D) The dose-response relationship for inhibition of DA release in the NAc shell.

Figure 4

In mutant mice having cocaine-insensitive DATs, DA release evoked by 1 p stimulation was still inhibited by cocaine (10 μM). (A) In 10 μM cocaine (middle trace) the DA signal was inhibited when compared to the control (left) or after washout of cocaine (right, wash). The dose-response relationship for cocaine inhibition (extreme right) was fitted with a curve through the data having IC₅₀ = 4 μM and Hill coefficient = 3.2. (B) Likewise, cocaine (10 μM) inhibits DA release evoked by 1 p stimulation from the NAc shell. The dose-response relationship for inhibition of DA release in the NAc shell by cocaine (extreme right) was fitted by a curve with IC50 = 5.2 μM and the Hill coefficient = 3.0. The traces were collected in the presence of 1 μM sulpiride, and 1 μM SKF83566.

Figure 5

In β2-subunit KO mice DA release evoked by 1 p stimulation was resistant to inhibition by cocaine (10 μM). (A) All the slices were bathed in 2 μM GBR12909 to block DATs and to remove complications arising from changes in DA reuptake. The control DA signal (left) evoked by 1 p stimulation shows a prolonged duration caused by inhibiting DA reuptake. In 10 μM cocaine (middle trace) there was slight inhibition of the DA signal. In 40 μM cocaine (right trace) there was a much greater inhibition of the DA signal. 1 μM sulpiride and SKF83566 were used to block local DA receptor influences within the striatum. (B) The dose-response relationship for cocaine inhibition of DA release in the dorsolateral striatum of β 2-nAChR KO mice. The curve through the data was produced with IC₅₀ = 26 μM and the Hill coefficient = 3 (red curve) (n = 4–6 slices). For comparison, the black curve from Figure 1 obtained with W-T mice is given to show the substantial decrease in cocaine-mediated inhibition when β 2^* nAChRs were absent. (C) Likewise, cocaine (10 μM) did not, but cocaine (40 μM) did, inhibit DA release evoked by 1 p stimulation from the NAc shell again in the presence of 2 μM GBR12909, 1 μM sulpiride, and 1 μM SKF83566. (D) The dose-response relationships for inhibition of DA release in the NAc shell by cocaine in β 2-subunit KO mice (red curve) (n = 4–5 slices) compared to the W-T mice (black curve).

Figure 6

Concentration-dependent multiple cocaine effects on the DA-release signal evoked by 1 p stimulation in the striatum measured by FCV. (A) In W-T mice DA signals were measured in the dorsolateral striatum while we bath applied cocaine ranging from 50 nM to 80 μM in the absence of GBR12909. At 50 nM to 1 μM, cocaine monotonically increased the amplitude and prolonged the duration of the evoked DA signal, as expected from cocaine's known inhibition of DATs (DAT effect). At cocaine concentrations above 1 μM, the DA peak decreased although the duration was further prolonged. This decrease is hypothesized to arise from cocaine inhibition of nAChRs (nAChR effect). (B) In β 2-nAChR knockout (KO) mice in the absence of GBR12909, the DA signal is not dependent on nAChRs. Note that the mid-range cocaine inhibition of the DA amplitude is absent in these measurements (i.e., the nAChR effect is absent). The difference between cocaine's influences in the absence of β 2-nAChRs is shaded in gray with the dotted curve representing the falling phase in (A). (A1,B1) Examples of the multiple effects induced by different concentrations of cocaine in WT and in β 2-nAChR KO mice. (C) In W-T mice, DA signaling in the NAc shell showed qualitatively similar cocaine effects as seen in the dorsal striatum. (D) In β 2-nAChR KO mice, the DA signal is not dependent on nAChRs. The difference between cocaine's influences in the absence of β 2-nAChRs is shaded in gray with the dotted curve representing the falling phase in (C). In (A–D), n = 4–7 slices for each data points.

Figure 7

Cocaine at high concentrations inhibits voltage-gated I_Na. (A) In 40 μM cocaine, I_Na was inhibited compared to the control or after cocaine washout. Holding potential was −90 mV and the testing potential was 0 mV for 10 ms given every 20 s. Extracellular and intracellular solutions are described in the Materials and Methods section. (B) The dose-response curve for cocaine inhibition of I_Na is shown on the right with a curve through the data with an IC₅₀ = 144 and a Hill coefficient of 1.

Figure 8

Cocaine reduces paired-pulse depression of DA release in W-T but not in β 2-nAChR KO mice. In this figure, P₁ was the 1 stimulus pulse-evoked DA signal shown in black and P₂ (shown in blue) was obtained by subtraction: the two paired pulses-evoked DA (red traces). (A,A1–A3) In W-T mice under control conditions (without inhibition of DATs), paired-pulse depression was strong, and the paired-pulse ratio (PPR, defined as P₂/P₁) was small. In 10 μM cocaine, the PPR significantly increased (n = 6). 1 μM sulpiride and SKF83566 were used in all these experiments to block local DA receptor interactions within the dorsolateral striatum. (B,B1–B3) In β 2-nAChR KO mice (without inhibition of DATs), 10 μM cocaine did not affect the PPR (n = 5). (C,C1–C3) In W-T mice with DAT inhibition by GBR12909 (5 μM) cocaine (10 μM) still enhances the PPR (n = 5). (D,D1–D3) In β 2-nAChR KO mice with inhibition of DATs, 10 μM cocaine did not affect the PPR (n = 5). ^*p < 0.01.

Figure 9

Cocaine increased the phasic to tonic DA ratio in the dorsal striatum of W-T and nAChR KO mice. Sulpiride and SKF83566 both at 1 μM were used to block DA receptor interactions within the striatum. (A,A1) The FCV measurements of DA signals evoked by lower frequency tonic stimulation (4 Hz, 4-pulses, left trace) and by phasic stimulation (20 Hz, 4-pulses, right trace) under control conditions had a phasic to tonic DA signal ratio close to 1. (A,A2) In 5 μM GBR12909 to inhibit DATs selectively (no cocaine), the DA signals evoked by tonic stimulation (left trace) and by phasic stimulation (right trace) showed a phasic to tonic ratio near 1. GBR12909 prolonged the DA signal duration but did not enhance the phasic to tonic DA ratio. (A,A3) In 10 μM cocaine, the DA signals evoked by tonic stimulation (left trace) and phasic stimulation (right trace) showed a larger phasic to tonic ratio. The DA signals were broader because cocaine inhibited DATs and, thus, prolonged the DA signal duration. (B,B1) Cocaine did not increase the phasic to tonic DA ratio in the dorsal striatum in β 2-nAChR KO Mice. The DA signal evoked by a tonic stimulation (4 Hz, 4-pulses, left trace) and phasic stimulation (20 Hz, 4-pulses, right trace) under control conditions. In β 2-nAChR KO mice the phasic to tonic DA signal ratio was larger than 1 and larger than in W-T mice. (B,B2) In 10 μM cocaine, the DA signals evoked by a tonic stimulation (left trace) and by a phasic stimulation (right trace) were prolonged, but the ratio of phasic to tonic signal was unchanged compared to the no cocaine condition. Cocaine did not dose-dependently enhance the phasic to tonic DA ratio in β 2-nAChR KO mice. Sulpiride and SKF83566 both at 1 μM were used to block DA receptor interactions within the striatum. (C) Summary graph showing the PPR under different conditions. ^*p < 0.05, ^**p < 0.01.

Figure 10

Diagram showing that cocaine may inhibit DAT-mediated DA uptake and DA release via nAChRs and Na channels in a dose-dependent manner. Depol., depolarization.