Nicotinic acetylcholine receptors containing the α6 subunit contribute to ethanol activation of ventral tegmental area dopaminergic neurons

Abstract

Nicotine and alcohol are often co-abused suggesting a common mechanism of action may underlie their reinforcing properties. Both drugs acutely increase activity of ventral tegmental area (VTA) dopaminergic (DAergic) neurons, a phenomenon associated with reward behavior. Recent evidence indicates that nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels activated by ACh and nicotine, may contribute to ethanol-mediated activation of VTA DAergic neurons although the nAChR subtype(s) involved has not been fully elucidated. Here we show that expression and activation of nAChRs containing the α6 subunit contribute to ethanol-induced activation of VTA DAergic neurons. In wild-type (WT) mouse midbrain sections that contain the VTA, ethanol (50 or 100 mM) significantly increased firing frequency of DAergic neurons. In contrast, ethanol did not significantly increase activity of VTA DAergic neurons in mice that do not express CHRNA6, the gene encoding the α6 nAChR subunit (α6 knock-out (KO) mice). Ethanol-induced activity in WT slices was also reduced by pre-application of the α6 subtype-selective nAChR antagonist, α-conotoxin MII[E11A]. When co-applied, ethanol potentiated the response to ACh in WT DAergic neurons; whereas co-application of ACh and ethanol failed to significantly increase activity of DAergic neurons in α6 KO slices. Finally, pre-application of α-conotoxin MII[E11A] in WT slices reduced ethanol potentiation of ACh responses. Together our data indicate that α6-subunit containing nAChRs may contribute to ethanol activation of VTA DAergic neurons. These receptors are predominantly expressed in DAergic neurons and known to be critical for nicotine reinforcement, providing a potential common therapeutic molecular target to reduce nicotine and alcohol co-abuse.

Keywords: Acetylcholine; Alcoholism; Dopamine; Nicotinic receptor; Ventral tegmental area.

Figure 1

Characteristics of DAergic neurons in VTA sagittal slices. Illustration of a mouse sagittal section containing the VTA (shaded region). B) Representative cell-attached recording from a putative DAergic neuron. DAergic neurons had characteristic low baseline firing frequencies (1-5 Hz) and as shown in C), expressed the hyperpolarizing activated cation current, I_h. Currents were elicited by a hyperpolarizing step from a holding potential of -60 mV to -120 mV as indicated. D) At the end of each recording, the content of each neuron was aspirated into the patch pipette and TH expression was verified by single-cell real-time PCR. A representative DNA agarose gel is shown illustrating a typical result for a DAergic neuron. Only neurons that clearly expressed TH and not GAD 65/67 were included in the analysis. As a negative control for the PCR, a sample containing no RNA was used (“(-) ctrl”).

Figure 2

A contribution of a6* nAChRs to ethanol activation of VTA DAergic neurons. Representative action potential firing frequency histogram from an A) WT or B) α6 KO VTA DAergic neuron before, during, and after 10 min bath application of 100 mM ethanol (EtOH). C) Representative action potential firing frequency histogram from a WT VTA DAergic neuron before, during, and after 10 min bath application of 100 mM ethanol in the presence of a-CTX-MII[E11A]. Action potentials were recorded in cell-attached mode. Representative action potential traces (bottom of each panel, a, b, c) are shown from the corresponding times on the histograms. D) Fold-change in average firing frequency at baseline (1 min. prior to alcohol application, dotted line) compared to last min of ethanol application for each genotype/condition. ^###p < 0.001 compared to baseline using a paired t-test; **p < 0.01, unpaired t-test. n = 5-8 neurons/condition.

Figure 3

α6* nAChR expression and activation is necessary for alcohol potentiation of DAergic neuron responses to ACh. Representative action potential firing frequency histogram from an A) WT or B) α6 KO VTA DAergic neuron before, during, and after 10-min bath application of 300 μM ACh. Representative action potential traces (bottom of each panel, a, b, c) are shown from the corresponding times on the histograms. Representative action potential firing frequency histogram from a C) WT or D) α6 KO VTA DAergic neuron before, during, and after 10-min bath co-application of 300 μM ACh and 50 mM ethanol. E) Representative action potential firing frequency histogram from a WT VTA DAergic neuron before, during, and after 10 min bath co-application of 300 μM ACh and 50 mM ethanol in the presence of 100 nM α-CTX-MII[E11A]. F) Fold-change in average DAergic neuron firing frequency in response to 300 μM ACh alone in WT (n = 8) or α6 KO slices (n = 6), in the presence of 50 mM ethanol (n = 12 and 11, respectively), or in the presence of 50 mM ethanol and α-CTX-MII[E11A] in WT slices (n = 8). ^#p < 0.05, ^##p<0.01 compared to baseline as in 1D. *p < 0.05, **p < 0.01, ***p < 0.001 response to ethanol compared between treatments/genotypes. ^p < 0.05 compared to ACh in α6 KO DAergic neurons.

Figure 4

Time course of ACh and ACh + ethanol modulation of DAergic neuron activity. The effects of A) ACh alone or B) with ethanol on average normalized AP frequency in WT and α6 KO VTA DAergic neurons under each condition are shown (from Figure 3). The effect of ACh with ethanol in the presence of α-CTX-MII[E11A] in WT VTA DAergic neurons is also shown (red circles). ACh ± ethanol was applied at the times indicated by the bar. Recording times between groups were aligned based on time of ethanol application to facilitate comparison.

Figure 5

Ethanol actions in mesolimbic circuitry. In the VTA, ethanol stimulates DAergic neurons at least, in part, via nAChR activation. Ethanol increases ACh release (red arrow, presumably through cholinergic projection from the LDT/PPTg) which in turn activates nAChRs on DAergic neurons driving activity. Neuronal nAChRs mediating this effect contain α4 and/or α6 subunits. In addition, ethanol potentiates ACh activation at these high affinity nAChRs (red plus sign). The effect of ethanol on additional nAChRs in the mesolimbic reward circuitry including those expressed at DAergic neuron presynaptic terminals and in GABAergic neurons is unknown (red question marks). Ultimately activation of nAChRs in combination with other effects of ethanol in the VTA is hypothesized to increase DA release in NAc (red arrow). Prefrontal cortex (PFC); Lateral dorsal tegmentum (LDT); Pedunculopontine tegmentum (PPTg).