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Published in final edited form as: Biochem Pharmacol. 2013 Jun 26;86(8):10.1016/j.bcp.2013.06.015. doi: 10.1016/j.bcp.2013.06.015

Nicotinic Acetylcholine Receptors containing the α6 subunit contribute to ethanol activation of ventral tegmental area dopaminergic neurons

Liwang Liu a, Rubing Zhao-Shea a, J Michael McIntosh b, Andrew R Tapper a
PMCID: PMC3842157  NIHMSID: NIHMS500482  PMID: 23811312

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.

1. Introduction

Nicotine and alcohol are the predominant co-abused drugs in the world. A significant proportion of alcoholics smoke and the majority of smokers (~60 %) binge drink or consume significant amounts of alcohol [1, 2] suggesting a shared mechanism of action between ethanol and nicotine, the addictive component of cigarette smoke. In the mesocorticolimbic “reward” circuitry of the brain, both drugs stimulate dopaminergic (DAergic) neurons in the ventral tegmental area (VTA), to elicit drug reinforcement [3-6].

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that, are activated by the endogenous neurotransmitter, ACh [7], as well as the tertiary alkaloid, nicotine. Twelve vertebrate genes encoding neuronal nAChR subunits have been identified (α2-α10, β2-β4) with five subunits coassembling to form a functional receptor [7]. High affinity nAChRs are heteromeric and consist of two or three α subunits coassembled with two or three β subunits. Low affinity receptors are mostly homomeric, predominantly consisting of α7 subunits. Several nAChR sub-types contribute to nicotine-mediated activation of DAergic neurons and reward/reinforcement including α4β2* (* indicates other unidentified subunits may coassemble with the indicated subunits), α6β2*, and α4α6β2* [8-13]. Of the critical VTA nAChR subunits, α6 is predominantly expressed in DAergic neurons [14, 15].

While ethanol is not a direct agonist of nAChRs, ethanol may induce an increase in ACh release from cholinergic neuron inputs into the VTA, thereby driving an increase in DAergic neuron activity via cholinergic signaling through nAChRs [16]. Interestingly, VTA infusion of α-conotoxin MII, a nAChR antagonist selective for α6, α3, or β3* nAChRs reduces ethanol induced NAc DA release, ethanol consumption and reinforcement in rodents [17, 18]. In addition, VTA DAergic neurons activated by acute ethanol express greater α6 nAChR subunit transcripts compared to DAergic neurons not activated by ethanol [19]. Interestingly, polymorphisms in CHRNA6, the gene encoding the α6 subunit has been associated with alcohol consumption in a representative sample of patients in the United States [20]. More recently, it has been shown that BAC transgenic mice expressing mutant α6 nAChR subunits that render α6* nAChRs hypersensitive to ACh consume more ethanol and are more sensitive to ethanol reward[21]. While these data implicate a role for α6* nAChRs in ethanol consumption and reinforcement, a direct involvement of this nAChR subtype in ethanol-induced activation of VTA DAergic neurons has not been demonstrated.

We sought to test the hypothesis that α6* nAChRs may be involved ethanol-induced activation of VTA DAergic neurons. Using a combination of pharmacology, genetics, and electrophysiology in a mouse midbrain slice preparation, we demonstrate that nAChRs containing the α6 subunit contribute to ethanol-mediated activation of VTA DAergic neurons.

2. Materials and Methods

Animals

α6 knockout (KO) homozygous mice and wild-type (WT) litter-mates (6-10 weeks of age) were used in all experiments. The α6 KO line has been back-crossed to the C57BL/6J strain at least nine generations. WT and KO animals were derived from heterozygous crosses. The genetic engineering of the α6 KO line has been described previously [14]. Animals were housed four/cage until the start of each experiment. Animals were kept on a standard 12-h light/dark cycle with lights on at 7:00 AM and off at 7:00 PM. Mice had access to food and water ad libitum. All experiments were conducted in accordance with the guidelines for care and use of laboratory animals provided by the National Research Council [22], as well as with an approved animal protocol from the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School.

2.1 Slice preparation

Mice were anesthetized by intraperitoneal (i.p.) injection of sodium pentobarbital (200 mg/kg, Vortech Pharmaceuticals, Dearborn, MI) and then decapitated. Brains were quickly removed and placed in an oxygenated ice-cold high sucrose artificial cerebrospinal fluid (SACSF) containing kynurenic acid (1 mM, Sigma, St. Louis, MO). Sagittal brain slices containing VTA and lateral dorsal tegmental brain regions (~180 μm) were made using a Leica VT1200 vibratome. The brain slices were incubated in oxygenated EBSS solution supplemented with glutathione (1.5 mg/ml, Sigma), N-ω-nitro-L-arginine methyl ester hydrochloride (2.2 mg/ml, Sigma), pyruvic acid (11 mg/ml, Sigma) and kynurenic acid (1 mM, Sigma) for 45 minutes at 34°C. Slices were transferred into oxygenated artificial cerebrospinal fluid (ACSF) at room temperature (24°C) for recording. SACSF solution contains (in mM, all chemicals purchased from Sigma): 250 sucrose, 2.5 KCl, 1.2 NaH2PO4•H2O, 1.2 MgCl2•6H2O, 2.4 CaCl2•2H2O, 26 NaHCO3, 11 D-Glucose. ACSF solution contains (in mM): 125 NaCl, 2.5 KCl, 1.2 NaH2PO4•H2O, 1.2 MgCl2•6H2O, 2.4 CaCl2•2H2O, 26 NaHCO3, 11 D-Glucose.

2.2 Electrophysiological recordings

Individual slices were transferred to a recording chamber continually superfused with oxygenated ACSF (30–32 °C) at a flow rate of ~ 2 ml/min. Cells were visualized using infrared differential interference contrast (IR–DIC) imaging on an Olympus BX-50WI microscope. Electrophysiological recordings were recorded using a Multiclamp 700B patch-clamp amplifier (Axon Instruments, Foster City, CA). Action potentials (APs) were obtained in the cell-attached configuration and gap-free acquisition mode in Clampex (Axon Instruments).

Ih currents in whole-cell configuration were elicited every 20 s by stepping from -60 mV to a test potential of -120 mV. APs and currents were filtered at 1 kHz using the amplifier's four-pole, low-pass Bessel filter, digitized at 10 kHz with an Axon Digidata 1440A interface and stored on a personal computer. The junction potential between the patch pipette and bath ACSF was nullified just prior to obtaining a seal on the neuronal membrane. At the end of recording, the cytoplasm was sucked into the recording pipette and the contents in the pipette were expelled into a microfuge-tube for single-cell PCR experiments to verify tyrosine hydroxylase (TH) expression as previously described [13].

Pipette solution contained (in mM, all chemicals purchased from Sigma): 121 KCl, 4 MgCl2•6H2O, 11 EGTA, 1 CaCl2•2H2O, 10 HEPES, 0.2 GTP and 4 mM ATP. Pipette solution was made with DEPC-treated distilled water to prevent RNA degradation.

ACSF was used for bath solution. Ethanol was added into ACSF. Ethanol, ACh, and α-conotoxin MII[E11A] were applied to each slice by gravity superfusion. α-conotoxin MII[E11A] was synthesized as previously described [23]. APs were recorded in the presence of a cocktail of inhibitors (Sigma) including atropine (1 μM) to block muscarinic receptors, bicuculline (20 μM) to block GABAA receptors, and CNQX (10 μM) to block AMPA receptors.

2.3 Data analysis

AP spikes were detected using a threshold detection protocol contained within pClampfit (pClamp v10.2, Axon Inst., Molecular Devices). Average fold change in AP frequency are presented as means ± standard errors of means (SEM). Paired t-tests were used to analyze differences between AP frequency at baseline (1 minute prior to drug application) and after 10 min application of drug. Unpaired t-tests were used to analyze data between genotypes or drug treatment unless otherwise indicated. Results were considered significant at p < 0.05.

3. Results

To test the involvement of α6* nAChRs in ethanol-mediated activation of VTA DAergic neurons, we recorded from VTA DAergic neurons in midbrain slices from mice that did not express CHRNA6, the gene encoding the α6 subunit (α6 KO mice) and their wild-type (WT) littermates. Slices containing the VTA were cut in the sagittal plane (Fig. 1A). DAergic neurons typically displayed a relative slow firing rate (1-5 hz, Fig. 1B) and a slow inward current upon hyperpolarization (Fig. 1C). DAergic neuron identity was verified after recording by detection of tyrosine hydroxylase (TH) mRNA as determine by single neuron RT-PCR (Fig. 1D). To test the effects of ethanol on DAergic neuron activity, AP frequency was monitored in cell-attached mode at baseline, during application of an intoxicating concentration of ethanol (50 or 100 mM), and after washout. Because the focus of our experiments was to uncover the contribution of nAChR activation in response to ethanol, recordings were made in the presence of a cocktail of inhibitors to block, muscarinic receptor, AMPA receptor, and GABAA receptor activity (see methods). No significant difference in baseline firing frequency of DAergic neurons was observed between WT and α6 KO animals (5.0 ± 1.8 and 4.0 ± 1.0 Hz, respectively). In WT VTA DAergic neurons, ten minute bath application of both ethanol concentrations produced an increase in AP frequency (~33 % and 35 % increase from baseline, p < 0.001, respectively, Fig 2A, D) that was completely reversed upon wash out. In contrast, ethanol did not significantly increase VTA DAergic neuron activity above baseline in α6 KO mice (Fig. 2B, D) in response to either concentration of ethanol. To simulate a more in vivo situation, responses to 100 mM ethanol were recorded in WT and α6 KO DAergic neurons in the absence of the inhibitor cocktail. In WT slices, 10 min bath application of 100 mM ethanol increased DAergic neuron AP frequency 1.42 ± 0.10 fold compared to baseline (n = 8). In contrast 100 mM ethanol increased AP frequency significantly less in α6 KO VTA DAergic neurons, (1.03 ± 0.057 fold, n = 7) compared to WT (p < 0.01, data not shown).

Figure 1.

Figure 1

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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, Ih. 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.

Figure 2

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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.

To further test the hypothesis that activation of α6* nAChRs was necessary for the observed ethanol-mediated increase in VTA DAergic neuron activity, we bath applied 100 nM α-conotoxin MII[E11A] (α-CTX-MII[E11A]), an α6* nAChR subtype-selective antagonist [23], prior to and during application of ethanol (Fig. 2C, D) in WT slices. In the presence of α-CTX-MII[E11A], both 50 mM and 100 mM ethanol failed to significantly increase DAergic neuron AP frequency above baseline (Fig. 2C, D). Together these data suggest that activation of nAChRs containing the α6 subunit is necessary for the observed effects of ethanol on VTA DAergic neurons in midbrain slices.

Previously, we demonstrated that ethanol potentiates the DAergic neuron response to ACh in midbrain slices[24]. To test the contribution of α6* nAChRs to this effect we recorded WT and α6 KO VTA DAergic neuron activity in response to bath application of 300 μM ACh alone or in combination with 50 mM ethanol. When applied alone, ACh increased AP frequency of VTA DAergic neurons in WT and α6 KO slices (~20 % and 12 %, respectively, Fig. 3A, B, F). Two-way repeated measures ANOVA revealed a main effect of ACh on AP frequency over time (F18,180 = 3.96, p < 0.001, Fig. 4A) but not an effect of genotype. Co-application of ACh and ethanol in WT slices robustly increased DAergic neuron activity and this increase was significantly greater than ACh alone (p < 0.05, Fig. 3C, F). In contrast, VTA DAergic neuron activity in response to co-application of ACh and ethanol in α6 KO slices was significantly decreased compared to WT after co-application (p < 0.001, Fig. 3D, F). In addition, two-way repeated measures ANOVA revealed a main effect of ACh plus ethanol on AP frequency over time (F13,156 = 4.63, p < 0.001) an effect of genotype (F1,12 =16.05, p <0.001), and a significant time × genotype interaction (F13, 156 = 5.60, p < 0.001, Fig. 4B). Interestingly, DAergic neuron activity in response to ACh and ethanol was also significantly reduced in α6 KO slices compared to ACh alone (Fig. 3F). Finally, we also recorded the effect of ACh and ethanol on WT VTA DAergic neuron activity in the presence of α-CTX-MII[E11A].

Figure 3.

Figure 3

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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.

Figure 4

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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.

DAergic neuron activity in response to ACh and ethanol was reduced by pre-application of α-CTX-MII[EllA] compared to activity in response to ACh and ethanol alone (Fig. 3E, F). Two-way repeated measures ANOVA also revealed a main effect of ACh plus ethanol on AP frequency over time (F13, 221 = 16.1, p < 0.001) an effect of α-CTX-MII[EllA] treatment (F1,17 =15.83, p <0.001), and a significant time × α-CTX-MII[EllA] treatment interaction (F13, 156 = 4.30, p < 0.001, Fig. 4B). Together, these data indicate that α6* nAChR activation is necessary for ethanol potentiation of ACh response in VTA DAergic neurons.

4. Discussion

Previous studies indicate that cholinergic signaling through neuronal nAChRs may play a role in ethanol reinforcement and ethanol-induced DA release in NAc [25]. For example, systemic or VTA infusion of the non-specific nicotinic antagonist mecamylamine has been shown to reduce ethanol consumption, reinforcement, and ethanol-mediated NAc DA release in rodents and also subjective euphoric responses to ethanol in humans [26-30].

Here we show that expression and activation of nAChRs containing the α6 subunit are necessary for ethanol activation of VTA DAergic neurons in mouse midbrain slices. Ethanol did not significantly increase DAergic neuron firing frequency in α6 KO slices and the increase in DAergic neuron activation observed in WT slices was significantly reduced by an α6* nAChR selective antagonist. One caveat to the results from the present study is that all experiments were done in midbrain slices where critical inputs necessary for ethanol action may be severed. In addition, drugs of abuse including ethanol induce burst firing of VTA DAergic neurons which is difficult to detect in slice preparations [31]. Finally, because the focus of our experiments was on cholinergic signaling through nAChRs in DAergic neurons, glutamatergic, GABAergic, and muscarinic signaling was blocked in the majority of experiments. This could influence DAergic neuron activity in response to ethanol [32-34]. Because of these methodological limitations, in vivo electrophysiology and single unit recordings preferably in awake behaving animals will need to be done to verify a role for α6* nAChR in ethanol activation of the VTA.

α6* nAChRs have recently emerged as unique mediators of nicotine reinforcement and VTA DAergic neuron activation [10-12]. They are almost exclusively expressed in midbrain DAergic neurons [14, 15, 35] but may also be expressed in VTA GABAergic presynaptic terminals [36]. Previous studies have shown that infusion of the non-mutated version of α-conotoxin MII, a nAChR antagonist selective for α3β2*, β3*, and α6* subtypes (as opposed to α-conotoxin MII[E11A] used in the present study that is selective for α6* nAChRs), reduces ethanol consumption and reinforcement in rats and NAc DA release in mice [17, 18, 37]. Our data suggest that behavioral effects of α-conotoxin MII infusion in the VTA are most likely influenced by direct blockade of ethanol-mediated DAergic neuron activation through α6* nAChRs. If α6* nAChRs are critical for ethanol-mediated activation of VTA DAergic neurons, then one would expect that α6 KO mice would exhibit reduced ethanol-mediated behaviors associated with reward such as consumption. However, a recent study found no difference in ethanol consumption or preference in a two bottle 24 hr access model of ethanol intake between α6 KO mice and WT littermates [38]. In contrast, Powers et al. determined that mice expressing α6* nAChRs hypersensitive to ACh consume more ethanol than WT littermates and condition a place-preference in response to sub-reward threshold doses of ethanol indicating a role for α6* nAChRs in ethanol reward behavior [21]. Because ethanol is a non-specific drug and interacts with multiple gene targets including those that modulate DAergic neuron activity [4, 39], caution is warranted in interpreting these apparent discrepant results. In the absence of α6* nAChR expression, compensatory mechanisms may occur such that ethanol reward is maintained [14, 40]. In addition, behavioral assays used to assess ethanol reward value such as conditioned place preference and two bottle choice tests may be influenced by ethanol effects on non-reward behaviors such as anxiety which may be altered in nAChR mouse models.

In the future, it will be critical to identify the relevant nAChR subunits that coassemble with α6 to form the nAChRs that mediate ethanol-induced activation of DAergic neurons. Previous studies indicate that the β3 subunit almost exclusively co-assembles with α6 subunits in DAergic neurons and the β2 subunit is required for functional α6* nAChRs [14, 41]. In addition, two distinct sub-types of α6* nAChRs have been identified: α4α6β2β3* and α6(non-α4)β2β3* [35, 42]. Both subtypes are expressed at the level of VTA DAergic neuron soma [12, 13, 43]. Interestingly, we have previously demonstrated that DAergic neurons more sensitive to ethanol-induced activation over-express transcripts encoding α4, α6, and β3 nAChR subunits compared to less alcohol sensitive DAergic neurons [19]. We have also shown that α4* nAChRs contribute to ethanol activation of VTA DAergic neurons to influence alcohol reward [24]. Combined with the findings from the results of Powers et al. discussed above, it is possible that α4α6β2β3 nAChRs may significantly contribute to ethanol-induced activation of VTA DAergic neurons. Based on these data, selective antagonists, partial agonist, or desensitizing agents that target α6* nAChR subtype might have therapeutic value to help reduce ethanol consumption or at least blunt acute ethanol-induced reinforcement. Several nAChR ligands that reduce ethanol intake in animal studies have been identified including cytisine, lobeline, sazetidine-A, CP-601932, PF-4575180, and varenicline [29, 44-46]. Of these compounds, at least cytisine and varenicline can interact with α6* nAChRs potentially contributing to their effects on ethanol consumption; whereas the ability of the other ligands to interact with α6* nAChRs has not been tested [47, 48].

Many questions remain regarding the mechanistic bases for ethanol effects on cholinergic signaling within the VTA that leads to activation of DAergic neurons and increased DA release in the NAc. It is hypothesized that ethanol consumption increases ACh concentrations in the VTA (Fig. 5)[16]. It is assumed that the increase in ACh mediated by ethanol originates from cholinergic projection from the lateral dorsal tegmentum or pedunculopontine nucleus but the actual origin of ACh and the mechanism by which ethanol increases ACh in the VTA is unknown. Regardless, if ethanol increases ACh concentrations in the VTA, then this could activate DAergic neurons via nAChRs. Ethanol also potentiates the response to ACh at α4 and α6* and/or α4α6* nAChRs expressed in DAergic neurons which, in turn, could drive activation of VTA DAergic neurons increasing DA release in NAc. Because α4* and α6* nAChRs are also robustly expressed in DAergic neuron terminals which receive ACh from tonically active cholinergic interneurons in striatum [11, 48-51], one would expect that ethanol should also potentiate DA release from DAergic presynaptic terminals but this has yet to be demonstrated. Finally, neuronal nAChRs are also robustly expressed in VTA GABAergic neurons in addition to DAergic neurons but the role of the receptors in ethanol effects in the VTA is unknown [52-54]. Interestingly, a recent study indicates that activation of β2* nAChRs in both VTA GABAergic and DAergic neurons is required for nicotine reinforcement raising the interesting possibility that ethanol action on GABAergic VTA neurons may also be critical for ethanol reward [55].

Figure 5.

Figure 5

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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).

It is important to point out that other brain regions and circuits that modulate VTA function and that express distinct nAChR subtypes also may influence ethanol intake. Of particular interest is the habenular-interpeduncular (Hb-IPN) pathway that robustly expresses unique nAChR subtypes including α3β4* nAChRs [56]. α3β4* nAChR antagonists or partial agonists reduce ethanol consumption in rodent models suggesting that this subtype may also contribute to ethanol intake [45, 57]. As emerging evidence indicates that the Hb-IPN pathway is involved in drug withdrawal symptoms and aversion [58-60], it is possible that, during initial stages of alcoholism driven by reward [61], α6* nAChRs in the VTA are critical. However, with chronic ethanol exposure, neuroadaptations occur such that ethanol consumption is dominated by avoidance of negative, affective withdrawal symptoms which may be mediated by distinct nAChR subtypes in the Hb-IPN pathway (or other circuits). Future experiments should directly test the involvement of the Hb-IPN circuit in ethanol intake and modulation of VTA activity.

Our data indicate that α6* nAChR contribute to ethanol activation of VTA DAergic neurons and are required for ethanol potentiation of ACh responses at VTA DAergic neuron nAChRs in midbrain slices. Because α6* nAChRs also are involved in nicotine reinforcement, these data point toward a common molecular link between ethanol and nicotine-mediated activation of VTA DAergic neurons and suggest α6* nAChRs as therapeutic targets to reduce ethanol consumption.

Acknowledgments

This study was supported by the National Institute on Alcohol Abuse and Alcoholism award number R01AA017656 (ART), the National Institutes of Mental Health and General Medical Sciences R01MH53631 and PO1 GM48677 (JMM). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

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Conflict of Interest: None

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