Role of α6-Nicotinic Receptors in Alcohol-Induced GABAergic Synaptic Transmission and Plasticity to Cholinergic Interneurons in the Nucleus Accumbens

Abstract

The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system, consisting of DA neurons in the ventral tegmental area (VTA) that project to the nucleus accumbens (NAc), underlies the reward properties of ethanol (EtOH) and nicotine (NIC). We have shown previously that EtOH and NIC modulation of DA release in the NAc is mediated by α6-containing nicotinic acetylcholine receptors (α6*-nAChRs), that α6*-nAChRs mediate low-dose EtOH effects on VTA GABA neurons and EtOH preference, and that α6*-nAChRs may be a molecular target for low-dose EtOH. However, the most sensitive target for reward-relevant EtOH modulation of mesolimbic DA transmission and the involvement of α6*-nAChRs in the mesolimbic DA reward system remains to be elucidated. The aim of this study was to evaluate EtOH effects on GABAergic modulation of VTA GABA neurons and VTA GABAergic input to cholinergic interneurons (CINs) in the NAc. Low-dose EtOH enhanced GABAergic input to VTA GABA neurons that was blocked by knockdown of α6*-nAChRs. Knockdown was achieved either by α6-miRNA injected into the VTA of VGAT-Cre/GAD67-GFP mice or by superfusion of the α-conotoxin MII[H9A;L15A] (MII). Superfusion of MII blocked EtOH inhibition of mIPSCs in NAc CINs. Concomitantly, EtOH enhanced CIN firing rate, which was blocked by knockdown of α6*-nAChRs with α6-miRNA injected into the VTA of VGAT-Cre/GAD67-GFP mice. The firing rate of CINs was not enhanced by EtOH in EtOH-dependent mice, and low-frequency stimulation (LFS; 1 Hz, 240 pulses) caused inhibitory long-term depression at this synapse (VTA-NAc CIN-iLTD) which was blocked by knockdown of α6*-nAChR and MII. Ethanol inhibition of CIN-mediated evoked DA release in the NAc was blocked by MII. Taken together, these findings suggest that α6*-nAChRs in the VTA-NAc pathway are sensitive to low-dose EtOH and play a role in plasticity associated with chronic EtOH.

Keywords: Alcohol; Alpha6 nAChRs (α6*-nAChRs); Cholinergic interneurons (CINs); Nicotine (NIC); Nicotinic acetylcholine receptors (nAChRs).

Fig. 1

Depletion of α6-nAChRs in midbrain DA neurons by α6-miRNA. A Illustration showing the experimental framework wherein AAV-flex-mRuby2-α6miRNA was injected into the VTA of DAT-Cre mice 3 weeks prior to experimentation. B Fluorescent in situ hybridization images of neurons (DAPI; blue) from the VTA showing knockdown of Chrna6 (red; α6-nAChRs) with DAergic neurons in the midbrain identified by TH (green) and distinguished from GAD67 + neurons (cyan). C α6-miRNA treatment depleted α6-nAChRs in TH + neurons, but not α4 or β3 nAChRs

Fig. 2

EtOH enhances evoked VTA GABA neuron IPSCs with block by α-conotoxins or knockdown of α6*-nAChRs. A Illustration showing the experimental framework wherein AAV-flex-mRuby2-α6miRNA was injected into the VTA 3 weeks prior and VTA GABA neurons were recorded in whole-cell, voltage-clamp mode in VGAT-Cre/GAD67-GFP mice. B Immunohistochemical panel showing that some VTA GABA neurons express α6*-nAChRs. An antibody against tyrosine hydroxylase (TH) was used along with the GAD67-GFP in these mice. Imaged using oil immersion 40 × objective (Olympus, UPlanFLN 1.30 numerical aperture). C Low-dose EtOH (5 mM) enhanced eIPSC amplitudes in VTA GABA neurons in VGAT-Cre/GAD67-GFP mice injected with scrambled α6-miRNA into the VTA, but not mice injected with α6-miRNA. There was no effect on paired-pulse ratio (50 ms), as previously reported [40]. Knockdown (KD) of α6*-nAChRs (D) or superfusion of MII (E) significantly reduced EtOH enhancement of VTA GABA neuron eIPSCs. Values in parentheses are n values. Asterisks ** indicate significance level p < 0.01

Fig. 3

EtOH inhibits miniature IPSCs with block by α-conotoxins. A Illustration showing the experimental framework wherein AAV-flex-mRuby2-α6-miRNA was injected into the VTA 3 weeks prior and NAc CINs were recorded in whole-cell, voltage-clamp mode in VGAT-Cre/GAD67-GFP mice. B Immunohistochemical image showing that putative CINs in VGAT-Cre/GAD67-GFP mice are innervated by VTA-NAc GABAergic projections expressing α6*-nAChRs. Imaged using oil immersion 60 × objective (Olympus, PlanApo 1.40 numerical aperture). C Illustration showing the experimental framework wherein CINs were recorded in ChAT-ChR2-eYFP mice. D Image showing CINs in ChAT-ChR2-eYFP mice with fluorescence imaging (Olympus, UPlanFLN 1.30 numerical aperture). E Patch clamp recording of CINs in ChAT-ChR2-eYFP mice using infrared imaging. F Representative 10-s recordings of CIN mIPSCs. G Ethanol (5–80 mM) reduced CIN mIPSC frequency. H Ethanol (5–80 mM) did not affect CIN mIPSC amplitude. I, J Summary of effects of MII on EtOH inhibition of CIN mIPSCs. MII significantly reduced EtOH inhibition of CIN mIPSC frequency (I), but not amplitude (J). Asterisks * and ** indicate significance levels p < 0.05 and p < 0.01

Fig. 4

EtOH markedly enhanced CIN firing rate with block by α-conotoxins or knockdown of α6*-nAChRs. A Illustration showing the experimental framework wherein AAV-flex-mRuby2-α6-miRNA was injected into the VTA 3 weeks prior and NAc CINs were recorded in cell-attached, voltage-clamp mode in VGAT-Cre/GAD67-GFP mice. B Immunohistochemical panel showing that CINs in VGAT-Cre/GAD67-GFP mice are innervated by VTA-NAc GABAergic projections expressing α6*-nAChRs. Imaged using oil immersion 40 × objective (Olympus, UPlanFLN 1.30 numerical aperture). C CINs visualized in ChAT mice were characterized by autoreceptor inhibition by the M2 agonist muscarine. Insets are 5-s representative spike recordings before (a), during (b), and after (c) muscarine (wash). Times for (a, b, c) are indicated on the representative ratemeter below. D In putative CINs inhibited by muscarine in VGAT-Cre mice, firing rate is enhanced by increasing doses of EtOH. E Block of EtOH enhancement of CIN firing rate at high doses by MII. F Block of EtOH enhancement of CIN firing rate in mice injected with AAV-flex-mRuby2-α6-miRNA into the VTA. G Summary of effects of α6-miRNA or MII on CIN baseline firing rate. H Summary of EtOH effects in mice treated with MII or α6-miRNA. Values in parentheses are n values. Asterisks *, **, and *** indicate significance levels p < 0.05, p < 0.01, and p < 0.001, respectively

Fig. 5

Tolerance to EtOH enhancement of CIN firing rate in EtOH-dependent mice. A This ratemeter shows a representative ratemeter recording of a CIN in a mouse during withdrawal from CIE exposure. B Chronic EtOH exposure produced tolerance to EtOH enhancement of CIN firing rate. Values in parentheses are n values. Asterisks *, **, and *** indicate significance levels p < 0.05, p < 0.01, and p < 0.001, respectively

Fig. 6

Role of α6*-nAChRs and atypical GABA receptors in CIN inhibitory plasticity. A Illustration showing the experimental framework, wherein Cre-dependent AAV-DIO-ChR2-mCherry was injected into the VTA in VGAT-Cre/GAD67-GFP mice and optically evoked IPSCs were recorded in NAc CINs in whole-cell, voltage-clamp mode. Activation of GABAergic inputs to NAc CINs from the VTA were obtained by blue light stimulation through the objective. B Inset shows representative oIPSCs evoked in NAc CINs before and after low frequency optical stimulation (LFS; 1 Hz, 240 pulses). LFS reduced GABAergic oIPSCs in NAc CINs, termed CIN-iLTD. Horizontal markers pre (green) and post (red) indicate times where comparisons between treatment conditions were performed. C Superfusion of MII or treatment with α6-miRNA reduced CIN-iLTD. D Superfusion of the GABA receptor ρ−1 antagonist TPMPA markedly reduced CIN-iLTD. E Chronic exposure to EtOH reduced CIN-iLTD and produced a mild LTP state. F Summary of drug and treatment effects on CIN-iLTD. Values in parentheses are n values. Asterisks ** and *** indicate significance levels p < 0.01 and p < 0.001, respectively, for comparisons to baseline. Hashtags #, ##, and ### represent significance levels p < 0.05, p < 0.01, and p < 0.001, respectively, for comparisons between control and treatment responses

Fig. 7

Role of α6*-nAChRs in EtOH inhibition of evoked dopamine release in the NAc. A Illustration showing the experimental framework wherein CIN-mediated DA release was recorded following blue light stimulation in ChAT-ChR2-eYFP mice. B, C Current vs time plots show representative DA release evoked by light stimulation before and after superfusion of 60 mM EtOH under control (B) vs MII (C) conditions. Insets show superimposed cyclic voltammograms at the peak of DA release and color plots show cyclic voltammograms over time before and after Con + EtOH vs MII + EtOH. Ethanol inhibits CIN-mediated DA release with block by MII. D Concentration response for EtOH (1–80 mM) effects on CIN-mediated DA release. D Ethanol significantly inhibited DA release at 60–80 mM. E Summary of EtOH effects on CIN-mediated DA release. MII significantly reduces EtOH inhibition of CIN-mediated DA release. Asterisks * and *** indicate significance levels p < 0.05, and p < 0.001, respectively

Fig. 8

Theoretical framework for the involvement of α6*-nAChRs in EtOH effects on DA release in the mesolimbic pathway. Dopamine neurons in the VTA project to the NAc (blue). Dopamine release in the NAc is modulated by CINs (brown). Dogma maintains that α6*-nAChRs are located on DAergic terminals in the NAc, and we have shown previously that they are also expressed on GABA terminals to some VTA GABA neurons (green). Based on our findings demonstrating enhancement of CIN firing rate by EtOH via α6*-nAChRs, we hypothesize that the enhancement of DA release by EtOH, and ultimately EtOH reward, is a result increased NAc CIN activity subsequent to reduction of VTA-NAc GABAergic transmission