α6 subunit‐containing nicotinic receptors mediate low‐dose ethanol effects on ventral tegmental area neurons and ethanol reward

Abstract

Dopamine (DA) neuron excitability is regulated by inhibitory GABAergic synaptic transmission and modulated by nicotinic acetylcholine receptors (nAChRs). The aim of this study was to evaluate the role of α6 subunit-containing nAChRs (α6*-nAChRs) in acute ethanol effects on ventral tegmental area (VTA) GABA and DA neurons. α6*-nAChRs were visualized on GABA terminals on VTA GABA neurons, and α6*-nAChR transcripts were expressed in most DA neurons, but only a minority of VTA GABA neurons from GAD67 GFP mice. Low concentrations of ethanol (1-10 mM) enhanced GABA receptor (GABA R)-mediated spontaneous and evoked inhibition with blockade by selective α6*-nAChR antagonist α-conotoxins (α-Ctxs) and lowered sensitivity in α6 knock-out (KO) mice. Ethanol suppression of VTA GABA neuron firing rate in wild-type mice in vivo was significantly reduced in α6 KO mice. Ethanol (5-100 mM) had no effect on optically evoked GABA R-mediated inhibition of DA neurons, and ethanol enhancement of VTA DA neuron firing rate at high concentrations was not affected by α-Ctxs. Ethanol conditioned place preference was reduced in α6 KO mice compared with wild-type controls. Taken together, these studies indicate that relatively low concentrations of ethanol act through α6*-nAChRs on GABA terminals to enhance GABA release onto VTA GABA neurons, in turn to reduce GABA neuron firing, which may lead to VTA DA neuron disinhibition, suggesting a possible mechanism of action of alcohol and nicotine co-abuse.

Alpha6-containing nicotinic acetylcholine receptor is a highly sensitive target of alcohol

“…Our new results reported here validate roles for α6*-nAChR subtypes in EtOHinduced behavioral alterations and more specifically illuminate mechanisms involved in effects of low dose EtOH on α6*-nAChRs and on mesolimbic DAergic neuron function. Effects on α6*-nAChR activity are consistent with EtOH action as a positive allosteric modulator and with those actions occurring at EtOH concentrations lower than those associated with EtOH-induced behavioral impairment, consistent with recentlydemonstrated, low dose EtOH-induced modulation of synaptic functions in mouse VTA GABA neurons (Steffensen et al, 2017).…”

“…However, voltammetry cannot spatially differentiate between single and multiple varicosities and it is possible that EtOH is not just enhancing DA release from individual sites. Another important factor that was not tested here is whether EtOH is having effects on cholinergic terminals that may involve other local circuitry influenced by α6*-nAChRs, including potentially VTA projection GABA terminals, which are known to innervate cholinergic interneurons and may express α6*-nAChRs at GABA terminals (Brown et al, 2012;Steffensen et al, 2017). Interestingly, when α-Ctx MII was combined with EtOH, we found a significant decrease in DA transient amplitude, but not frequency, suggesting that EtOH has effects on non-α6*-nAChRs.…”

“…Recently, we have found that low concentrations of EtOH (1-10 mM) enhance GABA A receptor (GABA A R)-mediated spontaneous and evoked inhibition of IPSCs via processes blocked by the α6*-nAChR-selective antagonist α-conotoxin MII (α-Ctx MII) and that there is lowered EtOH sensitivity and reward in nAChR α6 subunit knock-out (KO) mice (Steffensen et al, 2017). Moreover, we have established functional, heterologous expression of α6*-nAChRs by co-transfection of an α6/3 subunit chimera together with β2 and β3 subunits into the human SH-EP1 cell line (Chen et al, 2018).…”

Cocaine Directly Inhibits α6-Containing Nicotinic Acetylcholine Receptors in Human SH-EP1 Cells and Mouse VTA DA Neurons

“…Additionally, in mechanically dissociated VTA GABA neurons, 1 μM nicotine enhanced spontaneous inhibitory postsynaptic current (sIPSC) frequency, which was sensitive to an α6*-nAChR-selective antagonist (α-conotoxin MII, α-Ctx MII, 10 nM). This suggests that the nicotine-activated natural α6*-nAChRs are located on GABAergic presynaptic boutons of VTA GABA neurons as we recently described (Steffensen et al., 2018). Interestingly, cocaine (10 μM) prevented this nicotine-induced enhancement of sIPSCs, suggesting that cocaine also inhibits natural α6*-nAChRs.…”

“…Neurons with functional terminals were obtained by mechanical dissociation as described previously (Akaike and Moorhouse, 2003; Yang et al., 2011; Steffensen et al., 2018). In brief, one midbrain slice was transferred to a 35-mm culture dish (Falcon, Rutherford, NJ) filled with a standard external solution.…”