Alcohol and the Brain: Neuronal Molecular Targets, Synapses, and Circuits
Authors: Karina P. Abrahao, Armando G. Salinas, David M. Lovinger
Journal: Neuron (2017)

Ethanol is one of the most commonly abused drugs. Although environmental and genetic factors contribute to the etiology of alcohol use disorders, it is ethanol's actions in the brain that explain (1) acute ethanol-related behavioral changes, such as stimulant followed by depressant effects, and (2) chronic changes in behavior, including escalated use, tolerance, compulsive seeking, and dependence. Our knowledge of ethanol use and abuse thus relies on understanding its effects on the brain. Scientists have employed both bottom-up and top-down approaches, building from molecular targets to behavioral analyses and vice versa, respectively. This review highlights current progress in the field, focusing on recent and emerging molecular, cellular, and circuit effects of the drug that impact ethanol-related behaviors. The focus of the field is now on pinpointing which molecular effects in specific neurons within a brain region contribute to behavioral changes across the course of acute and chronic ethanol exposure. Ethanol is one of the most commonly abused drugs. Although environmental and genetic factors contribute to the etiology of alcohol use disorders, it is ethanol's actions in the brain that explain (1) acute ethanol-related behavioral changes, such as stimulant followed by depressant effects, and (2) chronic changes in behavior, including escalated use, tolerance, compulsive seeking, and dependence. Our knowledge of ethanol use and abuse thus relies on understanding its effects on the brain. Scientists have employed both bottom-up and top-down approaches, building from molecular targets to behavioral analyses and vice versa, respectively. This review highlights current progress in the field, focusing on recent and emerging molecular, cellular, and circuit effects of the drug that impact ethanol-related behaviors. The focus of the field is now on pinpointing which molecular effects in specific neurons within a brain region contribute to behavioral changes across the course of acute and chronic ethanol exposure. Humans consume and abuse ethanol, and thus understanding ethanol's effects on the nervous system necessarily involves knowing the pharmacology of the drug. This two-carbon molecule is only able to interact with other biomolecules via hydrogen bonding and weak hydrophobic interactions, limiting its potency. Thus, it is no surprise that ethanol has a reputation as a nonspecific drug. Indeed, ethanol's effects on brain function mainly occur across a range from the low millimolar range to 100 mM in naive and occasional users. Ethanol's effects at doses that produce blood ethanol concentrations (BECs) of ∼28 mg/dL (∼6 mM) can be reliably distinguished in humans and animals (Ando, 1975Ando K. The discriminative control of operant behavior by intravenous administration of drugs in rats.Psychopharmacologia. 1975; 45: 47-50Crossref Google Scholar, Schechter, 1980Schechter M.D. Ability of 3-carboxysalsolinol to produce ethanol-like discrimination in rats.Psychopharmacology (Berl.). 1980; 68: 277-281Crossref PubMed Google Scholar). Acute intoxication grows progressively stronger as BECs rise to higher levels associated with anxiolytic and euphoric effects (∼12 mM) and legal intoxication (∼18 mM), where slowed reaction times, motor incoordination, and cognitive impairment occur. At concentrations up to 50 mM, locomotor disruption, cognitive impairment, and sedation escalate. Above this level, strong sedation and respiratory depression can lead to coma or death (Alifimoff et al., 1989Alifimoff J.K. Firestone L.L. Miller K.W. Anaesthetic potencies of primary alkanols: implications for the molecular dimensions of the anaesthetic site.Br. J. Pharmacol. 1989; 96: 9-16Crossref PubMed Google Scholar). According to the 2015 National Survey on Drug Use and Health (NSDUH), injuries and fatalities due to acute intoxication (including toxicity due to respiratory depression), accidents, violence, and traffic fatalities affect tens of thousands of people (Bose et al., 2016Bose, J., Hedden, S.L., Lipari, R.N., Park-Lee, E., Porter, J., and Pemberton, M. (2016). Key substance use and mental health indicators in the United States: results from the 2015 National Survey on Drug Use and Health. Report of the Substance Abuse and Mental Health Services Administration. https://www.samhsa.gov/data/sites/default/files/NSDUH-FFR1-2015/NSDUH-FFR1-2015/NSDUH-FFR1-2015.pdf.Google Scholar, Mokdad et al., 2004Mokdad A.H. Marks J.S. Stroup D.F. Gerberding J.L. Actual causes of death in the United States, 2000.JAMA. 2004; 291: 1238-1245Crossref PubMed Scopus (3446) Google Scholar). Chronic ethanol exposure and alcohol use disorder (AUD) have an even greater negative impact on society, including failed relationships, loss of employment, psychiatric symptoms, overt neurotoxicity, liver failure, and severe cognitive disruption (Bose et al., 2016Bose, J., Hedden, S.L., Lipari, R.N., Park-Lee, E., Porter, J., and Pemberton, M. (2016). Key substance use and mental health indicators in the United States: results from the 2015 National Survey on Drug Use and Health. Report of the Substance Abuse and Mental Health Services Administration. https://www.samhsa.gov/data/sites/default/files/NSDUH-FFR1-2015/NSDUH-FFR1-2015/NSDUH-FFR1-2015.pdf.Google Scholar). These chronic problems consume considerable resources for psychiatric care, organ transplants, and long-term medical treatment. As tolerance to the acute effects of ethanol develops, humans can survive with BECs up to 8 times those that would kill an ethanol-naive person. Indeed, awake individuals with blood concentrations near 300 mM have been reported (Johnson et al., 1982Johnson R.A. Noll E.C. Rodney W.M. Survival after a serum ethanol concentration of 1 1/2%.Lancet. 1982; 2: 1394Abstract PubMed Google Scholar). Overall, the global consequences of AUD include 3.3 million annual deaths (5.9% of all deaths) and 5.1% of the burden of disease and injury, with an economic burden of ∼$250 billion annually in the United States (WHO, 2014WHOGlobal Status Report on Alcohol and Health, 2014. World Health Organization, 2014Google Scholar, Sacks et al., 2015Sacks J.J. Gonzales K.R. Bouchery E.E. Tomedi L.E. Brewer R.D. 2010 national and state costs of excessive alcohol consumption.Am. J. Prev. Med. 2015; 49: e73-e79Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Given this huge societal impact, the US Surgeon General recently issued a first of its kind report, "Facing Addiction in America: The Surgeon General's Report on Alcohol, Drugs, and Health," highlighting alcoholism and addiction (US DHHS, 2016U.S. Department of Health and Human Services (HHS) (2016). Facing addiction in America: the surgeon general's report on alcohol, drugs, and health. Office of the Surgeon General, November, 2016. https://addiction.surgeongeneral.gov.Google Scholar). Thus, a review of the current state of knowledge about ethanol effects on the brain is warranted. In light of this large societal impact, the field seeks to understand how ethanol alters brain function across a range of concentrations and time frames/phases of drinking. Indeed, several phenotypic phases of ethanol consumption and AUD that occur over weeks to years have been proposed (Koob and Volkow, 2016Koob G.F. Volkow N.D. Neurobiology of addiction: a neurocircuitry analysis.Lancet Psychiatry. 2016; 3: 760-773Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). We will focus on the latest findings from neurobiological studies examining acute and chronic ethanol effects on the brain, with emphasis on neuronal molecules, synapses, and brain circuits with important roles in behavioral effects of the drug. The entire scope of the neural actions of ethanol cannot be covered in this limited format (unfortunately including topics such as fetal alcohol effects, ethanol effects on glia, neuroinflammatory mechanisms, and extracellular matrix), but references for some topics are provided to allow the reader to gain a deeper understanding of the field. Ethanol distribution in the body and brain is similar to water, with equilibration throughout organs and cells within a few minutes of drinking. This property contributed to the idea that many of ethanol's effects involve its occupation of water-filled cavities in proteins and subsequent alteration of function. Considering the ubiquity of distribution and low drug potency, ethanol acts on numerous molecular targets in neurons and synapses throughout the brain. This lack of specificity can be daunting to those who study potent and specific drugs, including drugs of abuse with circumscribed primary molecular targets (e.g., opiates). However, even these target-specific drugs produce complex secondary neuroadaptations that contribute to drug use disorders. It is worth noting that the function of many molecules in mammalian neurons appears to be remarkably insensitive to ethanol (Yamakura et al., 2001Yamakura T. Lewohl J.M. Harris R.A. Differential effects of general anesthetics on G protein-coupled inwardly rectifying and other potassium channels.Anesthesiology. 2001; 95: 144-153Crossref PubMed Google Scholar). Thus, earlier ideas about ubiquitous molecular effects due to changes in membrane fluidity are not helpful in understanding how ethanol alters neuronal function (Peoples et al., 1996Peoples R.W. Li C. Weight F.F. Lipid vs protein theories of alcohol action in the nervous system.Annu. Rev. Pharmacol. Toxicol. 1996; 36: 185-201Crossref PubMed Google Scholar). Even if fluidity changes occur, these changes lead to altered neuronal function, and thus, we must examine the proteins that dictate neuronal function. Thankfully, the tools available to modern neuroscientists have enabled examination of ethanol effects at multiple levels. We can now determine how a given molecular effect on a specific neuronal or synaptic subtype contributes to ethanol-induced behavioral changes. Both bottom-up and top-down approaches are being used in such studies. The bottom-up approach builds from the identification of an ethanol-sensitive molecule followed by determination of its role in acute and chronic ethanol changes in physiology and behavior. On the other hand, top-down approaches begin with ethanol-related physiological or behavioral changes leading to the study of specific molecular mechanisms and brain circuits contributing to these effects. Recent theories have posited specific ethanol-binding sites on several proteins that may act directly or indirectly to produce a biological effect (Figure 1). To understand how ethanol affects the brain and behavior using a bottom-up approach, it is important to first distinguish between the direct and indirect effects of ethanol. To that end, the following criteria have been proposed to classify direct ethanol targets (Harris et al., 2008Harris R.A. Trudell J.R. Mihic S.J. Ethanol's molecular targets.Sci. Signal. 2008; 1: re7Crossref PubMed Scopus (145) Google Scholar, Trudell et al., 2014Trudell J.R. Messing R.O. Mayfield J. Harris R.A. Alcohol dependence: molecular and behavioral evidence.Trends Pharmacol. Sci. 2014; 35: 317-323Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar):1.The putative target protein should be affected by ethanol at both low and high concentrations.2.The molecular binding site and ethanol interaction should be characterized biochemically or modeled. Manipulation of the amino acids making up the putative binding site should alter the ethanol interaction and, consequently, the biological effect of ethanol.3.Structural biological evidence should indicate that ethanol inhabits the putative binding site.4.Genetic alteration of the target protein (e.g., knockout) should result in a readily discernable ethanol-related phenotype. These criteria allow for the clear identification of direct ethanol targets. However, the low-affinity and transient molecular interactions of ethanol make fulfillment of all four criteria challenging. Thus, some of the molecular targets we discuss will be referred to as "putative" direct targets to indicate only partial fulfillment of the preceding criteria. Targets that do not meet any of the criteria above or that do not have any molecular structures indicative of an ethanol-binding site are referred to as indirect targets. Low concentrations of ethanol can directly interact with several molecules (Cui and Koob, 2017Cui C. Koob G.F. Titrating tipsy targets: the neurobiology of low-dose alcohol.Trends Pharmacol. Sci. 2017; 38: 556-568Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). The best example of a direct ethanol target (though not brain exclusive) is alcohol dehydrogenase (ADH). Ethanol has been shown to interact with ADH at low millimolar concentrations, the binding site is well characterized, and manipulation of ADH results in biological effects (Goto et al., 2015Goto M. Kitamura H. Alam M.M. Ota N. Haseba T. Akimoto T. Shimizu A. Takano-Yamamoto T. Yamamoto M. Motohashi H. Alcohol dehydrogenase 3 contributes to the protection of liver from nonalcoholic steatohepatitis.Genes Cells. 2015; 20: 464-480Crossref PubMed Scopus (5) Google Scholar). Ethanol has rapid acute effects on the function of proteins involved in excitatory and inhibitory synaptic transmission (Figures 1 and 2). Ethanol generally potentiates cys-loop ligand-gated ion channels (LGICs) (e.g., GABAA and glycine receptors [GlyRs]) but inhibits ionotropic glutamate receptors (reviewed in Lovinger and Roberto, 2013Lovinger D.M. Roberto M. Synaptic effects induced by alcohol.Curr. Top. Behav. Neurosci. 2013; 13: 31-86Crossref PubMed Scopus (57) Google Scholar, Söderpalm et al., 2017Söderpalm B. Lidö H.H. Ericson M. The glycine receptor: a functionally important primary brain target of ethanol.Alcohol. Clin. Exp. Res. 2017; 41: 1816-1830Crossref PubMed Scopus (0) Google Scholar). The different ligand-binding and transmembrane domains of these proteins likely underlie this difference. The current thinking is that ethanol interacts with membrane-spanning domains within these proteins and the subsequent allosteric changes in conformation produced differ for the different LGIC subtypes (Möykkynen and Korpi, 2012Möykkynen T. Korpi E.R. Acute effects of ethanol on glutamate receptors.Basic Clin. Pharmacol. Toxicol. 2012; 111: 4-13PubMed Google Scholar, Olsen et al., 2014Olsen R.W. Li G.D. Wallner M. Trudell J.R. Bertaccini E.J. Lindahl E. Miller K.W. Alkana R.L. Davies D.L. Structural models of ligand-gated ion channels: sites of action for anesthetics and ethanol.Alcohol. Clin. Exp. Res. 2014; 38: 595-603Crossref PubMed Scopus (27) Google Scholar). However, more work is needed to understand the structural basis of these differences. Ethanol also modulates nicotinic acetylcholine receptor (nAChR) function in a subunit-specific manner (Davis and de Fiebre, 2006Davis T.J. de Fiebre C.M. Alcohol's actions on neuronal nicotinic acetylcholine receptors.Alcohol Res. Health. 2006; 29: 179-185PubMed Google Scholar, Hendrickson et al., 2013Hendrickson L.M. Guildford M.J. Tapper A.R. Neuronal nicotinic acetylcholine receptors: common molecular substrates of nicotine and alcohol dependence.Front. Psychiatry. 2013; 4: 29Crossref PubMed Scopus (44) Google Scholar, Rahman et al., 2016Rahman S. Engleman E.A. Bell R.L. Recent advances in nicotinic receptor signaling in alcohol abuse and alcoholism.Prog. Mol. Biol. Transl. Sci. 2016; 137: 183-201Crossref PubMed Scopus (0) Google Scholar) and potentiates 5HT3Rs (McBride et al., 2004McBride W.J. Lovinger D.M. Machu T. Thielen R.J. Rodd Z.A. Murphy J.M. Roache J.D. Johnson B.A. Serotonin-3 receptors in the actions of alcohol, alcohol reinforcement, and alcoholism.Alcohol. Clin. Exp. Res. 2004; 28: 257-267Crossref PubMed Scopus (0) Google Scholar). Primary ethanol-binding sites that fulfill the four criteria have yet to be identified for all of these LGICs, but there is evidence of direct interactions with several of the cys-loop LGICs (Howard et al., 2014Howard R.J. Trudell J.R. Harris R.A. Seeking structural specificity: direct modulation of pentameric ligand-gated ion channels by alcohols and general anesthetics.Pharmacol. Rev. 2014; 66: 396-412Crossref PubMed Google Scholar). For example, the structural basis for a direct interaction of ethanol with the prototypic G. violaceus LGIC has been determined and is thought to be a transmembrane cavity between two membrane-spanning domains (Sauguet et al., 2013Sauguet L. Howard R.J. Malherbe L. Lee U.S. Corringer P.J. Harris R.A. Delarue M. Structural basis for potentiation by alcohols and anaesthetics in a ligand-gated ion channel.Nat. Commun. 2013; 4: 1697Crossref PubMed Scopus (71) Google Scholar). Identifying the expression sites and cellular actions of the subunits of these ethanol-sensitive channels is an important next step in understanding how the molecular effect of ethanol translates into altered neuronal and circuit function. Studies of ethanol interactions with GlyRs are a good example of the bottom-up approach. These studies initially focused on the molecular mechanisms of ethanol's potentiation of GlyRs (Burgos et al., 2015Burgos C.F. Castro P.A. Mariqueo T. Bunster M. Guzmán L. Aguayo L.G. Evidence for α-helices in the large intracellular domain mediating modulation of the α1-glycine receptor by ethanol and Gβγ.J. Pharmacol. Exp. Ther. 2015; 352: 148-155Crossref PubMed Scopus (8) Google Scholar, Mascia et al., 1996Mascia M.P. Machu T.K. Harris R.A. Enhancement of homomeric glycine receptor function by long-chain alcohols and anaesthetics.Br. J. Pharmacol. 1996; 119: 1331-1336Crossref PubMed Google Scholar, Mihic, 1999Mihic S.J. Acute effects of ethanol on GABAA and glycine receptor function.Neurochem. Int. 1999; 35: 115-123Crossref PubMed Scopus (0) Google Scholar, Perkins et al., 2010Perkins D.I. Trudell J.R. Crawford D.K. Alkana R.L. Davies D.L. Molecular targets and mechanisms for ethanol action in glycine receptors.Pharmacol. Ther. 2010; 127: 53-65Crossref PubMed Scopus (34) Google Scholar) and moved to studies of how ethanol's effects on GlyRs contribute to changes in circuit function and behavior (Aguayo et al., 2014Aguayo L.G. Castro P. Mariqueo T. Muñoz B. Xiong W. Zhang L. Lovinger D.M. Homanics G.E. Altered sedative effects of ethanol in mice with α1 glycine receptor subunits that are insensitive to Gβγ modulation.Neuropsychopharmacology. 2014; 39: 2538-2548Crossref PubMed Scopus (13) Google Scholar, Blednov et al., 2012Blednov Y.A. Benavidez J.M. Homanics G.E. Harris R.A. Behavioral characterization of knockin mice with mutations M287L and Q266I in the glycine receptor α1 subunit.J. Pharmacol. Exp. Ther. 2012; 340: 317-329Crossref PubMed Scopus (0) Google Scholar, Blednov et al., 2015Blednov Y.A. Benavidez J.M. Black M. Leiter C.R. Osterndorff-Kahanek E. Harris R.A. Glycine receptors containing α2 or α3 subunits regulate specific ethanol-mediated behaviors.J. Pharmacol. Exp. Ther. 2015; 353: 181-191Crossref PubMed Scopus (12) Google Scholar). Ethanol's effects on GlyR function have been identified in several brain regions (Badanich et al., 2013Badanich K.A. Mulholland P.J. Beckley J.T. Trantham-Davidson H. Woodward J.J. 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Strychnine-sensitive glycine receptors on pyramidal neurons in layers II/III of the mouse prefrontal cortex are tonically activated.J. Neurophysiol. 2014; 112: 1169-1178Crossref PubMed Scopus (13) Google Scholar, Zhang et al., 2008Zhang L.H. Gong N. Fei D. Xu L. Xu T.L. Glycine uptake regulates hippocampal network activity via glycine receptor-mediated tonic inhibition.Neuropsychopharmacology. 2008; 33: 701-711Crossref PubMed Scopus (57) Google Scholar), but the site and basic mechanisms of glycine release remain unclear. The use of genetically engineered mice with alterations in receptor subunit expression or structure (knockouts and knockins) allow investigators to exploit the bottom-up approach and analyze the behavioral consequences of ethanol's effects on specific targets. Mice lacking the GlyR alpha 2 subunit show reduced ethanol intake, but GlyR alpha 3 knockout mice show increased intake (Mayfield et al., 2016Mayfield J. Arends M.A. Harris R.A. Blednov Y.A. Genes and alcohol consumption: studies with mutant mice.Int. Rev. Neurobiol. 2016; 126: 293-355Crossref PubMed Scopus (6) Google Scholar). More evidence of the cellular and brain region location of the GlyRs involved in these behaviors is needed. For example, glycine transporter blockade in the ventral medial prefrontal cortex (PFC) contributes to increased motor impulsivity during protracted abstinence from long-term ethanol exposure (Irimia et al., 2017Irimia C. Buczynski M.W. Natividad L.A. Laredo S.A. Avalos N. Parsons L.H. Dysregulated glycine signaling contributes to increased impulsivity during protracted alcohol abstinence.J. Neurosci. 2017; 37: 1853-1861Crossref PubMed Scopus (0) Google Scholar). Targeted conditional knockout mice may help us to better understand the contribution of GlyRs to ethanol intoxication, consumption, and AUD. Ethanol inhibition of NMDA receptor (NMDAR) function has been studied in depth, but the details of the ethanol-receptor interaction are poorly understood (Bell et al., 2016Bell R.L. Hauser S.R. McClintick J. Rahman S. Edenberg H.J. Szumlinski K.K. McBride W.J. Ethanol-associated changes in glutamate reward neurocircuitry: a minireview of clinical and preclinical genetic findings.Prog. Mol. Biol. Transl. Sci. 2016; 137: 41-85Crossref PubMed Scopus (5) Google Scholar). Interactions appear to involve the N-terminal and transmembrane 3 (TM3) domains of receptor subunits, as mutation of these sites alters ethanol inhibition of NMDARs (Smothers et al., 2013Smothers C.T. Jin C. Woodward J.J. Deletion of the N-terminal domain alters the ethanol inhibition of N-methyl-D-aspartate receptors in a subunit-dependent manner.Alcohol. Clin. Exp. Res. 2013; 37: 1882-1890Crossref PubMed Scopus (5) Google Scholar). Based on this work, a bottom-up approach using an ethanol-resistant TM3 domain mutation (Ronald et al., 2001Ronald K.M. Mirshahi T. Woodward J.J. Ethanol inhibition of N-methyl-D-aspartate receptors is reduced by site-directed mutagenesis of a transmembrane domain phenylalanine residue.J. Biol. Chem. 2001; 276: 44729-44735Crossref PubMed Scopus (0) Google Scholar, Smothers and Woodward, 2006Smothers C.T. Woodward J.J. Effects of amino acid substitutions in transmembrane domains of the NR1 subunit on the ethanol inhibition of recombinant N-methyl-D-aspartate receptors.Alcohol. Clin. Exp. Res. 2006; 30: 523-530Crossref PubMed Scopus (41) Google Scholar, Smothers and Woodward, 2016Smothers C.T. Woodward J.J. Differential effects of TM4 tryptophan mutations on inhibition of N-methyl-d-aspartate receptors by ethanol and toluene.Alcohol. 2016; 56: 15-19Crossref PubMed Google Scholar) found altered ethanol-associated behaviors (den Hartog et al., 2013den Hartog C.R. Beckley J.T. Smothers T.C. Lench D.H. Holseberg Z.L. Fedarovich H. Gilstrap M.J. Homanics G.E. Woodward J.J. Alterations in ethanol-induced behaviors and consumption in knock-in mice expressing ethanol-resistant NMDA receptors.PLoS ONE. 2013; 8: e80541Crossref PubMed Scopus (0) Google Scholar). Further investigation is needed to fully understand how this mutation and other alterations in NMDAR function can affect pharmacological and behavioral effects of ethanol. The putative direct interaction of ethanol with the large-conductance Ca2+-activated K+ channel (BK channel) has spurred research on a molecular target that affects neurons and circuit function using both bottom-up and top-down approaches. Early studies showed that acute ethanol enhances BK channel function (Dopico et al., 2014Dopico A.M. Bukiya A.N. Martin G.E. Ethanol modulation of mammalian BK channels in excitable tissues: molecular targets and their possible contribution to alcohol-induced altered behavior.Front. Physiol. 2014; 5: 466Crossref PubMed Scopus (20) Google Scholar). More recently, Bukiya et al., 2014Bukiya A.N. Kuntamallappanavar G. Edwards J. Singh A.K. Shivakumar B. Dopico A.M. An alcohol-sensing site in the calcium- and voltage-gated, large conductance potassium (BK) channel.Proc. Natl. Acad. Sci. USA. 2014; 111: 9313-9318Crossref PubMed Scopus (0) Google Scholar characterized an ethanol-sensing site in the channel-forming α subunit. Bottom-up studies have examined how changes in ethanol's effects on BK channels alter behavior. For example, ethanol potentiates α and αβ4 BK channel open probability, but the potentiation of an α-only-containing BK channel shows rapid tolerance (Martin et al., 2008Martin G.E. Hendrickson L.M. Penta K.L. Friesen R.M. Pietrzykowski A.Z. Tapper A.R. Treistman S.N. Identification of a BK channel auxiliary protein controlling molecular and behavioral tolerance to alcohol.Proc. Natl. Acad. Sci. 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The role of the BK channel in ethanol response behaviors: evidence from model organism and human studies.Front. Physiol. 2014; 5: 346Crossref PubMed Scopus (15) Google Scholar). A top-down approach revealed molecular mechanisms responsible for these behavioral effects. Blocking BK channel transport to the presynaptic plasma membrane alters ethanol-induced locomotor depression in C. elegans (Oh et al., 2017Oh K.H. Haney J.J. Wang X. Chuang C.F. Richmond J.E. Kim H. ERG-28 controls BK channel trafficking in the ER to regulate synaptic function and alcohol response in C. elegans.eLife. 2017; 6: e24733Crossref PubMed Scopus (0) Google Scholar), while Wnt/β-catenin-signaling-dependent trafficking of BK channels out of the membrane contributes to ethanol tolerance at the cellular level (Palacio et al., 2015Palacio S. Velázquez-Marrero C. Marrero H.G. Seale G.E. Yudowski G.A. Treistman S.N. Time-dependent effects of ethanol on BK channel expression and trafficking in hippocampal neurons.Alcohol. Clin. Exp. Res. 2015; 39: 1619-1631Crossref PubMed Scopus (6) Google Scholar, Pietrzykowski et al., 2004Pietrzykowski A.Z. Martin G.E. Puig S.I. Knott T.K. Lemos J.R. Treistman S.N. Alcohol tolerance in large-conductance, calcium-activated potassium channels of CNS terminals is intrinsic and includes two components: decreased ethanol potentiation and decreased channel density.J. Neurosci. 2004; 24: 8322-8332Crossref PubMed Scopus (0) Google Scholar, Velázquez-Marrero et al., 2016Velázquez-Marrero C. Burgos A. García J.O. Palacio S. Marrero H.G. Bernardo A. Pérez-Laspiur J. Rivera-Oliver M. Seale G. Treistman S.N. Alcohol regulates BK surface expression via Wnt/β-catenin signaling.J. Neurosci. 2016; 36: 10625-10639Crossref PubMed Scopus (1) Google Scholar). However, no data have associated these molecular events with ethanol-induced behaviors, including tolerance. Another ion channel with notable ethanol sensitivity is the G-protein-coupled inwardly rectifying K+ channel (GIRK). Ethanol enhances GIRK channel function (Bodhinathan and Slesinger, 2013Bodhinathan K. Slesinger P.A. Molecular mechanism underlying ethanol activation of G-protein-gated inwardly rectifying potassium channels.Proc. Natl. Acad. Sci. USA. 2013; 110: 18309-18314Crossref PubMed Scopus (0) Google Scholar, Glaaser and Slesinger, 2017Glaaser I.W. Slesinger P.A. Dual activation of neuronal G protein-gated inwardly rectifying potassium (GIRK) channels by cholesterol and alcohol.Sci. Rep. 2017; 7: 4592Crossref PubMed Scopus (2) Google Scholar), and genetic studies have identified a 43-amino-acid C-terminal region that is crucial for this action of ethanol (Lewohl et al., 1999Lewohl J.M. Wilson W.R. Mayfield R.D. Brozowski S.J. Morrisett R.A. Harris R.A. G-protein-coupled inwardly rectifying potassium channels are targets of alcohol action.Nat. Neurosci. 1999; 2: 1084-1090Crossref PubMed Scopus (179) Google Scholar). Mice carrying a missense mutation in the GIRK channel showed a loss of ethanol-induced analgesia (Kobayashi et al., 1999Kobayashi T. Ikeda K. Kojima H. Niki H. Yano R. Yoshioka T. Kumanishi T. Ethanol opens G-protein-activated in