Cellular and molecular neuroscience of alcoholism.

I. INTRODUCTION

Alcoholism, alcohol abuse, and the medical complications of excessive drinking are major world-wide health problems. In the United States, ~7% of adults are alcoholics, and [is greater than] 20% of hospitalized patients have a medical disorder related to heavy drinking (72). Recent advances in neuroscience have made it possible to investigate the pathophysiology of alcoholism at a cellular and molecular level. Evidence indicates ethanol affects hormone- and neurotransmitter-activated signal transduction, leading to short-term changes in regulation of cellular functions and long-term changes in gene expression. Such changes in the brain probably underlie many of the acute and chronic neurological events in alcoholism (202). In addition, genetic vulnerability also plays a role in alcoholism and, perhaps, in alcoholic medical disorders.

II. ACUTE AND CHRONIC RESPONSES TO ETHANOL

There are two major central nervous system (CNS) responses to alcohol abuse: severe intoxication and adaptive changes that develop in alcoholics because of prolonged drinking. Ethanol is both water soluble and lipid soluble and is readily distributed into the cytoplasm and lipid membranes of all cells in the body. There is no blood-brain barrier for ethanol; nuclear magnetic resonance studies in animals (265) and human volunteers (198) show that alcohol can be detected in the brain within a few minutes after drinking. Acute ethanol intercalates into cell membranes (297) and increases membrane fluidity (105), while chronic ethanol alters the lipid composition of cell membranes (323, 329). However, it is has never been clear how ethanol-induced disturbances in membrane order (104, 120, 298, 350) produce the characteristic short-term and long-term CNS effects of heavy drinking (201). These include such reversible clinical events as intoxication, memory loss during binge drinking (blackouts), tolerance to the intoxicating effects of ethanol in alcoholics, addiction (continued drinking despite adverse medical and socioeconomic complications), and a characteristic hyperexcitable alcohol withdrawal syndrome when alcohol abuse is discontinued (evidence of physical dependence).

Almost all of the important pathophysiological targets for ethanol in neural cells appear to be specific membrane proteins that mediate signal transduction (92, 201, 358). Ethanol does not appear to alter the activity of most soluble proteins. Not all membrane proteins are affected, but some signal transduction cascades are highly sensitive. Targets include certain ion channels, transporters, neurotransmitter receptors, G proteins, and enzymes that produce second messengers; interaction of ethanol with these target proteins leads to changes in activity of many enzymes, chaperones, and regulators of gene expression. In this review we consider first several membrane proteins that are specifically sensitive to ethanol, particularly because they have relevance for important clinical events in alcoholism and alcohol abuse. Then we discuss other regulatory signaling pathways that are also altered by ethanol and that may play a role in these events. It is also possible that over prolonged periods of time these ethanol-induced molecular changes contribute to the development of several alcoholic neurological disorders. Apart, from the role of thiamine deficiency in Wernicke's disease, however, the pathogenesis of the neurologic disorders in alcoholism is not well understood (41, 71), but today, alcohol toxicity appears to be more important than nutritional deficiency.

III. ETHANOL AND MEMBRANE PROTEINS

Alcohols with increasing carbon chain length have increasing solubility in cell membranes. Nevertheless, there is a "cut-off" in the biologic effect when alcohols of increasing chain length are studied in the same system (90, 164). Peoples and Weight (262) have recently shown that shorter chain length alcohols were increasingly potent inhibitors of neuronal N-methyl-D-aspartate (NMDA) receptor activated ion currents, but longer chain alcohols had no effect, despite greater solubility in membranes. These results suggest that there is a hydrophobic pocket in ethanol-sensitive membrane proteins. Because the cutoff response varies with different neurotransmitter receptor systems (164, 262), these hydrophobic sites are probably of different size in different membrane proteins. This is consistent with pioneering work by Franks and Lieb (92), who called attention to the molecular cut-off effects (90) and later documented highly specific hydrophobic binding sites on proteins that discriminate between optical isomers of anesthetic agents with identical lipid solubility (91). Taken together, these findings suggest the possibility of designing new drugs to compete with ethanol at selected hydrophobic sites to block or reverse specific adverse effects without affecting the function of other membrane proteins.

IV. N-METHYL-D-ASPARTATE RECEPTORS

The NMDA receptor is one of several major receptors for glutamate (155), the principal excitatory neurotransmitter in the brain. N-methyl-D-aspartate receptors in the hippocampus are involved in learning and memory (300) and are critical for long-term potentiation (LTP) and long-term depression (LTD) in models of synaptic plasticity (183). Specific NMDA receptor subunits mediate these NMDA receptor responses (89, 156, 300). N-methyl-D-aspartate receptor activation by glutamate promotes calcium influx through an ion channel that is part of the receptor (27). Calcium, in turn, regulates synaptic signaling (47) via activation of protein kinases, phosphatases, and proteases. Ethanol inhibits UP (222), perhaps by suppressing its induction (103). Lovinger et al. (174) were the first among several investigators (78, 126, 165) to discover that ethanol inhibits NMDA receptor activation at intoxicating blood alcohol levels. In Xenopus oocyte expression systems, ethanol inhibition is modulated by glycine (26) and may (26, 45, 188) or may not (218) vary with NMDA receptor subunit composition. Ethanol also inhibits kainate (79, 187) and DL-[Alpha]-amino-3-hydroxy-5-methylisoxazole-propionic acid (172, 187) responses, suggesting that non-NMDA receptors are also sensitive to ethanol (172). Indeed, ethanol modulates metabotropic glutamate receptors coupled to second messengers in cerebellar Purkinje cells (242). There is differential sensitivity to ethanol among NMDA receptor isoforms in the brain (211) or when expressed in Xenopus oocytes (218), and ethanol inhibition of NMDA receptor activation (188, 243, 262) appears to play a role in ethanol intoxication. For example, it is likely that ethanol inhibition of NMDA receptors accounts for alcoholic "blackouts" that, occur during heavy drinking. These startling episodes are characterized by hours of amnesia for events that occurred while intoxicated; they are best explained by transient ethanol inhibition of NMDA receptors in the hippocampus. Several agents prevent ethanol inhibition of LTP (43, 290, 322, 355, 375) and raise the possibility of new treatments for ethanol-induced memory loss in humans. Also, an agent like acamprosate, which appears to enhance NMDA receptor function (182), might also be useful. Perhaps this is related to the clinical impression that acamprosate helps to sustain abstinence in human alcoholics (253).

One of the adaptive CNS responses to chronic exposure to ethanol is an upregulation of NMDA receptors in human alcoholics (210) and in rats, particularly the hippocampus, measured by ligand binding (112, 304, 319) and NMDA receptor subunit immunoreactivity (334). Ethanol-induced upregulation could have serious neuropathological consequences (171) because overactivity of NMDA receptors appears to cause "excitotoxic" neuronal cell damage in several neurological disorders including ischemic strokes, hypoglycemia, and prolonged seizures (296). This was confirmed recently by demonstrating that antisense oligonucleotides to an NMDA receptor subunit protect specific neurons from excitotoxic cell death and reduce ischemic infarcts in the brain (344). Consistent with these findings, chronic exposure to ethanol causes increased NMDA receptor-mediated calcium flux (2, 134) and greater NMDA excitotoxicity in cultured neurons (2, 35, 125). These results suggest the possibility that memory deficits and neuronal cell loss in chronic alcoholics (118) might be due, in part, to chronic ethanol-induced upregulation of NMDA receptors. Unrestrained NMDA receptor activation is also implicated in alcohol withdrawal seizures because these receptors play a role in the pathogenesis of convulsions (150, 229). Therefore, new therapies directed against excessive glutamate release (294) and activation of NMDA receptors (289, 290) might prevent or reverse some complications of chronic alcohol abuse and withdrawal.

Paradoxically, ethanol inhibition of NMDA receptors might be of protective value for ischemic stroke in nonalcoholics where excitotoxic amino acids cause brain damage. Recent experiments show that ethanol can attenuate excitotoxic neuronal damage (36, 177, 356), presumably by blocking NMDA receptor activation and calcium influx (178, 356), leading to attenuation of stress-induced c-fos expression in vulnerable neurons as in the hippocampus (161, 299). However, unacceptably high concentrations of ethanol might be required for a protective effect in patients. Further research on agents, like ifenprodil (169), that mimic the acute interaction of ethanol with NMDA receptors might generate more effective anti-ischemic agents than available today.

V. [Gamma]-AMINOBUTYRIC ACID RECEPTORS

[Gamma]-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the brain, activating [GABA.sub.A] and [GABA.sub.B] receptors. The [GABA.sub.A] receptor is an oligomeric protein complex containing a receptor-operated chloride channel and specific allosteric binding sites for benzodiazepines, barbiturates, and other agents (248, 318). The function of [GABA.sub.A] receptors is potentiated at intoxicating concentrations of ethanol in heterogeneous neural preparations and cells stably transfected with GABA receptor subunits (122). Also, there is cross-tolerance between ethanol, benzodiazepines, and barbiturates (196, 325). Thus benzodiazepines are very helpful in treating the alcohol withdrawal syndrome (72) by substituting for alcohol. On the other hand, benzodiazepine inverse agonists such as the imidazobenzodiazepine Ro 15-4513 prevent the intoxicating effects of ethanol in rodents apparently by antagonizing ethanol potentiation of [GABA.sub.A] receptors (25, 196, 254, 324). Although Ro 15-4153 is not suitable for patients because it causes seizures, it is likely that new and safe drugs will be developed to block or reverse the acute intoxicating effects of ethanol by modulating [GABA.sub.A] receptor function. In addition, promising results with [Gamma]-hydroxybutyrate suggest that novel agents affecting GABA function may be useful in treating alcohol dependence (95).

The response of GABA receptors to ethanol varies in different regions of the brain (56, 272, 274, 286), but the molecular basis of this regional sensitivity is not well understood. In hippocampus, ethanol enhances [GABA.sub.A] receptor function only when [GABA.sub.B] receptors are blocked (347). In addition, molecular cloning has determined that the [GABA.sub.A] receptor complex is a multigene family (159, 248). Genes for a variety of [Alpha]-, [Beta]-, [Gamma]-, and [Delta]-subunits have been cloned, and [GABA.sub.A] receptors in brain appear to be assembled in multiple combinations of these subunits (159, 194). Moreover, the subunit composition of [GABA.sub.A] receptors changes under different biological conditions, introducing an additional element of [GABA.sub.A] receptor variability. Differences in subunit composition may account for developmental changes in receptor properties with maturation (159), receptor localization in cells (263), and differences in receptor pharmacology in neurons (6, 85, 122, 193). The role of specific subunits in determining ethanol sensitivity may best be studied in transfected cell lines (119, 336). It may be anticipated, therefore, that chronic ethanol-induced changes in [GABA.sub.A] receptor subunits (56, 69, 124, 207-209, 223, 370) will have significant functional consequences in the brain.

Ethanol potentiation of GABA-induced responses in cerebellar neurons (162) appears to be regulated by [Beta]-adrenergic receptor activation (166), suggesting a role for adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) phosphorylation in modulating sensitivity to ethanol. Other studies with mouse [GABA.sub.A] receptor subunits expressed in Xenopus oocytes by Wafford et al. (342) suggest that a specific [Gamma]-subunit confers sensitivity to ethanol potentiation of receptor activation, although this was not observed with [Gamma]-subunits expressed in Xenopus oocytes (312) or human embryonic cells (186). Recent results with mouse and bovine subunits in a clonal cell line suggest that the [Gamma]-subunit may be necessary but not sufficient for ethanol sensitivity (122). The [[Gamma].sub.2]-subunit exists as alternatively spliced short ([[Gamma].sub.2s]) and long ([[Gamma].sub.21]) forms; the long form contains 24 additional nucleotides that encode a phosphorylation site for protein kinase C (PKC) on an intracellular loop (342). Ethanol sensitivity appears to require PKC phosphorylation of the [[Gamma].sub.21]-subunit expressed in Xenopus oocytes (343), and perhaps, in hippocampal CA1 neurons (359). However, there is suggestive evidence that ethanol sensitivity of hippocampus [GABA.sub.A] receptors may also involve PKA (347). The [GABA.sub.A] receptors in mutant mice lacking the [Gamma]-isoform of PKC show reduced sensitivity to ethanol (121), but this may be related to impaired cerebellar function in PKC-[Gamma] mutants (42, 139). These results are all consistent with studies suggesting a critical role for phosphorylation in regulating the response of other membrane proteins to ethanol (see sect. IX, C-F). Further studies are needed to determine the stoichiometry of [GABA.sub.A] receptor subunit expression and the substrates for phosphorylation that confer ethanol sensitivity in the brain. Progress in this area will need to identify highly specific pharmacological targets for the therapy of alcoholism.

VI. SEROTONIN RECEPTORS

The serotonin (5-[HT.sub.3]) receptor (138) has increased mRNA expression in certain brain regions, particularly the hippocampus (330), and is structurally similar to the nicotinic acetylcholine and [GABA.sub.A] receptors. These three receptors are ligand-activated ion channels and are sensitive to ethanol (122, 175, 367). The 5-[HT.sub.3] receptor ionophore conducts monovalent cations, and low concentrations of ethanol potentiate 5-[HT.sub.3] receptor-stimulated currents in several neural preparations (170, 173). Serotonin receptor antagonists block the ability to discriminate between drinking water or ethanol in pigeons (111) and specifically reduce ethanol drinking in conditioned and alcohol-preferring rodents (135, 148, 333). Serotonin receptors have also been implicated in the control of appetite, and 5-[HT.sub.2c] receptor-deficient animals become overweight because of abnormal feeding behavior (331). Moreover, selected regions of the brain in alcohol-preferring rats have fewer serotonin 5-[HT.sub.2] receptors (190) and increased 5-[HT.sub.1A] receptors (191). Furthermore, serotonergic neurons and their axons appear to degenerate in alcohol-preferring rats (116) and in chronic alcoholics (117). Although 5-[HT.sub.2] receptors have been implicated in alcohol preference in rodents (46), the effect of 5-[HT.sub.2] antagonists on animal drinking behavior has been inconsistent (195, 256). Nevertheless, preliminary studies suggest that ondansetron, a serotonin receptor antagonist, reduces alcohol intake in normal men (137) and alcoholics with less severe drinking (311). These findings suggest that serotonin may play a role in alcohol intoxication and alcohol-seeking behavior. Because serotonin potentiates ethanol induced excitation in the ventral tegmental area (21), pharmacological agents like ifenprodil (192), which react with specific sites on serotonin receptors, may be of value in treating craving in alcoholics.

VII. VOLTAGE-DEPENDENT CHANNELS

A. Calcium Channels

In addition to receptor-activated calcium influx, intracellular concentrations of calcium are increased in neurons following depolarization through voltage-dependent calcium channels (284). At low concentrations, calcium ions are critical second messengers, but high concentrations lead to excitotoxicity and cell death (47). Recent evidence suggests that ethanol-induced upregulation of calcium channels may account for many features of the alcohol withdrawal syndrome, including intense neuronal hyperactivity and life-threatening seizures (201).

Studies with isolated neural cells have identified molecular mechanisms that may be responsible for these events. Voltage-dependent calcium channels consist of multi-subunit complexes characterized by pharmacological and neurophysiological criteria (12, 34). Acute ethanol inhibits voltage-dependent L-type calcium channels (19, 44, 80, 114, 123, 199, 224, 225, 292, 315, 335, 353), N channels (351, 352), and T channels (335) but has no effect on P-type calcium channels (115). Ethanol may act specifically on the channel Protein (353). Undifferentiated cells are most sensitive to ethanol inhibition (11, 225); this may involve the inhibitory G protein, [G.sub.i] (224). Chronic exposure of neural cells to ethanol, however, leads to increased depolarization-stimulated calcium influx (110, 199, 315) associated with an apparent increase in calcium channels measured by binding studies with labeled antagonists (82). Similar increases in brain calcium channel binding sites have also been found in alcohol-dependent animals (167) and Kupffer cells from the liver (109). This increase in voltage-dependent calcium channels requires PKC activity (205) and may be related to ethanol-induced increases in two PKC isoenzymes and PKC-mediated phosphorylation (204). Ethanol-induced upregulation of calcium channels persists for ~16 h after ethanol is removed (199), coinciding with the time of greatest risk for alcohol withdrawal seizures after alcoholics stop drinking (339). Increased voltage-dependent calcium channel activity could induce withdrawal symptoms by promoting neurotransmitter release (179), and enhancing NMDA receptor activation (363). Consistent with this hypothesis, treatment with calcium channel blockers reduces alcohol withdrawal tremors, seizures, and mortality in animals (17, 168) and human alcoholics (147). Moreover, treatment with some calcium channel blockers like nimodipine also reduces alcohol consumption in alcohol-preferring rats (65).

Ethanol regulation of calcium channels also appears to be under genetic control, producing selective upregulation in long-sleep mice (131). Ethanol-induced upregulation of calcium channels (18, 113) and voltage-activated calcium currents (264) is much greater in mice selectively bred for severe alcohol withdrawal seizures than in mice bred for mild signs of alcohol withdrawal. It remains to be determined whether genetic variation in ethanol regulation of calcium channels contributes to human alcohol withdrawal seizures and chronic alcoholic brain damage.

B. Potassium Channels

Ethanol appears to inhibit different kinds of potassium currents in a variety of neural preparations, but not all investigators agree (358). The expression of potassium channel subunits in the hippocampus has a spatial heterogeneity that varies with development (184), suggesting the…