BACKGROUND Proper ascertainment of the history of alcohol consumption by an individual is an important component of medical diagnosis of disease and influences the implementation of appropriate treatment strategies that include prescription of medication, as well as intervention for the negative physical and social consequences of hazardous/harmful levels of alcohol consumption. Biological (biometric) diagnostic tests that provide information on current and past quantity and frequency of alcohol consumption by an individual, prior to onset of organ damage, continue to be sought. METHODS Platelet monoamine oxidase B (MAO-B) protein was quantitated in 2 populations of subjects who had histories of different levels of alcohol consumption. Levels were assayed by immunoblotting or by ELISA. The development and evaluation of the new ELISA-based measure of platelet MAO-B protein levels is described. RESULTS One subject population constituted a nontreatment-seeking, cross-sectional subject sample, and the other population was a longitudinally followed, hospitalized group of subjects. An algorithm combining measures of platelet MAO-B protein with the plasma levels of carbohydrate-deficient transferrin (CDT) and with liver enzymes (aspartate aminotransferase or γ-glutamyltransferase [GGT]) can detect hazardous/harmful alcohol use (HHAU) with the highest sensitivity and specificity in the cross-sectional nontreatment-seeking population. In the treatment-seeking population, low MAO-B protein levels at admission are associated with heavy drinking prior to admission, and these protein levels increase over a period of abstinence from alcohol. CONCLUSIONS The platelet MAO-B protein measurement is particularly effective for male alcohol consumers. The combined use of MAO-B protein measures together with measures of CDT and GGT does, however, improve the diagnostic utility of both markers for ascertaining HHAU in women. Furthermore, measurement of changes in platelet MAO-B protein levels during treatment for alcohol dependence may help monitor the success of the treatment program.

This article summarizes content proceedings of a symposium held at the 2004 International Society for Biomedical Research on Alcoholism Congress in Mannheim, Germany. The chairs were Boris Tabakoff and Friedrich M. Wurst. The presentations were (1) Genetic associations with alcoholism and affective disorders, by Paula Hoffman; (2) Proteomic analysis of blood constituents in alcoholism, by Boris Tabakoff; (3) Contrasts between the responses of GGT and CDT to high alcohol intake, and a test of their combined use, by John Whitfield; (4) Direct ethanol metabolites such as ethyl glucuronide, fatty acid ethyl esters, phosphatidylethanol and ethyl sulfate: a new line of sensitive and specific biomarkers, by Friedrich Martin Wurst; and (5) Genetic studies of alcoholism subtypes in a Han Taiwanese population, by Ru-Band Lu.

This manuscript reviews the proceedings of a symposium organized by Drs. Antonio Noronha and Fulton Crews presented at the 2003 Research Society on Alcoholism meeting. The purpose of the symposium was to examine recent findings on when alcohol induced brain damage occurs, e.g., during intoxication and/or during alcohol withdrawal. Further studies investigate specific brain regions (where) and the mechanisms (why) of alcoholic neurodegeneration. The presentations were (1) Characterization of Synaptic Loss in Cerebella of Mature and Senescent Rats after Lengthy Chronic Ethanol Consumption, (2) Ethanol Withdrawal Both Causes Neurotoxicity and Inhibits Neuronal Recovery Processes in Rat Organotypic Hippocampal Cultures, (3) Binge Drinking-Induced Brain Damage: Genetic and Age Related Effects, (4) Binge Ethanol-Induced Brain Damage: Involvement of Edema, Arachidonic Acid and Tissue Necrosis Factor alpha (TNFalpha), and (5) Cyclic AMP Cascade, Stem Cells and Ethanol. Taken together these studies suggest that alcoholic neurodegeneration occurs through multiple mechanisms and in multiple brain regions both during intoxication and withdrawal.

In a previous analysis of existing antiseizure compounds, we suggested that a common diphenylureido moiety was responsible for the activity-dependent, Na(+) channel blocking actions of these drugs (L. D. Snell et al., 2000, J. Pharmacol. Exp. Ther. 292: 215-227). Thus the novel diphenylureido compound [N,N-(diphenyl)-4-ureido-5,7-dichloro-2-carboxyquinoline] DCUKA was developed to incorporate the diphenylureido pharmacophore into a structure that also acted as an NMDA receptor antagonist. DCUKA has previously been shown to have antiepileptic properties in animals, and in the present study the actions of DCUKA on Na(+) currents were characterized using transfected cells that stably expressed the rat brain Na(v)1.2 channel isoform. In whole-cell voltage-clamp recordings, DCUKA reduced Na(+) currents in a dose- and membrane potential-dependent fashion, with an apparent 1:1 stoichiometry of drug:channel interaction. Characterization of the effects of DCUKA on Na(+) channel function strongly suggested that DCUKA acts by enhancing Na(+) channel inactivation. Thus in the presence of DCUKA, Na(v)1.2 channels showed reduced availability in steady-state inactivation protocols, displayed use-dependent inhibition, and were slower to recover from inactivation than untreated channels, while DCUKA showed no significant interaction with the open state of the channel. As previously postulated for the anticonvulsants carbamazepine and phenytoin, these results could be well explained by a model in which the drug preferentially interacts with the fast inactivated state of the channel. Finally, DCUKA was generally more efficacious than carbamazepine in modifying sodium channel behavior. Thus the diphenylureido moiety identified by a structural analysis of classic anticonvulsants appears to be important to the inactivation-specific Na(+) channel inhibition by this class of antiseizure agents.

Chronic ethanol treatment of cultured neurons from various brain areas has been found to increase NMDA receptor function and to alter the levels of some NMDA receptor subunit proteins. Because the cultured neurons are exposed to ethanol during a period when the NMDA receptor is undergoing developmental changes in subunit expression, we wished to determine whether ethanol treatment alters this developmental pattern. We found that 3 days of treatment of cerebellar granule neurons with ethanol, which was previously reported to increase NMDA receptor function, resulted in a delay in the ‘developmental switch’ of the NR2A and NR2B subunits, i.e. the developmental decrease in NR2B and increase in NR2A protein expression. As a result, the level of NR2B was higher, and that of NR2A was lower, in the ethanol‐treated cells than in control cells. Cross‐linking experiments showed that the changes in total receptor subunit proteins levels were reflected in cell‐surface expressed proteins, indicating changes in the amount of functional receptors. These results were confirmed by a higher potency of glycine at the NMDA receptor in the ethanol‐treated cells, as determined by NMDA/glycine‐induced increases in intracellular Ca2+. The results suggest that the mechanism by which ethanol alters NMDA receptor expression in cultured neurons, where receptors are undergoing development, differs from the mechanism of ethanol's effect on NMDA receptors in adult brain. Changes in the proportion of NR2A and NR2B subunits may contribute to effects of ethanol on neuronal development.

A novel series of N-substituted 4-ureido-5,7-dichloro-quinolines were synthesized to contain pharmacophores directed at voltage-sensitive sodium channels (VSNaCs) and N-methyl-D-aspartate (NMDA) receptors. These compounds were shown to act in a use-dependent manner as antagonists of VSNaCs and to act as selective competitive antagonists at the strychnine-insensitive glycine recognition site of NMDA receptors. These agents had little or no effect on alpha-adrenergic receptors, other glutamate receptors, or sites other than the glycine site on the NMDA receptor, and did not block voltage-sensitive calcium channels in vitro. In vivo, the compounds were active in preventing or reducing the signs and symptoms of neurohyperexcitability and had anxiolytic properties. Unlike benzodiazepines, N-substituted 4-ureido-5, 7-dichloro-quinolines showed little interaction with the sedative effects of ethanol, but were effective in controlling ethanol withdrawal seizures. The combined actions of these compounds on VSNaCs and NMDA receptors also impart properties to these compounds that are important for preventing and reducing excitotoxic neurodegeneration, but these compounds lack the undesirable side effects of other agents used for these purposes.

Abstract: Ethanol, added to primary cultures of cerebellar granule neurons simultaneously with NMDA, was previously shown to inhibit the anti‐apoptotic effect of NMDA. The in vitro anti‐apoptotic effect of NMDA is believed to mimic in vivo protection against apoptosis afforded by innervation of developing cerebellar granule neurons by glutamatergic mossy fibers. Therefore, the results suggested that the presence of ethanol in the brain at a critical period of development would promote apoptosis. In the present studies, we examined the effect of chronic ethanol exposure on the anti‐apoptotic action of NMDA in cerebellar granule neurons. The neurons were treated with ethanol in vitro for 1‐3 days in the absence of NMDA. Even after ethanol was removed from the culture medium, as ascertained by gas chromatography, the protective effect of added NMDA was significantly attenuated. The decreased anti‐apoptotic effect of NMDA was associated with a change in the properties of the NMDA receptor, as indicated by a decrease in ligand binding, decreased expression of NMDA receptor subunit proteins, and decreased functional responses including stimulation of increases in intracellular Ca2+ and induction of brain‐derived neurotrophic factor expression. The latter effect may directly underlie the attenuated protective effect of NMDA in these neurons. The results suggest that ethanol exposure during development can have long‐lasting effects on neuronal survival. The change in the NMDA receptor caused by chronic ethanol treatment may contribute to the loss of cerebellar granule neurons that is observed in animals and humans exposed to ethanol during gestation.

The mechanism by which ethanol inhibits the function of the NMDA subtype of glutamate receptor has not been elucidated. One possibility that has been suggested is that NMDA receptor subunit composition influences the sensitivity of the receptor to ethanol. We have taken advantage of developmental changes in subunit composition of the NMDA receptor in cultured neurons to examine possible changes in the effect of ethanol. We found an increase in expression of the NR2A subunit, and a decrease in expression of the NR2B subunit of the NMDA receptor in primary cultures of cerebellar granule neurons over time in culture, with no significant change in NR1 expression. This change in NR2 subunit expression was associated with the expected changes in functional properties of the NMDA receptor (measured as the NMDA-induced increase in intracellular Ca2+), i.e., ifenprodil sensitivity and glycine potency were higher when there was a relatively greater proportion of NR2B in the cultured neurons. However, the potency of ethanol to inhibit NMDA receptor function was lower when there was a greater proportion of NR2B subunits. Previous studies showed that ethanol inhibition of NMDA receptor function in cerebellar granule neurons resulted from an ethanol-induced decrease in potency of the co-agonist, glycine, and that this effect of ethanol was blocked by inhibitors of protein kinase C. Our current results suggest that the lower potency of ethanol to inhibit the response of NMDA receptors when cerebellar granule neurons are expressing a greater proportion of NR2B subunits is a result of the higher affinity of the NMDA receptors for endogenous levels of glycine at this point in time.

Chronic ethanol treatment of mice has been shown to result in increased binding of dizocilpine and glutamate to hippocampal NMDA receptors. These changes were suggested to reflect an increase in NMDA receptor number that may underlie certain signs of the ethanol withdrawal syndrome. However, there was no change in binding of a competitive NMDA receptor antagonist, or of ligand binding to the glycine co-agonist site on the receptor after chronic ethanol treatment. Differential changes in the binding of particular ligands at the NMDA receptor suggested the possibility that chronic ethanol ingestion might selectively affect the expression of particular NMDA receptor subunits. Our current work demonstrates that chronic ethanol ingestion by mice, which results in the generation of physical dependence, also produces increases in the NMDA receptor NR1 subunit protein in the hippocampus and cerebellum (approximately 50% and 95%, respectively), and produces increases in the NR2A subunit protein in the hippocampus and cortex (approximately 25% and 40%, respectively). However, the mRNA levels for these subunits were not increased in the respective brain areas by the same ethanol treatment. The changes in NMDA receptor subunit expression in discrete areas of the brain may contribute to the previously observed changes in ligand binding and, possibly, signs of ethanol withdrawal.

Mice given an ethanol-containing liquid diet, as their sole source of nutrients and fluid, rapidly developed functional tolerance to and physical dependence on ethanol. The presence of physical dependence was demonstrated by measured signs of central nervous system hyperexcitability upon withdrawal of ethanol. The withdrawal hyperexcitability, which included tremors, handling-induced seizures and spontaneous clonic/tonic seizures, was more pronounced when mice consumed the ethanol-containing diet for 7 days, compared with 5 days. Daily treatment of the animals with either a ganglioside mixture (extracted bovine brain gangliosides, 250 or 500 mg/kg i.p.) or ganglioside GM1 (100 mg/kg i.p.) for the terminal two-thirds of the ethanol administration period resulted in a significant reduction in the ethanol withdrawal signs. On the other hand, tolerance to the hypnotic action of ethanol, tested 30 hr after withdrawal of ethanol, was unaffected by ganglioside treatment. Ganglioside GM1 given i.c.v. at a daily dose of 10 micrograms during the ethanol ingestion period was as effective as 100 mg/kg GM1 given i.p. in reducing signs of ethanol withdrawal. The daily administration of gangliosides during the feeding of the ethanol diet did not alter the animals' ethanol consumption, intoxication or blood ethanol levels at the time of ethanol withdrawal. A single dose of GM1 given either i.p. or i.c.v. 16 hr before withdrawal produced no effect on the measured ethanol withdrawal signs. Our prior work and the work of others has demonstrated a relationship between up-regulation of N-methyl-D-aspartate receptor numbers in brain and the manifestation of ethanol withdrawal signs. Daily administration of GM1, during the last 5 days of a 7-day period of ethanol ingestion, prevented the up-regulation of N-methyl-D-aspartate receptors in the hippocampus and reduced the ethanol withdrawal signs. Our data demonstrate that the daily administration of gangliosides during the period of ethanol consumption may prevent the development of ethanol physical dependence, while leaving ethanol tolerance intact.

Besides the N-methyl-D-aspartate (NMDA) receptor proteins NR1 and NR2, another complex of proteins which has been shown to contain ligand-binding sites characteristic of NMDA receptors is expressed in cerebellar granule cells. One of the proteins in the latter complex is the 71 kDa glutamate-binding protein (GBP). To determine the role of the GBP in the response to NMDA, primary cultures of cerebellar granule cells were treated with an antisense oligonucleotide complementary to mRNA for this protein. This treatment substantially reduced both mRNA and protein levels of the GBP, as well as the response of the cells to NMDA, measured as an increase in intracellular Ca2+ with fura-2 fluorescence. The antisense oligonucleotide treatment did not alter the Ca2+ responses to KC1 or kainate. Chronic ethanol exposure has previously been shown to increase NMDA receptor function and the density of binding sites for the NMDA receptor channel blocker, dizocilpine, in cerebellar granule cells. Chronic exposure of the cells to 100mM ethanol is now shown to result in significant increases in mRNA and protein levels for the GBP (45% and 100%, respectively). Ethanol treatment did not affect mRNA levels for NR1 or NR2A, caused only a small increase (20%) in protein levels for NR1, and resulted in a decrease (30%) in NR2A protein. Although a role of the NMDA receptor NR1/NR2 subunits cannot be ruled out, these results are compatible with the hypothesis of involvement of the GBP in the chronic ethanol-induced increase in NMDA receptor function in cerebellar granule cells.

Ethanol, acutely, is a potent inhibitor of the function of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. After chronic exposure of animals to ethanol, however, the NMDA receptor in brain is upregulated. This upregulation is associated with the occurrence of ethanol withdrawal seizures. When cultured cerebellar granule neurons are exposed chronically to ethanol, the resulting upregulation of NMDA receptor function renders the cells more susceptible to glutamate-induced neurotoxicity. The present studies show that chronic ethanol exposure produces an increase in NMDA receptor number in the cells, measured by ligand binding to intact cells. Glutamate-induced excitotoxicity, both in control and ethanol-exposed cells, is blocked by the same NMDA receptor antagonists previously shown to block ethanol withdrawal seizures in animals. In addition, glutamate neurotoxicity is blocked by acute (2-hr) pretreatment of cells with ganglioside GM1 or by chronic (3 days) treatment with the ganglioside. Acute ganglioside treatment does not interfere with the initial rise in intracellular calcium caused by glutamate, whereas this response is downregulated after chronic ganglioside treatment. These results suggest that therapeutic agents can be developed to block both ethanol withdrawal signs and the neuronal damage that accompanies ethanol withdrawal. Furthermore, chronic ganglioside treatment during ethanol exposure has the potential to prevent changes in the NMDA receptor that lead to withdrawal seizures and enhanced susceptibility to excitotoxicity.

Ethanol inhibits the function of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor in various neuronal systems, but the mechanism of the inhibition has not been elucidated. Previous work, using primary cultures of rat cerebellar granule cells, showed that both exposure to alcohol and activation of protein kinase C (PKC) by the phorbol ester PMA reduced the potency of the co-agonist, glycine, to enhance NMDA receptor function (measured as an increase in intracellular Ca2+), resulting in inhibition of the NMDA response at low glycine concentrations. Inhibition of NMDA receptor function by PMA and ethanol could also be overcome by PKC antagonists, implicating PKC in the inhibitory effect of ethanol. We have now compared the effects of ethanol and PKC activation of NMDA receptor function in primary cultures of rat cerebral cortical cells. The receptor in these cells was much less sensitive to ethanol inhibition, and the inhibition was not overcome by high concentrations of glycine. Furthermore, PMA treatment resulted in an increased response to NMDA at low glycine concentrations. The results indicate that PKC does not mediate ethanol inhibition of NMDA receptor function in cerebral cortical cells, and that the mechanism of ethanol inhibition can vary among brain regions and/or cell types. Possible determinants of the differing mechanisms of ethanol's actions include the subunit composition of the NMDA receptor and/or the isoforms of PKC present in the different cells.