Ethanol dependence and withdrawal selectively alter localized cerebral glucose utilization.

The 2-deoxyglucose technique was used to determine local cerebral glucose utilization (LCGU) in over 50 brain regions of rats physically dependent upon ethanol and compared to those of acutely intoxicated and those undergoing an overt ethanol-withdrawal syndrome. Dependent-intoxicated rats (average blood ethanol concentration 64 mM) had decreased LCGU in 13/54 regions, including those associated with the limbic system, cerebellum, and motor system. The ethanol withdrawal syndrome was associated with 17/50 gray regions showing an increase, including regions involved with motor function, auditory system, and mammillary bodies-anterior thalamus-cingulate cortex pathway. The most pronounced differences between these groups occurred in regions associated with motor function, cerebellar function, anterior thalamus, and median raphe. Comparisons between dependent-intoxicated and acutely intoxicated rats (average blood ethanol concentration 66 mM) revealed that acute intoxication was associated with a relatively greater reduction in LCGU in regions involved with sensory-related functions, mammillary bodies, and median raphe. With the development of dependence, adaptation occurred in these regions except for inferior colliculus and median raphe. Dependence was also associated with a relative decrease in LCGU in white matter, limbic system, and extrapyramidal motor system.

A comparison of calcium antagonists and diazepam in reducing ethanol withdrawal tremors.

The calcium antagonists nimodipine and dantrolene were compared with diazepam in an animal model of tolerance and physical dependence upon ethanol. Nimodipine and dantrolene were both effective in suppressing withdrawal tremors but diazepam was clearly superior to both agents. These results suggest that the ethanol withdrawal syndrome is only partially mediated by increased calcium flux.

Acute ethanol administration selectively alters localized cerebral glucose metabolism.

The effects of acute ethanol administration on glucose utilization in the CNS of rat were studied using the 2-deoxyglucose technique. Cerebral glucose utilization was determined for 53 brain regions at peak and descending blood ethanol concentrations averaging 14, 26 and 66 mM. Decreased glucose utilization was the predominant finding and was observed in 20% of the regions evaluated, with median raphe, vestibular nucleus, cerebellar vermis, and various structures associated with the auditory system showing the greatest reductions. The only structures that showed increased glucose utilization were the dentate region of the hippocampus and the superior olive, and this was only apparent at a blood ethanol concentration of 14 mM.

Actions of ethanol on voltage-sensitive sodium channels: effects of acute and chronic ethanol treatment.

The effects of acute and chronic ethanol treatment on neurotoxin-stimulated 22Na+ uptake and [3H]batrachotoxinin A20-alpha-benzoate binding to neuronal sodium channels were studied in rat forebrain synaptosomes. Fluorescence measurements were used to assess the intrinsic order or fluidity and the sensitivity to ethanol of rat forebrain synaptic plasma membranes at various intervals during and after chronic ethanol treatment. Acute ethanol administration had no significant effect on neurotoxin binding in the absence or presence of ethanol in vitro or on sodium uptake in the absence of ethanol in vitro. However, a single dose of ethanol produced a dose and time-dependent attenuation of the inhibitory effect of ethanol on sodium uptake, suggestive of acute tolerance. Chronic ethanol treatment reduced the influx of 22Na+ in the presence of batrachotoxin and diminished the inhibitory effect of ethanol in vitro on sodium uptake for up to 20 days after withdrawal, but the specific binding of the neurotoxin in the presence or absence of ethanol was unchanged. Synaptic plasma membranes from chronic ethanol-treated rats showed no change in intrinsic order but the disordering effect of ethanol was significantly smaller for up to 20 days after withdrawal. Results of this study demonstrate that brain tissue from ethanol-treated rats can adapt rapidly to some effects of ethanol and that chronic ethanol administration can reduce the effects of ethanol on physical and functional properties of neurons for a prolonged period of time.

Influence of thiamine deficiency on the response to ethanol in two inbred rat strains.

We investigated whether thiamine deficiency (TD), a frequent concomitant of chronic alcoholism, differentially modifies the response to ethanol in two inbred rat strains with highly different genetic susceptibilities to development of TD encephalopathy. Ethanol-induced (3 g/kg i.p.) behavioral impairment and hypothermia were studied after 2, 5 and 7 weeks of TD and after 6 weeks of repletion on normal diet. Controls of the M520/N (TD-sensitive) strain metabolized ethanol more rapidly, had a greater liver to body weight ratio, greater total body water, earlier and lower peak blood ethanol concentrations (BEC), diminished area under the BEC curve and lesser behavioral impairment and hypothermia (even at equivalent BEC values) than those of the F344/N (TD-resistant) strain. In both strains, TD resulted in reduced ethanol metabolic rate and liver to body weight ratio and equivalent ethanol-induced hypothermia and behavioral impairment at lower BEC. Lower and delayed peak BEC and unchanged area under the BEC curve suggest an increased volume of ethanol distribution during TD. Recovery appeared complete after 6 weeks of normal diet. Both strains lost an equivalent proportion of body weight during TD but M520/N rats had lesser decrements in ethanol metabolic rate, had greater reductions in liver weight, peak BEC and baseline body temperature and developed overt encephalopathy whereas F344/N rats did not. Therefore, in the chronic alcoholic, TD may modify ethanol's effects via pharmacokinetic and pharmacodynamic mechanisms. Relatively high ethanol tolerance of the strain with a genetic predisposition to TD encephalopathy is consistent with the hypothesized role of this avitaminosis in the pharmacogenetics of alcoholism.

Pineal function during ethanol intoxication, dependence, and withdrawal.

Pineal melatonin and serotonin content were determined during one to four days of continuous intoxication, and during the alcohol withdrawal syndrome. The nocturnal rise in pineal melatonin was blunted in continuously intoxicated animals, however this was found to be unrelated to duration of treatment. The initial dependent-intoxicated phase of the alcohol withdrawal syndrome produced a reduction of nocturnal pineal melatonin content with a concomitant elevation in pineal serotonin. The overt withdrawal phase of the alcohol withdrawal syndrome had no effect on pineal melatonin or serotonin content. This data suggests that ethanol may perturb pineal melatonin synthesis either directly, or indirectly by altered receptor function. Contrary to our expectations the pineal may not be a useful model to probe the physiology of increased noradrenergic neurotransmission produced by ethanol withdrawal.

The benzodiazepine antagonist Ro 15-1788 does not antagonize the ethanol withdrawal syndrome.

It has been suggested that the signs and symptoms of the ethanol withdrawal syndrome may be due to the increased production of an "inverse agonist" that binds to the central benzodiazepine (BZ) recognition site in the brain. Ro 15-1788 (a potent antagonist at the central BZ recognition site), diazepam, and Ro 15-1788 plus diazepam were administered to groups of rats undergoing overt ethanol withdrawal. Ro 15-1788 did not alter the severity of the ethanol withdrawal reactions, but antagonized the ameliorative effect of diazepam. The results of our studies suggest that (1) the ethanol withdrawal syndrome is not produced by an endogenous ligand acting on the central BZ recognition site, and (2) diazepam decreases the severity of the ethanol withdrawal syndrome, at least in part, by its action at the central BZ recognition site.

Effects of ethanol administration on parameters of immunocompetency in rats.

Ethanol administered to rats intragastrically in doses sufficient to cause dependency resulted in a rapid cell loss from the thymus and spleen. Cell loss from the peripheral blood was due primarily to a loss of lymphocytes, but a concomitant granulocytosis resulted in only small changes in the total leukocyte count. Lymphocyte proliferation to both T- and B-cell mitogens was severely compromised by ethanol treatment. The cell loss and functional lymphocyte impairment also occurred at half the ethanol dose required to induce dependency. Although cell numbers recovered relatively quickly after ethanol withdrawal, lymphocyte function, as measured by proliferation, recovered more slowly. Ethanol administration before or during immunization with sheep erythrocytes resulted in an impairment in the ability of animals to respond with a primary immune response to this antigen. These data suggest that ethanol given in quantities sufficient to produce dependence impairs in vitro and in vivo parameters of immunocompetency.

Cerebral 2-deoxyglucose uptake in rats during ethanol withdrawal and postwithdrawal.

The overt ethanol withdrawal syndrome is associated with a generalized increase in cerebral uptake of 2-deoxyglucose. Relatively high elevations of 2-deoxyglucose were observed in many structures associated with motor function, the mamillary body-anterior thalamus-cingulate cortex pathway, many thalamic nuclei, and the raphe. Overtly withdrawing rats had higher levels of 2-deoxyglucose than postwithdrawing animals that had been abstinent for 1-5 weeks in 96% of the gray areas evaluated. Postwithdrawal was associated with increased amounts of 2-deoxyglucose in comparison to controls in 80% of the gray areas evaluated. Postwithdrawal and control rats did not differ in some areas involved with motor function and some limbic structures, such as the mamillary body-anterior thalamus-cingulate cortex pathway. It is concluded that the ethanol-withdrawal syndrome results in alterations in cerebral physiology, some of which persist for at least 5 weeks postwithdrawal.

Alteration in calcium-binding activity in synaptosomal membranes from rat brains in association with physical dependence upon ethanol.

The effects of ethanol treatment on calcium-binding activity in synaptosomal membrane fraction from rat brains were studied. The synaptosomal membrane fraction from the hippocampus, the cortex and the cerebellum from control, single dose (6 g/kg), dependent intoxicated (prodromal) and dependent withdrawing (ethanol withdrawal syndrome) rats were used. Two different methods were used for determining the calcium activity in these membrane preparations: the calcium chelator fluorescence probe, chlortetracycline (CTC), was used to measure Ca2+ binding sites in the membranes, and radioactive calcium (45Ca) was used to measure the calcium binding to the synaptosomal membranes. Both methods provided similar conclusions; the calcium activity was higher during the dependent intoxicated phase of the ethanol withdrawal period. The synaptosomal membranes from the hippocampus showed more drastic changes in calcium-binding activity than the cortex and the cerebellum. These results suggest that ethanol dependence is associated with changes in calcium-binding activity in certain areas of the rat brain.

Cerebral alteration in calmodulin levels associated with the induction of physical dependence upon ethanol in rats.

Calmodulin levels were measured in different areas of brain in rats rendered physically dependent and after single doses (6 g/kg) of ethanol. After single doses of ethanol no changes in the calmodulin levels were found in the cortex, but those in the hippocampus and caudate nuclei were increased while those in the cerebellum were reduced. In the dependent intoxicated (prodromal) rats, calmodulin levels were elevated in all these regions except the cerebellum. In rats undergoing ethanol withdrawal syndrome, the calmodulin levels decreased in all regions of the brain except caudate nuclei.

Response to ethanol reduced by past thiamine deficiency.

Ethanol-induced intoxication and hypothermia were studied in rats approximately 7 months after severe thiamine deficiency, when treated rats appeared to have recovered their physical health. Previously induced thiamine deficiency without prior ethanol exposure significantly decreased the area under the curve plotted for the concentration of ethanol in blood and also decreased behavioral impairment and hypothermia due to ethanol exposure. Pathophysiologic changes resulting from thiamine deficiency may contribute to both the pharmacodynamic and pharmacokinetic tolerance to ethanol in chronic alcoholics.

Changes in cortical synaptosomal plasma membrane fluidity and composition in ethanol-dependent rats.

Synaptosomal plasma membranes (SPM) were examined from the following four groups of rats: controls; rats acutely treated with single doses of ethanol; a prodromal-detoxication group (dependent-intoxicated); rats undergoing overt ethanol-withdrawal syndrome. Estimates of the apparent microviscosity of SPM over a range of temperatures indicated that temperature-induced changes in SPM fluidity were smaller during the prodromal detoxication phase. The cholesterol:phospholipid molar ratio significantly increased in SPM from the prodromal-phase rats, but to a lesser extent in rats undergoing ethanol withdrawal syndrome. The fatty acid content of SPM phospholipids was not significantly changed in any of the treatment groups. Addition of cholesterol in vitro to control membranes altered the apparent microviscosity similarly to the changes found in SPM of ethanol-dependent rats. These studies suggest that physical dependence upon ethanol may be related to changes in synaptosomal membrane composition and viscosity.

Studies of neurotransmitter interactions after acute and chronic ethanol administration.

Evidence is accumulating suggesting that ethanol has a biphasic effect on several neurotransmitters in the brain and that interactions between two or more transmitters may contribute to the behavioral response obtained after ethanol administration. In the nigrostriatal complex where the most data have been derived, dopaminergic activity responds in a biphasic manner to ethanol treatment. At low doses, dopaminergic activity is elevated, while at high doses, activity is reduced. After chronic ethanol treatment, pre- and postsynaptic dopaminergic activity is hypoactive. Pharmacological data have suggested the possible involvement of acetylcholine (ACh) and gamma-aminobutyric acid (GABA) in the actions of ethanol on the striatal dopaminergic system. In support of this postulate, striatal high-affinity choline uptake, an index of ACh release, is elevated after high doses of ethanol and after ethanol withdrawal. GABA turnover exhibits a biphasic response to ethanol treatment. At low doses of ethanol, GABA turnover is reduced, while at high doses, turnover is unaffected. These latter effects correlate with known interrelationships of these transmitters in the nigrostriatal complex. The data suggest that an action of ethanol on one transmitter may influence the response of another transmitter to ethanol. To further address the interrelationship of transmitters, a high-performance liquid chromatographic method has been developed to study the activity of several transmitters simultaneously. This approach promises to shed more light on this important area.

Ethanol-withdrawal syndrome associated with both general and localized increases in glucose uptake in rat brain.

Glucose uptake was studied in the brains of rats undergoing an overt ethanol-withdrawal syndrome by 2-deoxy-D-[14C]glucose autoradiography. In addition to a general increase in glucose uptake, localized alterations were observed in sensorimotor cortex, globus pallidus, thalamus and cerebellum. The results suggest that the ethanol-withdrawal syndrome is associated with a general increase in glucose metabolism as well as localized increases in functionally distinct regions of sensory and motor brain regions.

Changes in erythrocyte ghost protein phosphorylation associated with physical dependence upon ethanol.

The effects of ethanol on phosphorylation in the erythrocyte ghost membranes was studied. Four groups of rats were used: controls; prodromal phase; overt withdrawal syndrome; and treated with a single dose (6g/kg) of ethanol. Spectrin (Band 2) phosphorylation was enhanced during prodromal phase (200 +/- 12%) and during overt ethanol withdrawal syndrome (150 +/- 18%), while a single dose of ethanol produced no significant change. Addition of micromolar calcium chloride solution to the phosphorylating medium produced pronounced decrease in spectrin phosphorylation in the ghost membranes isolated from controls and those treated with a single dose of ethanol, while this effect of calcium was less pronounced in the ghost membranes isolated from rats in the prodromal phase and in the withdrawal syndrome. This suggests that due to prolonged ethanol treatment membrane phosphorylating properties were less sensitive to the change in calcium concentration. The membrane polypeptide composition was unaffected upon ethanol treatment.

Reversal in central nervous system function during ethanol withdrawal in humans and experimental animals.

The ultimate objective in devising animal analogs of physical dependence on ethanol is to obtain meaningful imitations that have behavioral and biological similarities to human subjects during the ethanol withdrawal syndrome. The natural history of alcoholic disease in human subject and in experimental animals involves three periods, each characterized on the basis of temporal relationships, pattern of ethanol intake, blood ethanol concentrations, and/or a typical sequence in the onset and decay of the characteristic spectrum and continuum of overt behavioral, neurological, and biological signs and responses. These characteristics are expressions of different functional states of the central nervous system (CNS): 1) the baseline period or predrinking period reflects normal function of the CNS; 2) the induction period or drinking period is characterized by overt signs and responses of nonspecific, long-term CNS depression; and 3) the withdrawal period is characterized by a relatively rapid transition in the CNS function from depression during the prodromal detoxication phase to hyperexcitability observed during the withdrawal syndrome (dependence phase). The rapid transition from overt depression to overt hyperexcitability is a consequence of rapid removal of the drug from the system, and constitutes the basis of the reversal in the CNS function in both humans and experimental animals.