Bidirectional Alterations of GABAA Receptor Subunit Peptide Levels in Rat Cortex During Chronic Ethanol Consumption and Withdrawal

Abstract: The pharmacological properties of γ-aminobutyric acid_A (GABA_A) receptors are altered by prolonged exposure to ethanol both in vivo and in vitro. We have shown previously that prolonged ethanol exposure elicits selective alterations in various GABA_A receptor subunit mRNA levels in rat cerebral cortex. Some of these effects are rapidly reversed during ethanol withdrawal. The present study was conducted to determine the effects of prolonged ethanol exposure (dependence) and ethanol withdrawal on cerebral cortical peptide expression for several subunits. GABA_A receptor α1 subunit peptide levels were decreased by nearly 40%, whereas α4 subunit peptide levels were increased by 27% in both ethanol-dependent and withdrawn rats. These changes correlate well with observed alterations in mRNA levels following prolonged ethanol exposure in dependent rats, but do not match the effects on mRNA levels during ethanol withdrawal. β2/3 subunit peptide levels increased by ～32% in both ethanol-dependent rats and rats undergoing ethanol withdrawal. We observed a 30–60% increase in γ1 subunit peptide levels in both dependent rats and those undergoing withdrawal, also correlating with the previous report on ethanol-induced alterations in mRNA levels. Peptide levels for γ2 subunits did not differ from control values in either condition. These findings show that specific alterations in GABA_A receptor subunit peptide levels are associated with ethanol dependence in rats. GABA_A receptor subunit peptide expression is more stable than mRNA expression, and mRNA levels are not representative of peptide expression during ethanol withdrawal. These findings are consistent with the suggestion that alterations in GABA_A receptor gene expression underlie the functional properties of GABA_A receptors in ethanol-dependent rats and those undergoing ethanol withdrawal.     

Ethanol withdrawal in the rat: involvement of noradrenergic neurons

Ethanol dependence was induced in rats by maintaining them for 3 weeks on a liquid diet containing ethanol. When ethanol was abruptly replaced with sucrose in the diet, animals showed withdrawal symptoms. Eight hours later, the accumulation in brain and heart of ³H-norepinephrine synthesized from ³H-tyrosine, and of ³H-norepinephrine metabolites was greater than in animals not undergoing withdrawal. An injection of ethanol (3 g/kg) 1 $\text{1}\text{2}$ or 5 hours after the initiation of withdrawal resulted in less accumulation of newly synthesized ³H-norepinephrine and of ³H-norepinephrine metabolites in both brain and heart. If the rate of ethanol withdrawal was slow, i.e., the ethanol in the diet was replaced gradually with sucrose over a 3-day period, less accumulation of ³H-norepinephrine and ³H-norepinephrine metabolites occured in heart and brain than as a result of abrupt withdrawal. Also, no behavioral symptoms of withdrawal were observed. These results indicate that (a) gross withdrawal symptoms and the accompanying activation of noradrenergic neurons can be blocked during withdrawal by an acute dose of ethanol, and (b) ethanol withdrawal can be modified by altering the rate of withdrawal, a finding that may prove useful in clinical situations. We conclude that the withdrawal symptoms and the activation of noradrenergic neurons during withdrawal are caused by the sudden lack of ethanol in the system.     

Membrane solubility of ethanol in chronic alcoholism. The effect of ethanol feeding and its withdrawal on the protection by alcohol of rat red blood cells from hypotonic hemolysis

Membranes from ethanol-fed rats are resistant to the in vitro effects of ethanol on membrane structure and function. We have proposed that the resistance arises from adaptive changes in membrane composition which lower the solubility (partition coefficient) of ethanol in these membranes. The partition of ethanol (and other alcohols and anesthetics) into red blood cells protects the cells from hypotonic hemolysis. Here, we show that the protection by alcohols and anesthetics of red blood cells from ethanol-fed rats is greatly attenuated. This finding indicates that the membrane solubility of these agents is lowered in chronic alcoholism and thus explains the resistance to the acute effects of ethanol. The protection from hemolysis decreases over 2 weeks of ethanol-feeding and returns to normal values within 1 day after ethanol withdrawal. These changes are associated with a parallel increase in total and free serum cholesterol during ethanol feeding and a return to normal values within a day after withdrawal. However, we find only a slight increase in the cholesterol/phospholipid ratio of the red blood cell membranes during the development of ethanol tolerance. In rats fed a cholesterol and saturated fat diet, the increase in serum cholesterol is also associated with an attenuation of the protection from hypotonic hemolysis.     

Cerebral Metabolic State During the Ethanol Withdrawal Reaction in the Rat

Abstract: A severe ethanol withdrawal reaction was induced in rats by means of repeated intragastric intubation during a 4-day period. At the peak of the withdrawal reaction cerebral cortical tissue was frozen in situ for analysis of glycogen, glucose, phosphocreatine, creatine, ATP, ADP, AMP, lactate, pyruvate, GAB A, β-hydroxybutyrate, acetoacetate, cAMP and cGMP. Blood glucose concentration was also measured. The level of brain glycogen was decreased during ethanol withdrawal. Brain glucose concentration was increased, probably secondary to the increase in blood glucose concentration. The calculated NADH/NAD⁺ ratio was slightly increased during the withdrawal and brain ATP concentration and adenine nucleotide pool size were decreased. The adenylate energy charge remained unchanged. The overall changes in the metabolites were in agreement with the previously shown metabolic activation during ethanol withdrawal. The brain concentrations of ketone bodies (β-hydroxybutyrate and acetoacetate) during withdrawal did not deviate from controls, indicating that no abnormal ketone metabolism had developed as a consequence of the long-lasting ethanol intoxication. No changes were observed in the concentrations of GABA, cAMP, or cGMP in the rat cerebral cortex during ethanol withdrawal.     

Ethanol suppression of naloxone-induced withdrawal in morphine-dependent rats

The technique of morphine pellet implantation was used to produce physical dependence on morphine in male rats. The number of “wet dog” shakes occurring within a period of 30 minutes during naloxone-precipitated (1.0 mg/kg, s.c.) withdrawal in four-day morphine implanted rats was determined after either acute or chronic treatment with ethanol. An acute dose of ethanol administered prior to withdrawal had no significant effect on the withdrawal response whereas chronic administration of ethanol during the development of dependence on morphine significantly suppressed the naloxone-precipitated withdrawal response to 44–57 percent of the control response. Analysis of brain and plasma for morphine concentration four days following dependence development showed no significant differences between morphine controls and those animals treated with both morphine and ethanol. Pentobarbital, another central nervous system depressant, demonstrated no effect on the withdrawal response, whether administered acutely or chronically during the development of dependence.     

CHANGES IN BRAIN ALCOHOL DEHYDROGENASE ACTIVITY DURING CHRONIC ETHANOL INGESTION AND WITHDRAWAL

Abstract— Chronic ethanol ingestion in rats leads to a slow rise in brain alcohol dehydrogenase activity which levels off after 2 weeks at approximately twice the initial activity. The half-time of the rise is approximately 8 days. Abrupt withdrawal of the ethanol is followed by a rapid decline of the brain alcohol dehydrogenase activity to the normal level with a half-time of approximately 15 h. The difference in time constants between the rise in enzyme activity during ethanol-feeding and its decline following withdrawal suggests that the increased enzyme activity is at least in part the result of a reduced rate constant of enzyme degradation in the presence of ethanol. The effect of ethanol on brain alcohol dehydrogenase activity is not altered by supplementation of the diet with carbohydrate or vitamins. The effect is seen only in the cerebral hemispheres and not in the brain-stem. Acquisition of tolerance to ethanol during chronic ethanol ingestion and its extinction following withdrawal follow almost the same time courses as the changes in brain alcohol dehydrogenase activity.     

Anticonvulsive effects of kappa-opioid receptor modulation in an animal model of ethanol withdrawal

Although the neurochemical mechanisms contributing to alcohol withdrawal seizures are poorly understood, withdrawal seizures probably reflect neuronal hyperexcitability resulting from adaptation to chronic alcohol. Altered kappa-Opioid receptor (KOP-R) signaling has been observed in multiple seizure types; however, a role for this system in ethanol withdrawal seizures has not been systematically characterized. We hypothesized that pharmacological manipulations of the KOP-R would alter withdrawal in mice selectively bred for differences in ethanol withdrawal severity. Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice were made physically dependent using chronic ethanol vapor inhalation, and the effects of the KOP-R antagonist nor-binaltorphimine or agonist U-50,488H on withdrawal severity were examined. Pretreatment with nor-binaltorphimine significantly increased handling-induced convulsion (HIC) severity in withdrawing WSR mice, with no observable effects in withdrawing WSP mice. In contrast, U-50,488H significantly decreased HIC severity in WSP mice, with no effects in WSR mice. During extended withdrawal (i.e. hours 12+), a rebound hyperexcitability was observed in WSP mice given agonist. Thus, administration of a KOP-R antagonist increased withdrawal severity in mice normally resistant to withdrawal seizures, while a KOP-R agonist reduced convulsion severity in animals susceptible to withdrawal seizures. These observations are consistent with differences in the KOP-R system observed in these lines at the molecular level, and suggest the KOP-R system may be a promising therapeutic target for management of ethanol withdrawal seizures. Finally, these findings underscore the importance of determining the potential for rebound increases in withdrawal severity during later withdrawal episodes.     

Effects of delta sleep-inducing peptide on electrophysiological parameters of sleep during alcohol withdrawal in rats

In rats with the persistent alcohol motivation the electrophysiological sleep pattern was studied during ethanol intake, after 24 and 48 hours of alcohol withdrawal. It was established that during the voluntary ethanol intake rats may be divided into two groups: with comparative deficit (1st group) and comparative abundance (2nd group) of REM sleep. Alcohol withdrawal caused differential alterations of sleep-wakefulness cycle: in the 1st group of rats REM sleep was more suppressed while in the 2nd group--more increased in comparison to those during ethanol intake. In all animals the SWS depression, increase of awakenings, the aggravation of falling asleep and decrease of sleep depth were observed. DSIP (0.1 mg/kg, i.p. 1 hour before sleep recording) was found to regulate sleep disorders caused by ethanol withdrawal. It makes the neuropeptide possible to be recommended for ethanol withdrawal syndrome treatment in clinical practice.     

Changes in the aminopeptidase activity in the brain neurons of rats subjected long-term to alcoholic intoxication

A histochemical investigation was made of aminopeptidase activities using L-leucine-beta-naphthylamide and D, L-alanine-beta-naphthylamine as respective substrates in the rat's sensomotor cortex neurons during the period of ethanol withdrawal after a prolonged alcohol intoxication. Alcohol intoxication of the rat continued for 8 months. Different dynamics of aminopeptidase activities was shown depending on the substrate used during ethanol withdrawal. The results are discussed from the point of view of the existence of multiple forms of aminopeptidase in the brain and of their involvement in protein metabolism in pathological states.     

Ethanol and diazepam withdrawal convulsions are extensively codetermined in WSP and WSR mice

Selective breeding was used to produce lines of mice which differ markedly in their genetically-mediated vulnerability to handling-induced convulsions (HIC) associated with the ethanol withdrawal syndrome. These are known as the ethanol withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) selection lines. As a result of 5 generations of selective breeding with ethanol, a 3.4-fold difference between WSP and WSR mice was seen in HIC associated with ethanol withdrawal. When diazepam was used as the dependence-producing drug, a 2.4-fold difference emerged. After 6 more generations of selective breeding with ethanol, an approximate 10-fold difference was seen with ethanol, while with diazepam, this difference in HIC scores was also about 10-fold. This close parallel between ethanol and diazepam indicates that physical dependence on both drugs, as indexed by handling-induced convulsions, is extensively codetermined by the same genes, and thus by the same mechanisms, in these selectively-bred mice.     

Effects of PKA modulation on the expression of neuropeptide Y in rat amygdaloid structures during ethanol withdrawal

We recently reported that neuropeptide Y (NPY) protein levels and cAMP responsive element binding (CREB) protein phosphorylation are lower in amygdaloid structures during ethanol withdrawal after chronic exposure. Furthermore, we reported that normalization of CREB phosphorylation by infusing protein kinase A (PKA) activator into the central amygdala prevents anxiety-like effects in rats during ethanol withdrawal. Here we investigated whether normalization of CREB phosphorylation by infusing PKA activator (Sp-cAMP) into the central amygdala also normalizes the expression of NPY during ethanol withdrawal. Sprague-Dawley male rats were cannulated targeting the central amygdala and then treated either with Lieber-DeCarli ethanol diet or control diet for 15 days. Subsequently ethanol-fed rats were withdrawn for 0 and 24h. The control-diet fed and ethanol-withdrawn rats were infused twice with PKA activator or inhibitor (Rp-cAMP). The protein and mRNA levels of NPY were determined in amygdaloid structures using gold-immunolabeling and the in situ RT-PCR procedure. It was found that chronic ethanol treatment has no effect on mRNA and protein levels of NPY in the central, medial, or basolateral amygdala. On the other hand, ethanol withdrawal produced significant reductions in mRNA and protein levels of NPY in the central and medial but not in the basolateral amygdala. The reductions in mRNA and protein levels of NPY were normalized in the central amygdala by infusion with PKA activator in ethanol-withdrawn rats. On the other hand, PKA-inhibitor infusion does not have any effect on mRNA and protein levels of NPY in the central amygdala of ethanol-withdrawn rats, but significantly decreased the expression of NPY in the central amygdala of control-diet fed rats. These results suggest that the decreased cellular expression of NPY in the central amygdala may play an important role in the CREB-mediated regulation of anxiety-like behaviors during ethanol withdrawal.     

Chronic ethanol feeding produces an increase in muscarinic cholinergic receptors in mouse brain

Chronic ethanol administration (seven days) to mice produces physical dependence while subsequent withdrawal produces seizures and convulsions, especially during the first 24 hours. It is known that ethanol can inhibit brain acetylcholine (ACh) release. Our present study suggests that hippocampal (+25%) and cortical (+14%), but not striatal neurons, can respond to the ethanol-induced reduction in neurotransmitter (ACh) availability by increasing the number of muscarinic cholinergic receptors. It is possible that these supersensitive neurons are involved in the CNS hyperexcitability induced by withdrawal from chronic ethanol.     

Acute and Chronic Ethanol Consumption Effects on the Immunolabeling of Gq/11α Subunit Protein and Phospholipase C Isozymes in the Rat Brain

Abstract: The goal of this investigation was to examine whether postreceptor sites [G_q/11 protein and phospholipase C (PLC) isozymes] of the phosphoinositide signal transduction system are involved in neuroadaptational mechanisms in the brain during chronic ethanol consumption. It was observed that acute ethanol treatment has no effect on the immunolabeling of PLC-β₁, -γ₁, and -δ₁ and the α subunit of G_q/11 protein in the rat cortex as determined by western blotting using specific monoclonal antibodies. On the other hand, chronic ethanol consumption (15 days) resulted in a significant decrease in the immunolabeling of PLC-β₁, whereas under identical conditions, the immunolabeling of PLC-γ₁ and -δ₁ isozymes was not significantly altered. The decreased immunolabeling of PLC-β₁ during chronic ethanol consumption was not altered by 24 h of withdrawal after 15 days of ethanol consumption. The immunolabeling of the α subunit of G_q/11 protein was significantly decreased after 15 days of ethanol consumption but had returned to normal levels after 24 h of ethanol withdrawal. Also, chronic ethanol treatment resulted in a significant decrease in phosphatidylinositol 4,5-bisphosphate-specific PLC activity, which remained the same after 24 h of ethanol withdrawal. These results suggest that decreased PLC activity during ethanol consumption and its withdrawal may be due to decreased protein levels of the G_q/11 protein-coupled PLC-β₁ isozyme but not the PLC-γ₁ or -δ₁ isozyme in the rat cortex. It is possible that changes in the protein levels of the G_q/11 protein-coupled PLC-β₁ isozyme and in PLC activity in the brain may be involved in the cellular adaptation to chronic ethanol exposure.     

Brain benzodiazepine binding sites in ethanol dependent and withdrawal states

The brain benzodiazepine system has been implicated to be important in both the mechanism, and treatment of ethanol related syndromes. In this report evidence is presented which indicates that "peripheral type" benzodiazepine binding sites are probably more relevant than "central type" receptors for the neurochemical consequences of ethanol dependence and withdrawal states. Utilizing radioreceptor binding techniques 20-50% increases in the binding of [3H]RO-5-4864 (a "peripheral type" ligand) to brain membranes derived from rat cerebral cortex, cerebellum and hippocampus are observed in ethanol dependent rats. These increases persist for 3 days after cessation of ethanol. The number of [3H]RO-5-4864 binding sites in cerebellum returns to normal during 4-7 days after ethanol withdrawal. In all brain areas examined no changes were observed in the "central type" benzodiazepine receptor as judged by [3H]-ethyl-Beta-carboline-3-carboxylate, BCCE binding. Scatchard analysis revealed that the number of [3H]RO-5-4864 binding sites is increased in each brain area while the affinity was unchanged.     

Ethanol and nitrous oxide produce withdrawal-induced convulsions by similar mechanisms in mice

Twenty generations of selective breeding were used to produce lines (strains) of mice which differ markedly from one another in ethanol physical dependence development as indexed by handling-induced convulsions (HIC) induced by withdrawal from ethanol. These withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) selection lines now differ by over 10-fold in HIC scores after equivalent exposure to intoxicating levels of ethanol via inhalation. Since handling-induced convulsions can be readily elicited following withdrawal from nitrous oxide, we sought to determine if the very large differences in ethanol withdrawal-induced HIC bred into these selection lines would generalize to nitrous oxide. Following a 60 min exposure to 75% nitrous oxide (in O2), a greater than 10-fold difference in HIC scores, and a 2-fold difference in tremor incidence was seen upon withdrawal in WSP vs. WSR mice. These findings closely parallel those seen with ethanol, and demonstrate that a large degree of commonality exists in the genes and the mechanisms determining these withdrawal signs. HIC elicited by nitrous oxide withdrawal were readily suppressed by ethanol, and HIC elicited by ethanol withdrawal were promptly suppressed by 75% nitrous oxide in WSP mice. Nitrous oxide also suppressed HIC and tremor associated with nitrous oxide withdrawal.     

Interaction of nutrition and binge ethanol treatment on brain damage and withdrawal

To determine if nutrition plays a role in ethanol withdrawal and alcohol-induced brain damage, the effects of a 4-day ethanol binge treatment using ethanol in a nutritionally complete liquid diet compared to ethanol mixed with water were studied. The nutritionally complete diet group (ETOH-diet) received a complete diet of sugars, proteins and fats with vitamins and minerals with approximately 53% of calories from ethanol while the nutritionally deprived group (ETOH-H2O) received 100% of calories from ethanol. No difference in withdrawal behavior was found between the ETOH-diet and ETOH-H2O groups during the 72-hour period studied. In addition, no difference was seen for serum levels of magnesium and zinc taken at last dose or following 72 h of withdrawal. Serum alanine aminotransferase (ALT) and ammonia were increased in both groups with ETOH-diet showing a greater increase in ALT than ETOH-H2O. Both groups showed damage in the olfactory bulb, perirhinal, agranular insular, piriform and lateral entorhinal cortical areas as well as hippocampal dentate gyrus and CA-3. Interestingly, the ETOH-diet group displayed more damage at last dose in the posterior dentate and CA-3 of hippocampus than did the ETOH-H2O group. This study suggests that nutritional components and total caloric intake do not effect behavior during ethanol withdrawal and that a nutritionally complete diet may increase ethanol-induced brain damage.     

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.     

Pharmacological activation/inhibition of the cannabinoid system affects alcohol withdrawal-induced neuronal hypersensitivity to excitotoxic insults

Cessation of chronic ethanol consumption can increase the sensitivity of the brain to excitotoxic damages. Cannabinoids have been proposed as neuroprotectants in different models of neuronal injury, but their effect have never been investigated in a context of excitotoxicity after alcohol cessation. Here we examined the effects of the pharmacological activation/inhibition of the endocannabinoid system in an in vitro model of chronic ethanol exposure and withdrawal followed by an excitotoxic challenge. Ethanol withdrawal increased N-methyl-D-aspartate (NMDA)-evoked neuronal death, probably by altering the ratio between GluN2A and GluN2B NMDA receptor subunits. The stimulation of the endocannabinoid system with the cannabinoid agonist HU-210 decreased NMDA-induced neuronal death exclusively in ethanol-withdrawn neurons. This neuroprotection could be explained by a decrease in NMDA-stimulated calcium influx after the administration of HU-210, found exclusively in ethanol-withdrawn neurons. By contrast, the inhibition of the cannabinoid system with the CB1 receptor antagonist rimonabant (SR141716) during ethanol withdrawal increased death of ethanol-withdrawn neurons without any modification of NMDA-stimulated calcium influx. Moreover, chronic administration of rimonabant increased NMDA-stimulated toxicity not only in withdrawn neurons, but also in control neurons. In summary, we show for the first time that the stimulation of the endocannabinoid system is protective against the hyperexcitability developed during alcohol withdrawal. By contrast, the blockade of the endocannabinoid system is highly counterproductive during alcohol withdrawal.