Ethanol-dependent Rats Research Articles

Ethanol inhibition of NMDA currents in acutely dissociated medial septum/diagonal band neurons from ethanol dependent rats

The effect of acutely applied ethanol and the impact of chronic ethanol treatment, sufficient to induce tolerance and physical dependence, on N-methyl- d-aspartate (NMDA) receptor function were studied in acutely isolated neurons from the medial septum/diagonal band (MS/DB) of adult rats using whole cell, patch-clamp electrophysiology. There was a small positive correlation for capacitance and current amplitude activated by 100 μM NMDA for all groups. Also, cell membrane capacitance was significantly smaller for Ethanol Dependent (∼80–84%) than either Naive or Control cells. Therefore NMDA-activated responses were normalized for capacitance (current density, pA/pF) across all three groups. NMDA-activated (30–1000 μM) responses were significantly larger in cells from Control and Ethanol Dependent rats relative to those from Naives. In addition, estimated maximal responses were significantly larger for Ethanol Dependent cells, compared to either Control or Naive, respectively, while EC 50s and slopes were not significantly different. Acute 60 mM ethanol significantly inhibited responses to 100 μM NMDA in all three groups, however, mean ethanol inhibition was 12–25% smaller after ethanol dependence. There was no evidence of acute tolerance to ethanol inhibition for any group, but examination of patterns of inhibition for individual neurons showed a few cells were resistant to ethanol or exhibited progressive loss of ethanol inhibition. These results suggest that NMDA receptor function in acutely isolated MS/DB neurons is increased following in vivo chronic ethanol treatment, and shows resistance to acute ethanol inhibition suggesting NMDA receptor-mediated cellular tolerance.

Bidirectional alterations of GABA(A) receptor subunit peptide levels in rat cortex during chronic ethanol consumption and withdrawal.

The pharmacological properties of gamma-aminobutyric acidA (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 alpha1 subunit peptide levels were decreased by nearly 40%, whereas alpha4 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. Beta2/3 subunit peptide levels increased by approximately 32% in both ethanol-dependent rats and rats undergoing ethanol withdrawal. We observed a 30-60% increase in gamma1 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 gamma2 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.

Acamprosate. A review of its pharmacology and clinical potential in the management of alcohol dependence after detoxification.

Acamprosate (calcium acetylhomotaurinate), a synthetic compound with a similar chemical structure to that of gamma-aminobutyric acid, is thought to act via several mechanisms affecting multiple neurotransmitter systems; inhibition of neuronal hyperexcitability by antagonism of excitatory amino acid activity and reduction of calcium ion fluxes has been suggested as its predominant mechanism of action. The drug is the first agent specifically designed to maintain abstinence in alcohol (ethanol)-dependent patients after detoxification. Voluntary oral ethanol consumption in ethanol-preferring or ethanol-dependent rats is dose-dependently reduced by acamprosate: total fluid intake and food consumption are not affected. The drug does not potentiate the acute or chronic toxic effects of ethanol and has no hypnotic, antidepressant, anxiolytic or muscle-relaxant effects in animals. There is no evidence of abuse potential with acamprosate. Oral acamprosate 1.3 or 2 g/day in 3 divided doses administered for 3 to 12 months to alcohol-dependent patients after detoxification was more effective than placebo in preventing alcohol relapse according to abstinence rates, duration of abstinence, gamma-glutamyl transferase levels and/or a variety of other clinical or biological end-points. Concomitant psychosocial/behavioural therapies were used in some trials. Compared with those with placebo, the superior abstinence rates and durations of abstinence with acamprosate were maintained during 6- to 12-month post-treatment follow-up periods, and greater abstinence rates with acamprosate were confirmed in a pooled analysis of data from 11 randomised placebo-controlled trials involving a total of 3338 patients with alcohol dependence. The efficacy of acamprosate appears to be dose dependent and enhanced by the addition of disulfiram. Acamprosate was generally well tolerated in placebo-controlled trials. The most common adverse events were gastrointestinal (especially diarrhoea) or dermatological and were mostly mild and transient. The percentage of patient withdrawals because of adverse events was similar in acamprosate and placebo groups. No trials have compared the efficacy or tolerability of acamprosate with those of other treatment approaches (including opiate antagonists or selective serotonin reuptake inhibitors) aimed at maintaining abstinence in detoxified alcohol-dependent patients. Thus, acamprosate, as an adjunct to psychosocial/behavioural therapies, represents a novel advance for the management of alcohol-dependent patients in the postdetoxification period. Longer term and comparative trials with other active therapies are required to confirm these promising results.

Lanthanum and zinc sensitivity of GABA A-activated currents in adult medial septum/diagonal band neurons from ethanol dependent rats

The impact of chronic ethanol treatment, sufficient to induce tolerance and physical dependence, on GABA A receptor function was studied in acutely isolated neurons from the medial septum/nucleus diagonal band (MS/nDB) of adult rats using whole cell, patch-clamp recordings. In ethanol-naive Controls, GABA (0.3–300 μM) induced concentration-dependent increases in Cl − current with a threshold of 0.3–1 μM, a mean maximal current of 7645 ± 2148 pA at 100–300 μM, an EC 50 of 11.3 ± 1.3 μM and a slope of 1.53 ±0.07. GABA-activated currents in neurons from animals receiving two weeks of ethanol liquid diet treatment did not differ significantly on any of these measures. The rate of GABA A receptor desensitization (t 1/2 = 6.49 ± 1.19 s) estimated as the time required for loss of 50% of peak current during sustained application of 10 μM GABA, as well as the residual steady state current remaining following complete desensitization for controls was unchanged by chronic ethanol. The impact of chronic ethanol treatment on the GABA A receptor modulation by lanthanum and zinc which act as positive and negative allosteric modulators, respectively, was also evaluated. Test pulses of 3 μM GABA in control neurons showed maximal potentiation by 141 ± 30% at ~ 1000 μM lanthanum with an EC 50 of 107 ± 34 μM and a slope of ~ 1. Lanthanum potentiation remained the same following chronic ethanol treatment. Initial estimates based on fitted concentration response curves suggested that maximal inhibition of 3 μM GABA responses by zinc at the level of 70.2 ± 8.5% in control cells was significantly increased by chronic ethanol treatment to 95.3 ± 2.5%, although the IC 50 of 60.2 ± 25 μM was not changed. However, this difference was not supported by direct tests of maximal 3–10 mM zinc concentrations. These results suggest that chronic ethanol treatment, sufficient to induce tolerance and physical dependence, probably does not lead to readily detectible changes in GABA A receptor function in MS/nDB neurons.

The neurosteroid, 3 alpha-hydroxy-5 alpha-pregnan-20-one, protects against bicuculline-induced seizures during ethanol withdrawal in rats.

Prolonged alcohol consumption leads to the development of tolerance to and dependence on ethanol, resulting in a decreased response to the sedative/hypnotic effects of ethanol, and by negative symptomatology following abrupt termination of use. One symptom associated with ethanol withdrawal in humans, as well as laboratory animals, is enhanced susceptibility to seizures. This study investigated the effects of the neurosteroid, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-5 alpha-THP), on alterations in seizure sensitivity associated with ethanol withdrawal. 3 alpha-5 alpha-THP is a potent anxiolytic and anticonvulsant agent that acts via selective interactions with GABAA receptors. Extensive evidence suggests that some aspects of ethanol dependence and withdrawal are mediated by alterations in GABAA receptor function. Withdrawal from chronic ethanol exposure elicited dramatic increases in seizure susceptibility in male and female rats. Administration of 3 alpha-5 alpha-THP just before seizure threshold determinations blocked the increased seizure susceptibility induced by ethanol withdrawal. Ethanol-withdrawn animals were protected by 3 alpha-5 alpha-THP at a dose that had no effect on control animal seizure thresholds. Moreover, male and female rats displayed differential responses to the seizure-threshold lowering effects of ethanol withdrawal, as well as the protection by 3 alpha-5 alpha-THP pretreatment. These findings suggest that there are gender differences associated both with ethanol withdrawal as well as the protection by 3 alpha-5 alpha-THP in ethanol-dependent rats.

Ethanol withdrawal induces increased firing in inferior colliculus neurons associated with audiogenic seizure susceptibility

Ethanol withdrawal (ETX) in ethanol-dependent rats results in susceptibility to seizures, including generalized tonic-clonic audiogenic seizures (AGS). The inferior colliculus (IC) is strongly implicated in AGS initiation during ETX, but IC neuronal mechanisms subserving AGS are unclear. The present study examined IC (central nucleus) single neuronal firing during repeated (4 day) intragastric ethanol administration and during ETX. This involved microwire electrodes implanted chronically into freely moving rats and acoustic stimulation in intensities up to 105 dB SPL. During initial ethanol administration the animals were stuporous, and IC spontaneous neuronal firing and acoustically evoked firing at high stimulus intensities were significantly reduced. This firing reduction is consistent with the action of ethanol to enhance γ-aminobutyric acid (GABA)-mediated inhibition, which is prominent in IC neurons at high stimulus intensities. During ETX the animals were agitated, and spontaneous IC neuronal firing and acoustically evoked firing at all stimulus intensities were significantly increased during the period of AGS susceptibility. Previous studies indicate that IC neuronal responses are tightly regulated by GABA and glutamate. The IC firing increases during ETX in the present study may involve the down-regulation of GABA A receptors and supersensitivity of glutamate receptors reported to occur during ETX. Previous studies also indicate that focal blockade of GABA A receptors or activation of glutamate receptors produces AGS susceptibility in normal rats. Therefore, the IC neuronal firing increases observed in the present study may play a critical role in initiation of AGS during ethanol withdrawal.

BINDING CHARACTERISTICS OF THE CALCIUM CHANNEL ANTAGONIST [<sup>3</sup>H]-NITRENDIPINE IN TISSUES FROM ETHANOL-DEPENDENT RATS

Male Sprague-Dawley rats were made physically dependent on ethanol by exposure to ethanol vapour in inhalation chambers for 6-10 days. Animals were sacrificed immediately after removal from the chambers. After sacrifice, membrane preparations from cerebral cortex, heart, vas deferens and thigh skeletal muscle were prepared and studied to determine the characteristics of [3H]-nitrendipine binding. In membranes from cerebral cortex, the number of binding sites (Bmax) was increased 40% in preparations from ethanol-dependent rats compared to controls. Similar results were obtained in heart (Bmax increased 125%), vas deferens (Bmax increased 50%) and skeletal muscle (Bmax increased 33%). Binding affinity (Kd) in all these tissues was unchanged. Membranes from cortex and heart were studied more extensively. Displacement studies using other dihydropyridines and the effects of ionic composition on [3H]-nitrendipine binding provided no evidence to suppose that the binding sites induced by the development of ethanol dependence were from a population different from those in control tissues. The results suggest a generalised 'up-regulation' of the dihydropyridine-sensitive subclass of voltage operated calcium channels in excitable tissues from ethanol-dependent rats.

Increased sensitivity of the hippocampus in ethanol-dependent rats to toxic effect ofN-methyl- D-aspartic acid in vivo

Chronic administration of ethanol in animals leads to CNS tolerance and physical dependence. Subsequent withdrawal of ethanol causes hyperexcitability which is thought to be related to increased sensitivity ofN-methyl- d-aspartic acid (NMDA) receptors. The purpose of this study was to investigate sensitivity to NMDA in ethanol-treated animals by detecting damage after intrahippocampal injection of NMDA. Choline acetyltransferase (ChAT) and glutamate decar☐ylase (GAD) specific activity was used as markers of cholinergic and γ-aminobutyric acid neurons, respectively. Ethanol-dependent animals were more liable to die following intrahippocampal injection of either 120 or 240 nmol of NMDA. There was a significantly greater decrease in hippocampal GAD but not ChAT specific activity in the surviving animals. These data support the hypothesis that ethanol dependence is associated with increased sensitivity to NMDA which may be responsible for excitotoxic brain damage and death.

Chronic ethanol intoxication induces differential effects on GABAA and NMDA receptor function in the rat brain.

The effect of long-term treatment with ethanol was investigated on the function of gamma-aminobutyric acid A (GABAA) and N-methyl-d-aspartic acid (NMDA) receptors. Rats were rendered ethanol-dependent by repeated forced administration of a 20% ethanol solution (12 to 18 g/kg/day po) for 6 days and tested while still intoxicated or at different time intervals after withdrawal. t-[35S]Butylbicyclophosphorothionate (35S-TBPS) binding was increased by 30% in cortical homogenates of rats killed 1 to 3 hr after last ethanol administration, when compared with saline-treated animals. However, GABA-stimulated 36Cl- uptake and its enhancement by flunitrazepam was decreased in the ethanol-treated animals. 35S-TBPS binding and 36Cl- influx measured 9 to 24 hr following the last ethanol injection, when withdrawal signs were present, were unmodified with respect to saline-treated rats. Moreover, the effects of both isoniazid and FG 7142 on 35S-TBPS binding were unchanged in ethanol-dependent rats tested at 1 to 3 and 9 to 24 hr, compared with controls. In contrast, ethanol-withdrawn rats tested at 9 to 24 hr showed a dramatic enhancement in their sensitivity to the convulsant action of isoniazid (50 to 250 mg/kg, sc). The same animals were also more susceptible to the convulsant action of NMDA (0.5 to 5 micrograms/5 microliters/rat intracerebroventricularly) and kainic acid (12 mg/kg, ip), and this effect was paralleled by an enhancement (+25%) in the density of 3H-MK 801 recognition sites in the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

Marked decrease of A10 dopamine neuronal firing during ethanol withdrawal syndrome in rats

The electrophysiological activity of mesoaccumbens dopaminergic neurons was monitored during the ethanol-withdrawal syndrome in ethanol-dcpendent and in control rats. Spontaneous firing was reduced by about half in ethanol-dependent rats as compared to controls. Likewise, the number of spikes/burst was also reduced in ethanol-dependent rats. These results are consistent with the reduction in dopamine release observed during ethanol-withdrawal syndrome and may provide the basis for the aversive effects of the ethanol-withdrawal syndrome.

Dihydropyridine sensitive calcium channel upregulation and indices of myocardial cell dammage in isolated hearts from ethanol dependent rats: Leon J. Guppy and John M. Littleton. Pharmacology Group, Biomedical Division, Kings College London, Strand, London, WC2R 2LS, United Kingdom

Dihydropyridine sensitive calcium channel upregulation and indices of myocardial cell dammage in isolated hearts from ethanol dependent rats: Leon J. Guppy and John M. Littleton. Pharmacology Group, Biomedical Division, Kings College London, Strand, London, WC2R 2LS, United Kingdom

Ethanol withdrawal increases sensory responsiveness of single somatosensory cortical neurons in the awake, behaving rat.

This study investigated the effects of ethanol withdrawal on sensory responses of single neurons recorded in the somatosensory (SI) cortex of awake, behaving, ethanol-dependent rats. Eleven rats were fed an ethanol-containing liquid diet for 90-120 days, while 11 weight-matched controls were pair-fed an equivalent sucrose containing diet to equalize caloric intake. Single SI cortical neurons in the chronically treated rats were recorded continuously over several hours after withdrawal from ethanol, and after reintoxication induced by intraperitoneal injection of 10% (v/v) ethanol solution. Intoxication and withdrawal related changes in sensory responsiveness of these neurons were quantitatively measured by stimulating through electrodes chronically implanted under the skin of the forepaw. Sensory response histograms constructed from these stimuli showed a characteristic pattern, typically consisting of two early excitatory peaks (E1a and E1b), followed by an inhibition (I1), and in some neurons, a late excitatory response (E2). As withdrawal advanced, the sensory response histograms exhibited marked increases in the magnitudes of the E1a, I1, and E2 responses, coupled with a reduced spontaneous discharge rate. These changes are similar to, but quantitatively greater than, those which have previously been observed in normal and control rats during "immobile arousal" behaviors, which can be evoked when an experimenter holds the animals still, producing an immobile "aversive arousal." In withdrawing animals this same "holding" manipulation tended to markedly exacerbate and accelerate behavioral and neurophysiological signs of withdrawal. By contrast, the same manipulations had little effect when carried out during light intoxication or early stages of withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased dihydropyridine-sensitive calcium channels in rat brain may underlie ethanol physical dependence

Ethanol physical dependence can be viewed as a state of latent hyperexcitability in brain which is exposed on withdrawal of the drug. This hyperexcitability may reflect an increased sensitivity to Ca 2+ of central neurones. Dihydropyridine (DHP) binding sites which represent a subtype of neuronal Ca 2+-channel, are increased in brains from ethanol-dependent rats as are functional effects of the DHP Ca 2+-channel activator, BAYK8644. These effects are reversed by DHP Ca 2+ inhibitors, which also prevent the ethanol physical withdrawal syndrome. These results suggest that an increase in DHP-sensitive Ca 2+-channels on central neurons may represent the molecular basis for ethanol physical dependence.

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.

Supression of an ethanol withdrawal syndrome in rats by 3-hydroxybutyrate

An ethanol withdrawal syndrome was elicited by witholding ethanol from physically dependent, male Sprange-Dawley rats. Ethanol dependence has been induced by intragastric administration of ethanol at a dosage of 9 to 15 grams per kilogram per day over a four-day period. Oral administration of 3-hydroxybutyrate, a compound which is elevated in blood of ethanol dependent rats and is a substrate of both the cerebral small-pool and large-pool Krebs-cycle, was effective in suppressing the tremulous component of the ethanol withdrawal syndrome. 3-Hydroxybutyrate did not function as a central nervous system depressant at the dose levels employed.

Age related differences in dopamine-stimulated adenylate cyclase sensitivity to “in vivo” chronic ethanol treatment

The effect of chronic ethanol consumption on adenylate cyclase activity was measured in striatal membranes derived from aged male rats. The results indicate that the cyclic AMP generating system of old rats has a different sensitivity to ethanol effect compared to the adult animals. In young animals the basal adenylate cyclase activity was enhanced by alcohol consumption while the DA stimulated cyclic AMP production was reduced. In contrast, in 24 month old rats ethanol reduced the basal adenylyl cyclase and enhanced the response to DA indicating a supersensitivity of adenylate cyclase linked DA receptors. This observation was further supported by 3H-Spiperone binding studies. In fact, a higher B max was measured in striatal membranes of aged ethanol-dependent rats in comparison to control.

Chronic ethanol changes opiate receptor function in rat striatum

Ethanol produces supersensitivity of striatal σ-opiate receptor sites labeled by [ 3H]DADLE and [ 3H]etorphine. The impairment may be ascribed to the diminished enkephalin release detected in rat striatum after chronic ethanol consumption. On the contrary, a lower affinity of striatal μ-opiate receptors results after same ethanol exposure. In fact, the K d values of [ 3H]Met-enkephalin and [ 3H]DHM are enhanced when measured in striata of ethanol-dependent rats. The diverse sensitivity of the various classes of opiate receptors to ethanol may be ascribed to different ethanol effects on enekephalinergic transmission.

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.

Effect of ethanol on synaptosomal (Na + + K +)-ATPase in control and ethanol-dependent rats

Rats were made dependent upon ethanol by feeding them liquid diets containing ethanol. Synaptosomal plasma membranes (SPM) were isolated from cerebral cortex and midbrain regions of isocaloric-fed control and ethanol-dependent rats. No major alcohol-induced alteration in in vitro (Na + + K +)-ATPase activity was found in SPM of either brain area. At 37 °C, ethanol (0.10 to 0.98 m) added to incubations caused a dose-dependent inhibition of (Na + + K +)-ATPase activity. The degree of inhibition found was independent of the diet administered or whether ethanol was present in the diet. At temperatures between 14 and 22 °C, 0.48 m ethanol caused a temperature-dependent decrease in activity. Arrhenius plots for SPM (Na + + K +)-ATPase showed that in control and ethanol-dependent rats fed the Lieber de Carli diet, 0.48 m ethanol did not alter the transition temperature of this enzyme. Activation energies both above and below the transition temperature were decreased by the addition of ethanol to incubations. These results indicate that (Na + + K +)-ATPase, a membrane-bound enzyme that is sensitive to its lipid environment and to the presence of ethanol, is not altered by the chronic administration of ethanol to rats.

Suppression of an ethanol withdrawal syndrome in rats by butyrate, lactate and β-hydroxybutyrate

Butyrate, lactate and β-hydroxybutyrate, compounds which may be elevated in blood of ethanol dependent rats and substrates of the cerebral small-pool Krebs-cycle, were tested for their ability to surpress an ethanol withdrawal syndrome. Male Sprague-Dawley rats were rendered physically dependent on ethanol by intragastric administration of ethanol at a dosage of 9 to 15 grams per kilogram per day over a 4-day period. Oral administration of a mixture of butyrate, lactate and β-hydroxybutyrate was effective in suppressing the tremulous component of the ethanol withdrawal syndrome.