Development Of Ethanol Dependence Research Articles

Corticotropin releasing factor, but not alcohol, modulates norepinephrine release in the rat central nucleus of the amygdala

Alcohol misuse and dependence is a widespread health problem. The central nucleus of the amygdala (CeA) plays important roles in both the anxiety associated with alcohol (ethanol) dependence and the increased alcohol intake that is observed during withdrawal in dependent animals. We and others have shown the essential involvement of the corticotropin releasing factor (CRF) system in alcohol's synaptic effects on the CeA and in the development of ethanol dependence. Another system that has been shown to be critically involved in the molecular underpinnings of alcohol dependence is the norepinephrine (NE) system originating in the locus coeruleus. Both the CRF and NE systems act in concert to facilitate a stress response: central amygdalar afferents release CRF in the locus coeruleus promoting widespread release of NE. In this study, we are the first to use fast-scan cyclic voltammetry to classify local electrically-evoked NE release in the CeA and to determine if acute alcohol and CRF modulate it. Evoked NE release is action potential dependent, is abolished after depletion of monoaminergic vesicles, differs pharmacologically from dopamine release, is insensitive to acute alcohol, and decreases in response to locally applied CRF. Taken together, these results indicate that NE release in the CeA is released canonically in a vesicular-dependent manner, and that while acute alcohol does not directly alter NE release, CRF decreases it. Our results suggest that CRF acts locally on NE terminals as negative feedback and potentially prevents hyperactivation of the CRF-norepinephrine stress pathway.

Role of HPA axis on the development of ethanol dependence in mice

Role of HPA axis on the development of ethanol dependence in mice

Ethanol reinforcement elicits novel response inhibition behavior in a model of ethanol dependence

Reduced impulse control is a known risk factor for drug abuse vulnerability. Chronic experience with drugs of abuse is suggested to enhance impulsivity and thereby perpetuate addiction. However, the nature of this relationship (directionality, causality) with respect to alcohol use disorder is unclear. The current study aims to evaluate changes in impulsivity during the development of ethanol dependence and subsequent protracted abstinence from excessive ethanol. A widely validated chronic intermittent ethanol-vapor exposure (CIE; 14 h vapor-ON/10 h vapor-OFF) was used to generate ethanol dependence and impulsivity was tested using a differential reinforcement of low rates 15 second (DRL15) schedule using either SUCROSE (palatable modified sucrose pellets) or ETHANOL (10%v/v with 0.125% w/v saccharine sodium, 1.5% w/v sucrose). Reduced efficiency of earning reinforcers (expressed as % reinforcers/responses) is indicative of reduced response inhibition or increased impulsivity. Neither CIE, nor forced abstinence altered efficiency in SUCROSE-CIE rats compared to matched SUCROSE-NonCIE. In contrast, efficiency of reinforcement increased in ETHANOL-CIE compared to ETHANOL-NonCIE during during protracted abstinence. Interestingly, ETHANOL-CIE rats escalated the ETHANOL (not SUCROSE) intake during the weeks of CIE exposure; this effect attenuated over the weeks of protracted abstinence. Taken together, the reinforcer-type is key to identifying changes due to ethanol dependence. In contrast to other drugs of abuse, evidence for reduced impulsivity was obtained in the ethanol dependence model. Finally, the behavioral adaptations under DRL15 appear to be targeted towards maximization of ethanol intake and thus, may overlap with neurological underpinnings of escalation and relapse. Support or Funding Information AA020098, AA06420 and DA034140 Ethanol dependent rodents exhibit escalation of ethanol intake under a differential reinforcement of low rates schedule. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ethanol Withdrawal-Induced Impaired Recognition Is Reversed by Chronic Exposure to Stress and the Acute Administration of Corticosterone in Mice.

The present study was designed to ascertain the effects of repeated exposure to stress and the acute administration of corticosterone (1, 3, 10 mg/kg, intraperitoneally (i.p.)) on the ethanol withdrawal-induced impairment of novel object recognition in mice. Mice were chronically treated with 3% ethanol for 7 d, with or without exposure to restraint stress for 1 h/d. A significant decrease in cognitive function was observed in the ethanol plus no stress group at 48 h after the discontinuation of ethanol treatment. This impaired recognition was recovered in the ethanol plus stress group. Moreover, we investigated the effects of acute pretreatment with corticosterone, which is a corticosteroid-type hormone produced in the cortex of the adrenal glands, on the impaired recognition after the discontinuation of ethanol treatment in mice. The impaired recognition in the 3% ethanol alone-treated group at 48 h after the discontinuation of ethanol treatment was recovered by treatment with the middle dose (3 mg/kg) of corticosterone, but not with the low or high doses (1, 10 mg/kg). These results suggest that chronic stress during the development of ethanol dependence may reduce the impaired recognition after the discontinuation of ethanol treatment. Moreover, acute pretreatment with the middle dose of corticosterone also recovered the impaired recognition after the discontinuation of ethanol treatment in mice. Adequate regulation of the hypothalamic-pituitary-adrenal (HPA) axis by corticosterone may improve the impaired recognition after the discontinuation of ethanol treatment.

Group 1 mGlu-family proteins promote neuroadaptation to ethanol and withdrawal-associated hippocampal damage

BackgroundGroup 1 mGlu-family proteins (i.e., mGlu) consist of mGlu1 and mGlu5 and their activity may influence voluntary ethanol intake. The present studies sought to examine the influence of these receptors on the development of ethanol dependence using in vitro and in vivo models of chronic, intermittent ethanol (CIE). MethodsRat hippocampal explants were exposed to CIE with or without the addition of mGlu1 antagonist (7-hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt; 0.5, 1, and 3μM) or mGlu5 antagonist (E)-2-methyl-6-styryl-pyridine (SIB-1893; 20, 100, and 200μM) to assess sparing of withdrawal-induced cytotoxicity. In a separate study, adult male rats were administered CIE with or without the addition of oral administration of group 1 mGlu antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP; 3mg/kg). Blood ethanol levels (BELs) were determined at 0930h on Day 2 of Weeks 1, 2, and 3. Withdrawal behavior was monitored during Day 6 of the third consecutive withdrawal. ResultsCIE produced significant hippocampal cytotoxicity. These effects were attenuated by co-exposure to CPCCOEt (3μM) with ethanol in the CA3. By contrast, these effects were blocked by SIB-1893 (20μM) in each primary cell layer. Oral administration of MPEP with ethanol significantly attenuated behavioral effects of subsequent withdrawal and reduced BELs. ConclusionsThese data demonstrate that ethanol activates group 1 mGlu-family proteins to promote withdrawal-associated cytotoxicity in vitro and physical dependence in vivo. These findings suggest that group 1 mGlu-family proteins may be therapeutic targets for treatment of alcohol use disorders.

Ethanol withdrawal is required to produce persisting N-methyl-d-aspartate receptor-dependent hippocampal cytotoxicity during chronic intermittent ethanol exposure

Chronic intermittent ethanol consumption is associated with neurodegeneration and cognitive deficits in preclinical laboratory animals and in the clinical population. While previous work suggests a role for neuroadaptations in the N-methyl-d-aspartate (NMDA) receptor in the development of ethanol dependence and manifestation of withdrawal, the relative roles of ethanol exposure and ethanol withdrawal in producing these effects have not been fully characterized. To examine underlying cytotoxic mechanisms associated with chronic intermittent ethanol (CIE) exposure, organotypic hippocampal slices were exposed to 1–3 cycles of ethanol (50 mM) in cell culture medium for 5 days, followed by 24 h of ethanol withdrawal, in which a portion of slices were exposed to competitive NMDA receptor antagonist (2R)-amino-5-phosphonovaleric acid (APV; 40 μM). Cytotoxicity was assessed using immunohistochemical labeling of neuron-specific nuclear protein (NeuN; Fox-3), a marker of mature neurons, and thionine (2%) staining of Nissl bodies. Multiple cycles of CIE produced neurotoxicity, as reflected in persisting losses of neuron NeuN immunoreactivity and thionine staining in each of the primary cell layers of the hippocampal formation. Hippocampi aged in vitro were significantly more sensitive to the toxic effects of multiple cycles of CIE than were non-aged hippocampi. This effect was not demonstrated in slices exposed to continuous ethanol, in the absence of withdrawal, or to a single exposure/withdrawal regimen. Exposure to APV significantly attenuated the cytotoxicity observed in the primary cell layers of the hippocampus. The present findings suggest that ethanol withdrawal is required to produce NMDA receptor-dependent hippocampal cytotoxicity, particularly in the aging hippocampus in vitro.

Novel Subunit-Specific Tonic GABA Currents and Differential Effects of Ethanol in the Central Amygdala of CRF Receptor-1 Reporter Mice

The central nucleus of the amygdala (CeA) is an important integrative site for the reinforcing effects of drugs of abuse, such as ethanol. Activation of corticotropin-releasing factor type 1 (CRF1) receptors in the CeA plays a critical role in the development of ethanol dependence, but these neurons remain uncharacterized. Using CRF1:GFP reporter mice and a combined electrophysiological/immunohistochemical approach, we found that CRF1 neurons exhibit an α1 GABA(A) receptor subunit-mediated tonic conductance that is driven by action potential-dependent GABA release. In contrast, unlabeled CeA neurons displayed a δ subunit-mediated tonic conductance that is enhanced by ethanol. Ethanol increased the firing discharge of CRF1 neurons and decreased the firing discharge of unlabeled CeA neurons. Retrograde tracing studies indicate that CeA CRF1 neurons project into the bed nucleus of the stria terminalis. Together, these data demonstrate subunit-specific tonic signaling and provide mechanistic insight into the specific effects of ethanol on CeA microcircuitry.

Enhanced GABAergic transmission in the central nucleus of the amygdala of genetically selected Marchigian Sardinian rats: Alcohol and CRF effects

The GABAergic system in the central amygdala (CeA) plays a major role in ethanol dependence and the anxiogenic-like response to ethanol withdrawal. Alcohol dependence is associated with increased corticotropin releasing factor (CRF) influence on CeA GABA release and CRF type 1 receptor (CRF1) antagonists prevent the excessive alcohol consumption associated with dependence. Genetically selected Marchigian Sardinian (msP) rats have an overactive extrahypothalamic CRF1 system, are highly sensitive to stress, and display an innate preference for alcohol. The present study examined differences in CeA GABAergic transmission and the effects of ethanol, CRF and a CRF1 antagonist in msP, Sprague Dawley, and Wistar rats using an electrophysiological approach. We found no significant differences in membrane properties or mean amplitude of evoked GABAA-inhibitory postsynaptic potentials (IPSPs). However, paired-pulse facilitation (PPF) ratios of evoked IPSPs were significantly lower and spontaneous miniature inhibitory postsynaptic current (mIPSC) frequencies were higher in msP rats, suggesting increased CeA GABA release in msP as compared to Sprague Dawley and Wistar rats. The sensitivity of spontaneous GABAergic transmission to ethanol (44 mM), CRF (200 nM) and CRF1 antagonist (R121919, 1 μM) was comparable in msP, Sprague Dawley, and Wistar rats. However, a history of ethanol drinking significantly increased the baseline mIPSC frequency and decreased the effects of a CRF1 antagonist in msP rats, suggesting increased GABA release and decreased CRF1 sensitivity. These results provide electrophysiological evidence that msP rats display distinct CeA GABAergic activity as compared to Sprague Dawley and Wistar rats. The elevated GABAergic transmission observed in naïve msP rats is consistent with the neuroadaptations reported in Sprague Dawley rats after the development of ethanol dependence.

Central Neuropeptide Y Modulates Binge-Like Ethanol Drinking in C57BL/6J Mice via Y1 and Y2 Receptors

Frequent binge drinking has been linked to heart disease, high blood pressure, type 2 diabetes, and the development of ethanol dependence. Thus, identifying pharmaceutical targets to treat binge drinking is of paramount importance. Here we employed a mouse model of binge-like ethanol drinking to study the role of neuropeptide Y (NPY). To this end, the present set of studies utilized pharmacological manipulation of NPY signaling, immunoreactivity (IR) mapping of NPY and NPY receptors, and electrophysiological recordings from slice preparations of the amygdala. The results indicated that central infusion of NPY, a NPY Y1 receptor (Y1R) agonist, and a Y2R antagonist significantly blunted binge-like ethanol drinking in C57BL/6J mice (that achieved blood ethanol levels >80 mg/dl in control conditions). Binge-like ethanol drinking reduced NPY and Y1R IR in the central nucleus of the amygdala (CeA), and 24 h of ethanol abstinence after a history of binge-like drinking promoted increases of Y1R and Y2R IR. Electrophysiological recordings of slice preparations from the CeA showed that binge-like ethanol drinking augmented the ability of NPY to inhibit GABAergic transmission. Thus, binge-like ethanol drinking in C57BL/6J mice promoted alterations of NPY signaling in the CeA, and administration of exogenous NPY compounds protected against binge-like drinking. The current data suggest that Y1R agonists and Y2R antagonists may be useful for curbing and/or preventing binge drinking, protecting vulnerable individuals from progressing to the point of ethanol dependence.

Leuprolide – A luteinizing hormone releasing hormone agonist attenuates ethanol withdrawal syndrome and ethanol-induced locomotor sensitization in mice

Ethanol inhibits the synthesis, content and release of hypothalamic luteinizing hormone releasing hormone (LHRH), and LHRH modulates the activity of several neurotransmitters that experience adaptive changes on chronic exposure to ethanol, and also implicate in ethanol dependence. Hence, it was contemplated that LHRH agonist such as leuprolide may influence the behavioral consequences of withdrawing ethanol in dependent state. In the present study, ethanol dependence was produced in mice by providing ethanol liquid diet for 10 days; and its withdrawal on day 11 led to physical signs of hyperexcitability with its peak at 6th h. Acute treatment with leuprolide (20 ng/mouse, i.c.v.), 10 min prior to peak, significantly attenuated hyperexcitability. Such effect of leuprolide was evident even in castrated mice, and castration significantly increased the hyperexcitability in ethanol withdrawal state. Chronic treatment with leuprolide (10 ng/mouse, twice daily, i.c.v.) till day 10 significantly reduced the signs of hyperexcitability in ethanol withdrawal state. In another set of experiment, ethanol (2.4 g/kg, i.p.) was administered on day 1, 4, 7, 10 and 15, which caused gradual increase in locomotor activity indicating ethanol-induced sensitization. Leuprolide (20 ng/mouse, i.c.v.), 10 min prior to the challenge dose of ethanol (2.4 g/kg, i.p.) on day 15 significantly attenuated the expression of sensitization to hyperlocomotor effect of ethanol. Similarly, administration of leuprolide (20 ng/mouse, i.c.v.), 10 min prior to ethanol on day 1, 4, 7 and 10 not only reduced the gradual increase in locomotor activity but also attenuated the sensitized locomotor response on day 15, indicated attenuation of development of sensitization. Leuprolide per se did not affect physical signs and locomotor activity in control group. In conclusion, the present study demonstrated that leuprolide treatment attenuates expression and development of ethanol dependence and sensitization in mice.

Plasticity and impact of the central renin–angiotensin system during development of ethanol dependence

Pharmacological and genetic interference with the renin-angiotensin system (RAS) seems to alter voluntary ethanol consumption. However, understanding the influence of the RAS on ethanol dependence and its treatment requires modeling the neuroadaptations that occur with prolonged exposure to ethanol. Increased ethanol consumption was induced in rats through repeated cycles of intoxication and withdrawal. Expression of angiotensinogen, angiotensin-converting enzyme, and the angiotensin II receptor, AT1a, was examined by quantitative reverse transcription polymerase chain reaction. Increased ethanol consumption after a history of dependence was associated with increased angiotensinogen expression in medial prefrontal cortex but not in nucleus accumbens or amygdala. Increased angiotensinogen expression also demonstrates that the astroglia is an integral part of the plasticity underlying the development of dependence. The effects of low central RAS activity on increased ethanol consumption were investigated using either spirapril, a blood-brain barrier-penetrating inhibitor of angiotensin-converting enzyme, or transgenic rats (TGR(ASrAOGEN)680) with reduced central angiotensinogen expression. Spirapril reduced ethanol intake in dependent rats compared to controls. After induction of dependence, TGR(ASrAOGEN)680 rats had increased ethanol consumption but to a lesser degree than Wistar rats with the same history of dependence. These data suggest that the central RAS is sensitized in its modulatory control of ethanol consumption in the dependent state, but pharmacological or genetic blockade of the system appears to be insufficient to halt the progression of dependence.

Memantine inhibits ethanol-induced NMDA receptor up-regulation in rat hippocampal neurons

The present study examined the effect of memantine, an uncompetitive NMDA receptor antagonist, on ethanol-induced NMDA receptor up-regulation. Primary glutamatergic rat hippocampal neurons were exposed to ethanol and memantine for 5 days. The ethanol-sensitive NMDA receptor subunits NR1, NR2A and NR2B were quantified by Western immunoblot analysis. Exposure to ethanol (50 mM) caused an increase in the levels of NR1 (137 ± 11% of untreated control, P = 0.009), NR2A (128 ± 14%, P = 0.022) and NR2B (136 ± 19%, P = 0.012). Coincubation with memantine (10 μM) completely blocked the ethanol-induced up-regulation of NR1 (102 ± 4%), NR2A (95 ± 7%) and NR2B (105 ± 13%). No effect of memantine on NR subunit expression was observable, except for NR2A, where a decrease (79 ± 6%, P = 0.034) was noted. Neither ethanol nor memantine alone or in combination were toxic in the concentrations tested. These results may provide a molecular explanation for beneficial effects of memantine on ethanol-induced glutamatergic hyperexcitability reflected in the ethanol withdrawal syndrome and on the development of ethanol dependence.

Chronic intermittent ethanol (CIE) administration in rats decreases levels of neurosteroids in hippocampus, accompanied by altered behavioral responses to neurosteroids and memory function

The administration of ethanol on a chronic intermittent regimen (CIE) involving multiple withdrawal episodes is a model for ethanol dependence. After CIE, rats exhibited reduced seizure threshold, increased anxiety, tolerance to GABAergic sedative-hypnotic drugs, and changes in GABA A receptor function and subunit composition in hippocampus. Previous studies have shown that acute and chronic ethanol may induce changes in the levels of the neurosteroid 3α-hydroxysteroid-5α-pregnan-20-one (3α, 5α-THP) in the brain. Therefore, the current study analyses the correlation between chronic intermittent ethanol effects on the level of 3α, 5α-THP in hippocampus of CIE rats and the behavioral changes in sensitivity to neurosteroids. After CIE, the levels for 3α, 5α-THP were significantly reduced in hippocampus of rats. The mRNA levels for the enzymes 5α-reductase and 3α-HSD in hippocampus were also reduced. In vivo, (in contrast to a tolerance to the hypnotic effect of steroids), CIE rats showed increased sensitivity to the anticonvulsant and to the anxiolytic effect of the steroid alphaxalone. Perhaps, this is a response to lowered levels of endogenous neuroactive steroids. CIE rats also showed impairment of hippocampus-dependent memory function. These results suggest that changes in neurosteroids level and in vivo sensitivity to these compounds are involved in the development of ethanol dependence in the CIE model.

Chronic Ethanol Consumption Differentially Alters GABAA Receptor α1 and α4 Subunit Peptide Expression and GABAA Receptor‐Mediated 36Cl− Uptake in Mesocorticolimbic Regions of Rat Brain

Gamma-aminobutyric acid type A (GABA(A)) receptors in the brain are modulated by chronic ethanol exposure via the regulation of their function and expression throughout the central nervous system. Recent studies show that chronic ethanol exposure alters subsequent ethanol self-administration, effects that are believed to be mediated by subcortical regions of the rat brain including the amygdala (AMG), the nucleus accumbens (NAC), and the ventral tegmental area (VTA). We evaluated GABA(A) receptor subunit expression using subunit specific (alpha1 and alpha4) immunoblotting of small tissue punches from AMG, NAC, and VTA. GABA(A) receptor-mediated 36 Cl- uptake was measured in these brain areas after chronic ethanol consumption for 2 weeks. Regional differences in the effect of chronic ethanol on alpha1 and alpha4 subunit expression were found. In the AMG, alpha1 and alpha4 subunit expressions were significantly decreased by 21.1 +/- 5.5% and 22.0 +/- 7.1%, respectively. In the NAC, there was a decrease of 28.1 +/- 1.3% in alpha4 subunit expression (p < 0.0001), but no change in alpha1 subunit expression was observed. In the VTA, there were no changes in alpha1 and alpha4 subunit expressions. Muscimol-stimulated Cl- uptake was enhanced in the extended AMG, but not the extended NAC of ethanol-dependent rats. The muscimol concentration response curve was left-shifted with a 74% decrease (p < 0.01) in the EC50 and a 42% increase (p < 0.05) in the Emax in the AMG of ethanol-dependent rats. These results suggest that chronic ethanol exposure alters GABA(A) receptor expression in the AMG and NAC. Decreased expression of alpha4 subunits is associated with increases in GABA(A) receptor function in the AMG, but not the NAC. These changes may contribute to alcohol drinking behavior and the development of ethanol dependence.

Ethanol Dependence Has Limited Effects on GABA or Glutamate Transporters in Rat Brain

Neuroadaptations of GABAergic and glutamatergic systems appear to play an important role in both the acute as well as chronic effects of ethanol. Chronic ethanol intake leads to the development of ethanol tolerance and dependence that is associated with a decrease in GABAergic and an increase in glutamatergic function. The present report assessed the involvement of GABA and glutamate transporters in the chronic ethanol-induced adaptations of these two neuronal systems. Male and female rats were made ethanol dependent by 2-week administration of ethanol in a liquid diet. Levels of GABA (GAT-1, GAT-3) and glutamate (GLT-1, EAAC-1) transporters were assayed by immunoblotting. Transporter function was assessed by [3H]GABA and [3H]glutamate uptake assays. Ethanol dependence did not alter levels of GABA or glutamate transporters in cerebral cortex compared with pair-fed control values. There were increases in some, but not all, transporter levels in hippocampus and hypothalamus with the development of ethanol dependence. A decreased rate of uptake was observed for GABA in cerebral cortex. There was no change in maximal GABA uptake or in glutamate uptake (Vmax). These results suggest that alterations in GABA and glutamate transporters have only a limited role in neuroadaptations to chronic ethanol intake in rats. However, the observed alterations were region-specific, supporting the complex responses to chronic ethanol exposure and suggesting that neuroadaptations of GABAergic and glutamatergic systems vary across the brain.

Ethanol Dependence Has Limited Effects on GABA or Glutamate Transporters in Rat Brain

Background: Neuroadaptations of GABAergic and glutamatergic systems appear to play an important role in both the acute as well as chronic effects of ethanol. Chronic ethanol intake leads to the development of ethanol tolerance and dependence that is associated with a decrease in GABAergic and an increase in glutamatergic function. The present report assessed the involvement of GABA and glutamate transporters in the chronic ethanol-induced adaptations of these two neuronal systems. Methods: Male and female rats were made ethanol dependent by 2-week administration of ethanol in a liquid diet. Levels of GABA (GAT-1, GAT-3) and glutamate (GLT-1, EAAC-1) transporters were assayed by immunoblotting. Transporter function was assessed by [3H]GABA and [3H]glutamate uptake assays. Results: Ethanol dependence did not alter levels of GABA or glutamate transporters in cerebral cortex compared with pair-fed control values. There were increases in some, but not all, transporter levels in hippocampus and hypothalamus with the development of ethanol dependence. A decreased rate of uptake was observed for GABA in cerebral cortex. There was no change in maximal GABA uptake or in glutamate uptake (Vmax). Conclusions: These results suggest that alterations in GABA and glutamate transporters have only a limited role in neuroadaptations to chronic ethanol intake in rats. However, the observed alterations were region-specific, supporting the complex responses to chronic ethanol exposure and suggesting that neuroadaptations of GABAergic and glutamatergic systems vary across the brain.

Cytotoxic effect of alcohol-withdrawal on primary cultures of cortical neurones

Physical dependence on alcohol was observed previously at the cellular level in cultured IM-9 human lymphoblast cells. To answer the question whether physical dependence can also develop in neurones and to investigate the neuronal processes involved in the development of alcohol dependence and withdrawal symptoms, cultures of cortical neurones were adapted to alcohol. Morphological characteristics of neurones were not altered during the chronic (3-day) repeated (once per day) ethanol (50–100 mM) treatment, whereas obvious signs of neuronal damage were seen after the following 24 h of alcohol-withdrawal. The extent of the damage, quantitated by measuring the release of lactate dehydrogenase (LDH) into the culture media, was dependent on the concentration of ethanol in the medium during adaptation. LDH-release induced by alcohol-withdrawal was significantly reduced by re-addition of ethanol, as well as by administration of non-competitive (MK-801) or NR2B selective (threo-ifenprodil) N-methyl- d-aspartate (NMDA) receptor antagonists. The sigma ligand haloperidol and the L-type voltage sensitive calcium channel blocker nimodipine were also effective, whereas the effect of the gamma-aminobutyric acid type A (GABA A) receptor agonist muscimol was not significant. Furthermore, chronic ethanol treatment potentiated the NMDA induced neurotoxicity and the ability of acute alcohol to inhibit LDH-release in response to NMDA. According to these results, (i) the phenomenon of alcohol-dependence can be observed at the level of neurones and (ii) NMDA receptors seem to play a central role in the development of ethanol dependence and in neurotoxicity induced by alcohol-withdrawal.

Ovariectomy has minimal effects on neuroadaptations associated with ethanol dependence in female rats

We previously found gender selective alterations in gene expression for GABA A and NMDA receptors associated with the development of ethanol dependence. Males and females have a differing hormonal environment, including steroid hormone derivatives (neuroactive steroids) that exert effects at GABA A and NMDA receptors. Therefore, we explored whether the removal of ovarian steroids would alter gender differences in response to chronic ethanol exposure. We found that ovariectomy reduced ethanol drinking levels by 15%, comparable to earlier observations between intact female and male rats. However, investigation of the effects of chronic ethanol exposure on intact versus ovariectomized female rats uncovered few differences in chronic ethanol-induced alterations in selected GABA A or NMDA receptor subunit peptide levels. In general, findings for both groups of females were similar to previous observations. There was no reduction in GABA A receptor α1 subunit levels in cerebral cortex in either intact or ovariectomized female rats, in contrast to the significant reduction observed in male rats. In addition, both intact and ovariectomized female rats had increased levels of the NMDA NR1 subunit in cerebral cortex and hypothalamus, but not in hippocampus, whereas ethanol dependent male rats displayed significant increases in the NR1 subunit only in hippocampus. Radioligand binding analysis with [ 35S]TBPS found no differences in modulation of the GABA A receptor by neuroactive steroids between ethanol dependent male, intact female or ovariectomized female rats. Seizure susceptibility was not different between intact or ovariectomized female rats during ethanol withdrawal. We did observe differential effects on brain allopregnanolone and plasma corticosterone levels between ethanol dependent intact and ovariectomized female rats, suggesting that ovarian steroids influence HPA axis adaptations to prolonged ethanol exposure. Overall, these data suggest that ovarian steroids do not significantly impact the gender selective alterations of GABA A and NMDA receptors associated with ethanol dependence.

Effects of prolonged exposure to ethanol in vivo on functional parameters and sensitivity to nitrendipine in the isolated rat heart.

This study investigated the possibility that previously reported marked upregulation of binding sites for the dihydropyridine calcium channel antagonist nitrendipine in heart tissue during the development of ethanol dependence in the rat may represent functional L-type voltage-operated calcium channels (L-VOCCs). Isolated hearts obtained from adult Sprague-Dawley rats, which received intoxicating concentrations of ethanol for 6-10 days, by inhalation, were perfused with Krebs-Henseleit solution in the Langendorff mode. Basic measurements of cardiac function were compared with hearts from control rats not exposed to ethanol vapor. In another study, concentration-response curves were constructed for nitrendipine at concentrations in the range of 10(-10)-10(-6) M for hearts obtained from control and ethanol-exposed animals. Changes in measured cardiac parameters such as R-wave amplitude, heart rate, diastolic and systolic pressure, and (+)dP/dt(max) and coronary flow were recorded. All comparisons were made between preparations set to a similar left ventricular end-diastolic pressure. Under these conditions, there were no significant differences in R-wave amplitude, but isolated hearts from ethanol-dependent rats showed significantly greater indices of myocardial contraction than did controls. These included increased systolic and developed ventricular pressure and increased (+)dP/dt(max). Coronary flow also was significantly greater in hearts from ethanol-dependent rats compared with controls. Heart rate was higher in the alcohol-exposed group, but this difference did not achieve significance. When nitrendipine was added to the perfusate at concentrations between 10(-10) and 10(-6) M, hearts from ethanol-dependent animals showed a greater sensitivity to the effects of the drug on heart rate and systolic pressure. Effects on R-wave amplitude and (+)dP/dt(max) were less clear but also suggested a greater sensitivity to nitrendipine in hearts from ethanol-exposed rats. Effects on coronary flow were small and did not differ significantly between preparations from control and ethanol-dependent rats. The results suggest that the increase in Bmax of DHP binding previously observed in hearts from ethanol-dependent animals might represent an increase in L-VOCCs, which alters physiologic function, and pharmacologic responses in the isolated heart. These changes may represent the exposure of an adaptive mechanism designed to overcome the generally depressant effects of ethanol on cardiac function in vivo.

NMDA receptor characterization and subunit expression in rat cultured mesencephalic neurones.

1. NMDA-induced changes in free intracellular Ca2+ concentration ([Ca2+]i) were determined in individual cultured rat mesencephalic neurones by the fura-2 method. mRNA expression encoding NMDA receptor subunits (NR1, NR2A-D) was examined by RT-PCR. 2. NMDA (1-100 microM, plus 10 microM glycine) induced a concentration-dependent increase in [Ca2+]i (EC50 = 5.7 microM). The effect of NMDA was virtually insensitive to tetrodotoxin (0.3 microM) and nitrendipine (1 microM), but dependent on extracellular Ca2+. 5,7-Dichlorokynurenic acid (10 microM), a specific antagonist at the glycine binding site on the NMDA receptor, abolished the NMDA response. 3. Memantine, an open-channel blocker, and ifenprodil, a preferential non-competitive NR1/NR2B receptor antagonist diminished the NMDA effect with an IC50 value of 0.17 and 1 microM, respectively. Ethanol at 50 and 100 mM caused about 25 and 45%-inhibition, respectively. 4. Agarose gel analysis of the PCR products followed by ethidium bromide fluorescence or CSPD chemiluminescence detection revealed an almost exclusive expression of the NR1 splice variants lacking exon (E) 5 and E22. The 3' splice form without both E21 and E22 exceeded that containing E21 by approximately 4 fold. The relative amounts of NR2A, NR2B, NR2C corresponded to approximately 1:2:1. NR2D mRNA was also detectable. 5. In conclusion, mesencephalic neurones bear ethanol-sensitive NMDA receptors which might be involved in the development of ethanol dependence and withdrawal. The high affinity of NMDA to this receptor, its sensitivity to ifenprodil and memantine may suggest that the mesencephalic NMDA receptor comprises the NR1 splice variant lacking E5, NR2B, and NR2C, respectively.