The decreased cellular expression of neuropeptide Y protein in rat brain structures during ethanol withdrawal after chronic ethanol exposure

Background: Neuropeptide Y (NPY) has been implicated in the alcohol-drinking behaviors of rodents. This study investigated the possible involvement of NPY in the neuroadaptational mechanisms to chronic ethanol exposure and its withdrawal.
Methods: Male Sprague-Dawley rats were treated either with Lieber-DeCarli ethanol diet or control diet for 15 days, and ethanol-fed rats were withdrawn for 0 and 24 hr. The protein expression of NPY was determined in cortical, hippocampal, amygdaloid, striatal, and hypothalamic structures by using the gold-immunolabeling histochemical procedure.
Results: It was found that ethanol withdrawal, but not ethanol treatment, produced significant reductions in NPY protein levels in (1) layers IV and V of the frontal and parietal cortex, (2) layer II of the piriform cortex, (3) the central and medial nuclei of the amygdala, and (4) the paraventricular nucleus of the hypothalamus in rat brain. Chronic ethanol exposure and its withdrawal had no effect on the NPY protein levels in layers II, III, and VI of the frontal and parietal cortex or cingulate gyrus, in hippocampal (CA1, CA2, CA3, and dentate gyrus) and striatal (caudate putamen and globus pallidus) structures, or in the ventro-medial hypothalamus and basolateral amygdala. However, chronic ethanol exposure and its withdrawal produced significant reductions in NPY protein levels in the arcuate nucleus of the hypothalamus and in layers IV and V of the cingulate gyrus.
Conclusions: These results suggest that the decreased protein levels of NPY in the central and medial nuclei of the amygdala, as well as in the cortical and hypothalamic structures, during ethanol withdrawal may play an important role in the neuromechanisms of some ethanol withdrawal symptoms.     

Gene Expression in the Neuropeptide Y System During Ethanol Withdrawal Kindling in Rats

Background: Multiple episodes of ethanol intoxication and withdrawal result in progressive, irreversible intensification of the withdrawal reaction, a process termed “ethanol withdrawal kindling.” Previous studies show that a single episode of chronic ethanol intoxication and withdrawal causes prominent changes in neuropeptide Y (NPY) and its receptors that have been implicated in regulating withdrawal hyperexcitability. This study for the first time examined the NPY system during ethanol withdrawal kindling. Methods: Ethanol withdrawal kindling was studied in rats receiving 16 episodes of 2 days of chronic ethanol intoxication by intragastric intubations followed by 5 days withdrawal. The study included 6 groups: 4 multiple withdrawal episode (MW) groups [peak withdrawal plus (MW+)/minus (MW?) seizures, 3‐day (MW3d), and 1‐month (MW1mth) withdrawal], a single withdrawal episode group (SW), and an isocalorically fed control group. Gene expression of NPY and its receptors Y1, Y2, and Y5 was studied in the hippocampal dentate gyrus (DG) and CA3/CA1, as well as piriform cortex (PirCx), and neocortex (NeoCx). Results: MW+/? as well as SW groups showed decreased NPY gene expression in all hippocampal areas compared with controls, but, in the DG and CA3, decreases were significantly smaller in the MW? group compared with the SW group. In the MW+/? and SW groups, Y1, Y2, and Y5 mRNA levels were decreased in most brain areas compared with controls; however, decreases in Y1 and Y5 mRNA were augmented in the MW+/? groups compared with the SW group. The MW+ group differed from the MW? group in the PirCx, where Y2 gene expression was significantly higher. Conclusion: Multiple withdrawal episodes reversibly decreased NPY and NPY receptor mRNA levels at peak withdrawal, with smaller decreases in NPY mRNA levels and augmented decreases in Y1/Y5 mRNA levels compared with a SW episode. Multiple withdrawal‐induced seizures increased the Y2 mRNA levels in PirCx. These complex changes in NPY system gene expression could play a role in the ethanol withdrawal kindling process.     

Nifedipine alleviates alterations in hippocampal kindling after repeated ethanol withdrawal

BACKGROUND: Current clinical treatment of alcohol detoxification commonly includes pharmacotherapy to lessen the potential for seizures, especially in those patients undergoing repeated treatment. Basic research continues to study the alcohol withdrawal-related "kindling" of seizures both to understand the mechanisms involved and to identify alternative treatments. Ethanol withdrawal has been shown to result in the delay of electrical kindling at several brain sites, which suggests a long-lasting disruption of neuronal function. METHODS: This study focused on the participation of the L-type voltage-gated calcium channels in this process by the treatment of animals during withdrawal with nifedipine, an agent that blocks these channels. Animals were randomly assigned to ethanol (ethanol-exposed/ethanol-naive) and drug treatment (nifedipine/vehicle) groups. Subjects receiving ethanol were exposed to five cycles of 3 days' ethanol exposure, with each exposure cycle separated by a 1-day withdrawal period. Drug treatment was administered twice during each withdrawal period. Twenty days after completion of ethanol exposure, animals received daily kindling stimulations to hippocampal area CA3 until the kindling criterion was attained. RESULTS: Ethanol-exposed animals that received vehicle treatment during ethanol withdrawal required more daily stimulations to become fully kindled than did ethanol-naive controls. This delay in seizure development was most pronounced in the progression from focal seizure behaviors to more generalized seizures. Animals that received the same ethanol exposure but that were treated with nifedipine required significantly fewer stimulations than did ethanol-exposed animals that received vehicle. Ethanol-exposed/ nifedipine-treated animals did not differ from ethanol-naive controls that received vehicle or nifedipine. CONCLUSIONS: Alcohol withdrawal-related alterations in seizure-sensitive neural circuitry such as the hippocampus persist long after cessation of ethanol exposure. Furthermore, the L-type voltage-gated calcium channels are involved in this effect in that blockade of these channels during acute withdrawal alleviates alterations in seizure mechanisms on a long-term basis.     

Sex Differences in Rats in the Development of and Recovery From Ethanol Dependence Assessed by Changes in Seizure Susceptibility

BACKGROUND: Previous investigations have found sex differences in rats in response to chronic ethanol exposure. The most dramatic differences were observed with anticonvulsant treatment during ethanol withdrawal, when seizure susceptibility is significantly increased. Sex differences in this response were found for both GABAergic and glutamatergic compounds. This study was aimed at exploring whether sex also influences the timing for the development of and recovery from ethanol dependence. METHODS: Ethanol was administered in a liquid diet, with pair-fed animals receiving dextrose, substituted isocalorically for the ethanol. Ethanol dependence and withdrawal were assessed by measurement of seizure thresholds after abrupt removal of the ethanol diet. Seizure thresholds were determined by slow, tail vein infusion of the gamma-aminobutyric acidA-receptor antagonist bicuculline. RESULTS: Male and female rats displayed differences in timing for both onset and recovery from ethanol dependence, as determined by changes in ethanol withdrawal seizure susceptibility. Female rats were slower to develop dependence and quicker to recover compared with male rats. Furthermore, acute ethanol administration did not alter seizure susceptibility in pair-fed control animals, but it was anticonvulsant in ethanol-withdrawn rats. Ethanol-withdrawn female rats showed a greater response to acute ethanol administration than did male rats. CONCLUSIONS: This set of experiments uncovered additional sex differences in one measure of ethanol dependence and withdrawal. Proposed mechanisms for the development of ethanol dependence involve alterations in subunit assembly of gamma-aminobutyric acidA and NMDA receptors or various posttranslational modifications. In consideration of these findings, whatever mechanisms underlie the development of ethanol dependence, there is a different sequence of events in male compared with female rats. Studies are ongoing to determine associations between behavioral measures of ethanol dependence/withdrawal and selective neuronal adaptations.     

Neuronal Degeneration in Rat Cerebrocortical and Olfactory Regions During Subchronic "Binge" Intoxication with Ethanol: Possible Explanation for Olfactory Deficits in Alcoholics

Severe, repetitive (“binge”) ethanol intoxication in adult rats (intragastric delivery 3 times daily for 4 days in a modification of the Majchrowicz method) precipitates neuronal degeneration in selected cerebral cortical regions involved in memory and olfaction, confirming the results of Switzer and colleagues (Anat. Rec. 202 186a, 1982). Neuronal damage was visualized with the de Olmos cupric silver technique for degenerating neurons and processes (argyrophilia), and was quantitated by total counts and densities of argyrophilic cells/fields. The specificity of the degeneration provides a neuropathological basis for the olfactory memory deficits in chronic alcoholics. In highly intoxicated rats, argyrophilia was most extensive among hippocampal dentate gyrus granule cells, pyramidal neurons in layer 3 of the entorhinal cortex, and olfactory nerve terminals in the olfactory bulb. Degenerating pyramidal neurons were also consistently seen in the insular cortex and olfactory cortical regions, such as the piriform and perirhinal cortices. There were few argyrophilic neurons in the CA regions of the hippocampus and none in the cerebellum—regions generally shown to have cell loss in long-term ethanol feeding models—but degenerating mossy fibers in the CA2 region were observed. Degeneration was maximal before the peak period of abstinence symptoms in this model, because argyrophilic densities were no greater36 hr, compared with 8 hr after the last ethanol dose. High blood ethanol levels were required, because argyrophilia, absent from isocaloric controls, also was only evident in ethanol-intoxicated rats with mean blood ethanol levels for days 2 to 4 above 300 mg/dl; however, it increased substantially between 350 and 550 mg/dl. The resemblance of the argyrophilic distribution to the regional neuropathology that occurs in experimental seizures indicates that the ethanol-induced degeneration may have an excitotoxic basis. Progressive reductions in the seizure threshold (e.g., kindling phenomena that have been documented during binge ethanol intoxication) might be associated with excitotoxic hyperactivity during the repetitive nadirs between high blood and brain ethanol peaks. However, direct toxic actions of ethanol or its metabolites could also be involved. Overall, the model should be useful for studying mechanisms of ethanol-induced selective cortical and olfactory brain damage.     

Repeated Ethanol Withdrawal Delays Development of Focal Seizures in Hippocampal Kindling

Clinical studies report an increase in the prevalence of alcohol withdrawal-related seizures in patients with a history of multiple detoxifications. In order to investigate the alcohol withdrawal-related alterations in neural activity that lead to this increase in seizure propensity, basic researchers have examined both spontaneous and elicited seizures in animals undergoing withdrawal from chronic ethanol. This study was designed to further examine alcohol withdrawal-related seizure activity in a rodent model by assessing the development of electrical kindling after chronic ethanol exposure administered in multiple or single treatment episodes. Laboratory rats were exposed to either five periods of 3 days of ethanol, one 15-day period of continuous ethanol, or a period of control handling with no ethanol exposure. Ten days after a final withdrawal episode, all animals were surgically prepared with recording and stimulating electrodes. Twenty days after final withdrawal from ethanol or an equivalent period of similar handling, daily electrical stimulation of hippocampal area CA3 was initiated. Animals exposed to ethanol required more daily stimulations to become fully kindled than did ethanol-naive controls, with those animals experiencing five withdrawals requiring the most stimulations overall and more stimulations to progress from focal to generalized seizure behaviors. These results indicate that chronic ethanol exposure and withdrawal alter neuronal mechanisms important for hippocampal kindling in a manner that persists long after cessation of ethanol exposure, and they indicate that this effect is increased by exposure to repeated withdrawal episodes.     

The effects of short-term chronic ethanol intoxication and ethanol withdrawal on the molecular composition of the rat hippocampus by FT-IR spectroscopy

Background: The numerous adverse effects of ethanol abuse and ethanol withdrawal on biological systems are well documented. Conversely, the understanding of the molecular mechanisms underlying these pathological effects is still incomplete. This study was undertaken to investigate the effects of short‐term chronic ethanol administration and ethanol withdrawal on the molecular structure and function of hippocampal tissue, a brain region important for mnemonic processes and known to be highly susceptible to ethanol intoxication. Methods: Ethanol was administered to adult Wistar rats by intragastric intubation for 15 days with a stepwise increase in the daily dose from 6 to 12 g/kg body weight, with the highest dose delivered for the last 2 days only. The total daily dose of ethanol was divided into 3 equal portions administered 4 hours apart. Animals were sacrificed by decapitation at 4, 24, and 72 hours after the last ethanol administration to examine potential effects of ethanol intoxication and ethanol withdrawal. Ethanol‐related molecular changes were monitored by Fourier transform infrared (FT‐IR) spectroscopy. Results: Significant changes in the hippocampal content, structure, and function of lipids, proteins, and nucleic acids were recorded under ethanol intoxication. Seventy‐two hours after the cessation of ethanol administration, during the late phase of withdrawal, alterations in the macromolecules’ content and conformational changes in protein and nucleic acid structure ameliorated, while the changes in macromolecular ratios, lipid order, and dynamics aggravated. Conclusions: Our results suggest that 15 days of binge‐like drinking resulting in the high blood alcohol concentration (varying in the dose‐dependent manner between 253 and 606 mg/dl) produced a strong physical dependence manifested mainly by the changes in lipid profiles pointing toward withdrawal‐induced oxidative stress. These results show that ethanol withdrawal may cause equal to or even more severe brain damage than the ethanol itself, which should be considered when designing antialcohol therapies.     

Loss of BETA2/NeuroD leads to malformation of the dentate gyrus and epilepsy

BETA2/NeuroD is a homologue of the Drosophila atonal gene that is widely expressed during development in the mammalian brain and pancreas. Although studies in Xenopus suggest that BETA2/NeuroD is involved in cellular differentiation, its function in the mammalian nervous system is unclear. Here we show that mutant mice homozygous for a deletion at the BETA2/NeuroD locus fail to develop a granule cell layer within the dentate gyrus, one of the principal structures of the hippocampal formation. To understand the basis of this abnormality, we analyzed dentate gyrus development by using immunocytochemical markers in BETA2/NeuroD-deficient mice. The early cell populations in the dentate gyrus, including Cajal-Retzius cells and radial glia, are present and appear normally organized. The migration of dentate precursor cells and newly born granule cells from the neuroepithelium to the dentate gyrus remains intact. However, there is a dramatic defect in the proliferation of precursor cells once they reach the dentate and a significant delay in the differentiation of granule cells. This leads to malformation of the dentate granule cell layer and excess cell death. BETA2/NeuroD null mice also exhibit spontaneous limbic seizures associated with electrophysiological evidence of seizure activity in the hippocampus and cortex. These findings thus establish a critical role of BETA2/NeuroD in the development of a specific class of neurons. Furthermore, failure to express BETA2/NeuroD leads to a stereotyped pattern of pathological excitability of the adult central nervous system.     

The Kindling Model of Alcohol Dependence: Similar Persistent Reduction in Seizure Threshold to Pentylenetetrazol in Animals Receiving Chronic Ethanol or Chronic Pentylenetetrazol

Rats on a chronic intermittent ethanol (CIE) regimen showed a persistent reduction in seizure threshold to the convulsant drug pentylenetetrazol (PTZ). CIE rats were given ethanol by intubation on an alternate day schedule and tested at selected intervals for seizure threshold with PTZ. A significant reduction in seizure threshold, a sign of withdrawal, was observed 20 hr after the first dose. The severity of withdrawal intensified on repetition of the ethanol administration and depression-hyperexcitability cycle, with the seizure threshold reaching a maximum decrease after 12 doses and remaining reduced up to 60 doses. The reduction in seizure threshold persisted for at least 40 days of no alcohol following the 60th dose. The long-lasting decrease in seizure threshold following CIE treatment resembled the “kindling” phenomenon produced by chronic administration of PTZ (25 mg/kg, 3 times/week). The CIE rats developed, in addition, a tolerance to the anticonvulsant action of ethanol, which occurred well after the decrease in PTZ seizure threshold, and a tolerance to the hypothermic effect of ethanol, which developed rapidly. PTZ kindled rats that had never been exposed to ethanol also exhibited tolerance to the hypothermic effect of ethanol. We propose that kindling contributes to the mechanism of the development of dependence on central nervous system depressants like benzodiazepines, barbiturates, and alcohol, drugs that act on the γ-aminobutyric acid-A receptor chloride ion channel complex. Repeated episodes of depression and withdrawal hyperexcitability are postulated to produce kindling during the repeated withdrawal episodes. In chronic ethanol abuse, the withdrawal stress/hyperexcitability would be repeated many times and lead to the development of dependence as measured by the persistent increase in withdrawal severity, manifested as heightened sensitivity to seizures (kindling). The long-lasting decreases in seizure threshold following both chronic pentylenetetrazol and chronic intermittent ethanol suggest the possibility that some common mechanisms, such as the involvement of γ-aminobutyric acid-A receptors, may underlie the two kindling phenomena.     

The 5-HT3 Antagonist MDL-72222 Exacerbates Ethanol Withdrawal Seizures in Mice

Ethanol-dependent mice were treated with the 5-HT₃ antagonist MDL 72222 after withdrawal from ethanol. Treatment with unit doses (0, 5.6, 10, and 17.0 mg/kg) of MDL 72222 at 0, 4, and 7 hr after withdrawal dose-dependently exacerbated the severity of ethanol withdrawal seizures. Treatment with a single dose (17 mg/kg) of MDL 72222 at 5 hr after withdrawal also exacerbated the severity of ethanol withdrawal seizures. Ethanol naive mice treated with MDL 72222 (56 mg/kg) did not display any seizures. Treatment with another 5-HT₃ antagonist, ICS 205-930 (23 and 46 mg/kg), or the 5- HT₂ receptor antagonist ketanserin, did not affect ethanol withdrawal seizures. The findings suggest MDL 72222 selectively enhances sensitivity to withdrawal seizures following chronic ethanol exposure.     

Induction of cyclooxygenase-2 in brain during acute and chronic ethanol treatment and ethanol withdrawal

Prostaglandins play an important role in the regulation of nervous system function including thermoregulation, autonomic nervous system function, hypothalamic regulation of pituitary function, and neuronal excitation. Prostaglandin synthesis is catalyzed by cyclooxygenase (COX; prostaglandin synthase) which occurs as two isozymes, COX-1 and COX-2. COX-1 and COX-2 are constitutively expressed in brain whereas COX-2 type is also inducible in brain by excitatory neurotransmission. Ethanol intoxication and the hyperexcitability of ethanol withdrawal may be influenced by inducible proteins, thus we investigated COX-2 in the rat brain during acute and chronic ethanol treatment, ethanol withdrawal, and after peripheral administration of excitatory amino acids. Kainic acid or NMDA treatment increased COX-2 immunoreactivity in the cortex, hippocampus, and amygdala. An acute dose of ethanol (5 g/kg, intragastric-i.g.) increased COX-2, particularly in the CA4 region of the hippocampus and agranular insular cortex. Chronic ethanol treatment (4 days-intragastric) robustly induced COX-2 in limbic cortex, isocortex, and amygdala. Particularly dense immunocytochemical staining was found in perirhinal and piriform cortices, dentate gyrus, and tenia tecta. During ethanol withdrawal, COX-2 expression increased further in some regions, peaking in most areas 16 hr after the last dose of ethanol. These results indicate that COX-2 immunoreactivity is: 1) increased in the brain during acute ethanol exposure that increases further during chronic treatment; 2) sensitive to excitatory amino acid receptor stimulation; and 3) dramatically increased during ethanol withdrawal. These studies suggest that COX-2 induction may be involved in the acute and chronic effects of ethanol.     

Tyrosine Hydroxylase Activity in the Brain and Adrenal Gland of Rats Following Chronic Administration of Ethanol

The effects of chronic administration and ethanol withdrawal on the activity of tyrosine hydroxylase were examined in the adrenal gland and six brain regions, including the frontal cortex, hippocampus, locus coeruleus, striatum, substantia nigra, and hypothalamus. Ethanol was administered to rats by oral intubation every 8 hr for 48 hr in amounts based upon their state of intoxication. One hr after the last intubation, tyrosine hydroxylase activity was not significantly different from control values in either the adrenal gland or brain. Forty hr following the last ethanol intubation, tyrosine hydroxylase activity was significantly increased above control values in both the adrenal gland and locus coeruleus. Tyrosine hydroxylase activity in the remaining five brain areas was unaffected by ethanol treatment. Immunotitration studies indicate that the increases in tyrosine hydroxylase activity found in the adrenal gland and locus coeruleus 40 hr after ethanol administration were due to an increase in enzyme protein. These data indicate that high blood ethanol concentrations maintained over a period of time (48 hr) do not affect adrenal gland or brain tyrosine hydroxylase activity. However, withdrawal from ethanol following 48 hr of treatment does produce an increase in tyrosine hydroxylase activity in the adrenal gland and locus coeruleus, similar to that seen following other stresses.     

The Neurosteroid, 3α-Hydroxy-5α-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, 3α-hydroxy-5α-pregnan-20-one (3α-5α-THP), on alterations in seizure sensitivity associated with ethanol withdrawal. 3α-5α-THP is a potent anxiolytic and anticonvulsant agent that acts via selective interactions with GABA_A receptors. Extensive evidence suggests that some aspects of ethanol dependence and withdrawal are mediated by alterations in GABA_A receptor function. Withdrawal from chronic ethanol exposure elicited dramatic increases in seizure susceptibility in male and female rats. Administration of 3α-5α-THP just before seizure threshold determinations blocked the increased seizure susceptibility induced by ethanol withdrawal. Ethanol-withdrawn animals were protected by 3α-5α-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 3α-5α-THP pretreatment. These findings suggest that there are gender differences associated both with ethanol withdrawal as well as the protection by 3α-5α-THP in ethanol-dependent rats.     

Chronic ethanol-induced subtype- and subregion-specific decrease in the mRNA expression of metabotropic glutamate receptors in rat hippocampus

BACKGROUND: Chronic ethanol consumption is known to induce adaptive changes in the hippocampal glutamatergic transmission and alter NMDA receptor binding and subunit expression. Metabotropic glutamate (mGlu) receptors have been shown to function as modulators of neuronal excitability and can fine tune glutamatergic transmission. This study was aimed to determine whether chronic ethanol treatment could change the messenger RNA (mRNA) expression of mGlu receptors in the hippocampus. METHODS: Male Sprague Dawley rats were fed a Lieber-DeCarli liquid diet with 5% (w/v) ethanol or isocaloric amount of maltose for 2 months. Quantitative in situ hybridization was carried out using coronal brain sections through the hippocampus. RESULTS: The results revealed decreases in mRNA expression of several mGlu receptors in different subregions of the hippocampus. In the dentate gyrus, mGlu3 and mGlu5 receptor mRNA levels were significantly lower in the ethanol-treated rats than in the control rats. In the CA3 region, the mRNA expression of mGlu1, mGlu5, and mGlu7 receptors showed substantial decreases after ethanol exposure. The mGlu7 receptor mRNA levels were also declined in the CA1 region and the polymorph layer of the dentate gyrus. No changes were found in mRNA expression of mGlu2, mGlu4, and mGlu8 receptors. CONCLUSIONS: Considering the involvement of hippocampal mGlu receptors in learning and memory processes as well as in neurotoxicity and seizure production, the reduced expression of these receptors might contribute to ethanol withdrawal-induced seizures and also may play a role in cognitive deficits and brain damage caused by long-term ethanol consumption.     

Ethanol-Induced Suppression of Hippocampal Long-Term Potentiation Is Blocked by Lesions of the Septohippocampal Nucleus

Systemic ethanol increases synaptic inhibition and suppresses long term potentiation (LTP) in the dentate gyrus of the rat hippocampus. Local application of ethanol directly into the dentate gyrus of anesthetized rats increased the perforant path to dentate feed-forward inhibition, but had no effect on LTP. Local application of ethanol to the medial septum, a subcortical structure with major input to the dentate, increased recurrent inhibition. Selective disruption of septodentate input produced by lesions of the septohippocampal nucleus blocked the effects of systemic ethanol on LTP. These findings are the first to demonstrate that septodentate input is necessary for ethanol to increase recurrent inhibition and suppress LTP in the dentate gyrus and suggest an important role for extrahippocampal modulation of both short- and long-term plasticity in the hippocampus.     

Ethanol exposure and withdrawal sensitizes the rat hippocampal CA1 pyramidal cell region to beta-amyloid (25-35)-induced cytotoxicity: NMDA receptor involvement

Abstract : Background: Millions of Americans suffer from Alzheimer's Disease (AD), which is characterized by significant neurological impairment and an accumulation in brain tissue of senile plaques consisting of beta amyloid (Aβ) peptide. The hippocampus, a region primarily responsible for learning and memory, appears to be particularly susceptible to AD‐related injury and chronic alcohol abuse. Although certain risk factors for AD are known, it is unclear if alcohol abuse or dependence may contribute to neuropathology in AD. Recent research suggests that low‐to‐moderate consumption of alcohol may protect against development of AD, while alcohol dependence may increase risk of developing AD. Therefore, the current studies aimed to investigate the effects of exposure to 50 or 100 mM ethanol (EtOH) and withdrawal on hippocampal injury induced by Aβ peptide treatment. Methods: The present studies exposed organotypic hippocampal slice cultures to 50 or 100 mM ethanol (EtOH) for 10 days, after which the slices underwent ethanol withdrawal (EWD) in the presence of varying concentrations of Aβ 25‐35 (0.1, 1, 10 μM), or 35‐25 (200 μM), a negative control reverse sequence peptide. Cellular injury, as evidenced by uptake of propidium iodide (PI), was assessed for each subregion of the hippocampal complex (CA1, CA3, and dentate gyrus). Results: Cellular injury in the CA1 pyramidal cell layer was significantly increased during withdrawal from exposure to 100 mM, but not 50 mM, EtOH. Exposure to Aβ in ethanol‐naïve cultures did not produce significant cytotoxicity. However, exposure to Aβ during EWD from 100 mM produced marked increases in CA1 pyramidal cell region cytotoxicity, effects reversed by cotreatment with a nontoxic concentration of the NMDA receptor channel blocker MK‐801 (20 μM). Conclusions: These data suggest that withdrawal from exposure to a high concentration of EtOH produces marked cellular injury in the hippocampus, particularly the CA1 subregion. Further, this EtOH exposure and withdrawal regimen sensitizes the hippocampus to the toxic effects of Aβ treatment in a manner reflecting over activity of NMDA receptor function.     

Development of ethanol withdrawal-related sensitization and relapse drinking in mice selected for high- or low-ethanol preference

Background: Previous studies have shown that high alcohol consumption is associated with low withdrawal susceptibility, while at the same time, other studies have shown that exposure to ethanol vapor increases alcohol drinking in rats and mice. In the present studies, we sought to shed light on this seeming contradiction using mice selectively bred for High‐ (HAP) and Low‐ (LAP) Alcohol Preference, first, assessing these lines for differences in signs of ethanol withdrawal and second, for differences in the efficacy of intermittent alcohol vapor exposure on elevating subsequent ethanol intake. Methods: Experiment 1 examined whether these lines of mice differed in ethanol withdrawal‐induced CNS hyperexcitability and the development of sensitization to this effect following intermittent ethanol vapor exposure. Adult HAP and LAP lines (replicates 1 and 2), and the C3H/HeNcr inbred strain (included as a control genotype for comparison purposes) received intermittent exposure to ethanol vapor and were evaluated for ethanol withdrawal‐induced seizures assessed by scoring handling‐induced convulsions (HIC). Experiment 2 examined the influence of chronic intermittent ethanol exposure on voluntary ethanol drinking. Adult male and female HAP‐2 and LAP‐2 mice, along with male C57BL/6J (included as comparative controls) were trained to drink 10% ethanol using a limited access (2 h/d) 2‐bottle choice paradigm. After stable baseline daily intake was established, mice received chronic intermittent ethanol vapor exposure in inhalation chambers. Ethanol intake sessions resumed 72 hours after final ethanol (or air) exposure for 5 consecutive days. Results: Following chronic ethanol treatment, LAP mice exhibited overall greater withdrawal seizure activity compared with HAP mice. In Experiment 2, chronic ethanol exposure/withdrawal resulted in a significant increase in ethanol intake in male C57BL/6J, and modestly elevated intake in HAP‐2 male mice. Ethanol intake for male control mice did not change from baseline levels of intake. In contrast, HAP‐2 female and LAP‐2 mice of both sexes did not show changes in ethanol intake as a consequence of intermittent ethanol exposure. Conclusions: Overall, these results indicate that the magnitude of ethanol withdrawal‐related seizures is inversely related to inherited ethanol intake preference. Additionally, intermittent ethanol vapor exposure appears more likely to affect high‐drinking mice (C57BL/6J and HAP‐2) than low drinkers, although these animals are less affected by ethanol withdrawal.     

Seizures during Ethanol Withdrawal Are Blocked by Focal Microinjection of Excitant Amino Acid Antagonists into the Inferior Colliculus and Pontine Reticular Formation

Physical dependence on ethanol can result in seizure susceptibility during ethanol withdrawal. In rats, generalized tonic-clonic seizures are precipitated by auditory stimulation during the ethanol withdrawal syndrome. Excitant amino acids (EAAs) are implicated as neurotransmitters in the inferior colliculus and the brain stem reticular formation, which play important roles in the neuronal network for genetic models of audiogenic seizures (AGSs). Ethanol blocks the actions of EAAs in various brain regions, including the inferior colliculus. In this study, dependence was produced by intragastric administration of ethanol for 4 days. During ethanol withdrawal, AGSs were blocked by systemic administration of competitive or noncompetitive NMDA antagonists 3-((±)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) or dizocilpine (MK-801). Focal microinjections of NMDA or non-NMDA antagonists into the inferior colliculus or the pontine reticular formation also inhibited AGSs. MK-801 was the most potent anticonvulsant systemically. When injected into the inferior colliculus, CPP had a more potent anticonvulsant effect than either MK-801 or the non-NMDA antagonist 6-cyano-7-nitroquinoxa-line-2,3-dione. The inferior colliculus was more sensitive than the pontine reticular formation to the anticonvulsant effects of both competitive NMDA and non-NMDA antagonists. The results of the present support the idea that continued ethanol administration may lead to development of supersensitivity to the action of EAAs in inferior colliculus and pontine reticular formation neurons. This may be a critical mechanism subserving AGS susceptibility during ethanol withdrawal.     

Somatostatin: An endogenous antiepileptic

The neuropeptide somatostatin (SST) is highly expressed in brain regions associated with seizures. In hippocampus, SST expression and release is regulated by seizures, and SST-containing neurons within the hilus of the dentate gyrus are sensitive to seizure-induced death. In vivo and in vitro studies suggest that the loss of SST function in the dentate could contribute to epileptogenesis and seizure susceptibility. SST also has inhibitory actions in the CA1 and CA3 hippocampus indicating this peptide is an important homeostatic regulator throughout the hippocampus. In vivo studies show SST has robust antiepileptic properties with the major site of action being hippocampus. In rodents, somatostatin receptor subtype 2 (SST₂) and SST₄ appear to mediate the majority of the antiepileptic actions of SST, with SST₂ predominate in rat and SST₄ in mouse. Thus SST receptors may be appropriate targets for new antiepileptic drugs (AEDs), although validation in human tissue is lacking.     

Selective Changes in GABAergic Transmission in Substantia Nigra and Superior Colliculus Caused by Ethanol and Ethanol Withdrawal

One of ethanol's actions after acute exposure is anticonvulsant activity whereas withdrawal from chronic ethanol exposure increases convulsant activity. An increase in neuronal transmission in the GABAergic pathways from striatum to the substantia nigra (SN) and a decrease in GABAergic transmission from SN to superior colliculus (SC) both appear to play a major role in inhibiting seizure propagation. If this is the case, then the changes in seizure sensitivity caused by ethanol may be expected to affect GABAergic transmission in opposite ways in SN and SC. We measured the effects of in vitro ethanol on pre- and postsynaptic indices of GABA transmission using SN and SC tissue from both ethanol-naive rats and rats given ethanol in their drinking water for 24 days and then withdrawn for 24 hr, a treatment that decreases seizure latency. While ethanol inhibited 3H-GABA release from slices of SC at low concentrations (20-100 nM), much higher concentrations were required to inhibit release from SN (100-500 mM). In fact, release from SN was increased by low concentrations of ethanol. Ethanol in vitro (20-1000 mM) also inhibited specific binding of 35S-TBPS to the GABAA receptor but this effect was similar in both potency and efficacy in SC and SN. Next, the in vitro effects of ethanol were measured in rats that had consumed an average of 9.8 g ethanol/kg body weight/day and were then withdrawn for 24 hr. Ethanol inhibition of 3H-GABA release from SC was significantly less in ethanol-treated rats compared to controls whereas the inhibitory effect of ethanol was increased in SN from ethanol-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)