Administration of low doses of MK-801 during ethanol withdrawal in the developing rat pup attenuates alcohol's teratogenic effects

BACKGROUND: Alcohol exposure during development can produce severe and long-lasting central nervous system damage and consequent behavioral alterations. Recent evidence suggests that NMDA receptor-mediated excitotoxicity during periods of withdrawal may contribute to this damage. We have demonstrated that blocking the NMDA receptor with MK-801 during alcohol withdrawal can attenuate ethanol's adverse effects on behavioral development in the rat. This study examined the dose dependency of MK-801's ability to mitigate ethanol's teratogenic effects. METHODS: Neonatal rat pups were exposed to 6.0 g/kg of ethanol in a binge-like manner on postnatal day (PD) 6, a period of brain development equivalent to a portion of the human third trimester. Alcohol administration was accomplished with an artificial rearing procedure. Twenty-one hours after ethanol treatment, pups were injected intraperitoneally with one of four doses of MK-801 (0.05, 0.1, 0.5, or 1.0 mg/kg) or saline vehicle. An artificially reared control and a normally reared control group were included. On PD 18-19, activity level was monitored, and on PD 40-42, serial spatial discrimination reversal learning was assessed. RESULTS: Alcohol exposure on PD 6 produced significant increases in activity level and deficits in reversal learning. These alcohol-induced behavioral alterations were significantly attenuated in subjects treated with one of the three lower doses (0.05-0.5 mg/kg) of MK-801 during withdrawal. The performance of ethanol-exposed subjects treated with the high dose of MK-801 (1.0 mg/kg) did not differ from that of the Ethanol Only group. CONCLUSIONS: These data suggest that alterations in NMDA receptor activation during alcohol withdrawal contribute to the neuropathology and consequent behavioral alterations associated with developmental alcohol exposure. These data have important implications for pregnant women and newborns undergoing ethanol withdrawal.     

MK-801 can exacerbate or attenuate behavioral alterations associated with neonatal alcohol exposure in the rat, depending on the timing of administration

BACKGROUND: We have reported that administration of MK-801, an NMDA receptor antagonist, during ethanol withdrawal in the developing rat attenuates ethanol's adverse effects on behavioral development. In the present study, we altered the timing of MK-801 delivery in relation to the last alcohol dose to determine if its protective effects were specific to the ethanol withdrawal phase. METHODS: Five groups of rats were artificially reared and exposed to alcohol in a binge-like manner on postnatal day (PD) 6, producing peak blood alcohol levels of 335 mg/dl that cleared to 0 mg/dl by 33 hours. Four groups received MK-801 at various times after alcohol treatment (0, 9, 21, or 33 hr post-ethanol). The fifth alcohol-treated group received saline. Two artificially reared control groups were included: one was injected with saline and the other injected with 0.5 mg/kg MK-801. Finally, a normally reared suckle control group was also included. Activity level and performance on a spatial discrimination reversal-learning task were evaluated at PD 18 and PD 40, respectively. RESULTS: Administration of MK-801 at the same time as ethanol treatment (0 hr) produced a high rate of mortality. Ethanol exposure on PD6 increased activity level relative to controls. Administration of MK-801 at 0 hr exacerbated this ethanol-induced overactivity, whereas administration of MK-801 at 21 and 33 hr reduced the severity of ethanol-related overactivity. Similarly, ethanol exposure on PD 6 significantly increased the number of errors committed on a spatial discrimination reversal-learning task. MK-801 injections 9 hrs after ethanol exacerbated this effect, whereas MK-801 treatment 33 hrs after ethanol attenuated this effect. Thus, MK-801 administration at the time of ethanol treatment was highly toxic, whereas during the withdrawal period it was protective. CONCLUSION: These data are consistent with the hypothesis that ethanol exposure in the neonatal rat inhibits the NMDA receptor, producing a subsequent rebound in NMDA receptor activation and possible excitotoxicity during withdrawal. Both the acute inhibitory effects of ethanol and the excitatory effects of withdrawal may contribute to fetal alcohol effects.     

Administration of memantine during ethanol withdrawal in neonatal rats: effects on long-term ethanol-induced motor incoordination and cerebellar Purkinje cell loss

Background: Alcohol consumption during pregnancy can damage the developing fetus, illustrated by central nervous system dysfunction and deficits in motor and cognitive abilities. Binge drinking has been associated with an increased risk of fetal alcohol spectrum disorders, likely due to increased episodes of ethanol withdrawal. We hypothesized that overactivity of the N‐methyl‐D‐aspartate (NMDA) receptor during ethanol withdrawal leads to excitotoxic cell death in the developing brain. Consistent with this, administration of NMDA receptor antagonists (e.g., MK‐801) during withdrawal can attenuate ethanol’s teratogenic effects. The aim of this study was to determine whether administration of memantine, an NMDA receptor antagonist, during ethanol withdrawal could effectively attenuate ethanol‐related deficits, without the adverse side effects associated with other NMDA receptor antagonists. Methods: Sprague–Dawley pups were exposed to 6.0 g/kg ethanol or isocaloric maltose solution via intubation on postnatal day 6, a period of brain development equivalent to a portion of the 3rd trimester. Twenty‐four and 36 hours after ethanol, subjects were injected with 0, 10, or 15 mg/kg memantine, totaling doses of 0, 20, or 30 mg/kg. Motor coordination was tested on a parallel bar task and the total number of cerebellar Purkinje cells was estimated using unbiased stereology. Results: Alcohol exposure induced significant parallel bar motor incoordination and reduced Purkinje cell number. Memantine administration significantly attenuated both ethanol‐associated motor deficits and cerebellar cell loss in a dose‐dependent manner. Conclusions: Memantine was neuroprotective when administered during ethanol withdrawal. These data provide further support that ethanol withdrawal contributes to fetal alcohol spectrum disorders.     

Brain Neuronal Degeneration Caused by Episodic Alcohol Intoxication in Rats: Effects of Nimodipine, 6,7-Dinitro-quinoxaline-2,3-dione, and MK-801

Rats repeatedly intoxicated with alcohol (ethanol, three times daily) over a 4-day period display neuronal degeneration in the dentate gyrus; entorhinal, piriform, insular, orbital, and perirhinal cortices; and in the olfactory nerve fibers and terminals in the olfactory bulb. Postulating a role for excitotoxicity, we have attempted to prevent the degeneration using antagonists that are neuroprotective in this type of brain damage. In an initial study, continuous subcutaneous infusion of a high dose of the glutamate/NMDA receptor antagonist MK-801 (2 mg/kg/day) by itself caused extensive neuronal degeneration in several brain regions and severe behavioral intoxication that precluded survival if combined with high blood alcohol levels (～300 mg/dl). Moreover, the lower, nonneurotoxic blood alcohol levels (～150 mg/dl) that were compatible with survival worsened the MK-801-induced brain damage. In a subsequent experiment, daily intraperitoneal injections of a lower dose of MK-801 (1 mg/kg/day) resulted in no MK-801 toxicity and, when combined with neurotoxic levels of alcohol, no reduction in alcohol-induced neurotoxicity. Nimodipine, a voltage-gated Ca²⁺ channel blocker, reduced the neuronal damage in the dentate gyrus, but greatly increased it in the piriform cortex when administered intragastrically at 600 mg/kg/day; it provided no protection from alcohol-dependent degeneration when given intragastrically at 100 mg/kg/day. Continuous intracere-broventricular delivery of 0.24 to 0.29 mg/day of 6,7-dinitro-quinoxa-line-2,3-dione, a glutamate/α-amino-3-hydroxy-5-methyl-4-isoxazole receptor antagonist, failed to diminish alcohol-dependent neuronal damage in any brain region. We conclude that brain damage from episodic “binge” alcohol intoxication is not primarily mediated by excitotoxic mechanisms, implying that other, nonexcrtotoxic pathophysiological mechanisms, are involved. Furthermore, MK-801, far from protecting from the alcohol-induced damage, at high doses causes widespread neuropathology that is significantly potentiated by alcohol.     

The effects of NMDA and GABAA pharmacological manipulations on ethanol sensitivity in immature and mature animals

BACKGROUND: Sensitivity to the hypnotic effects of ethanol dramatically increases with age (Silveri and Spears, 1998). Little is known regarding the neural mechanisms that might underlie this relative resistance of young animals to ethanol. The present study used a psychopharmacological approach to examine the influence of NMDA (N-methyl-D-aspartate) and GABA (gamma-Aminobutyric acid) receptor systems in modulating age differences in ethanol responsiveness between young [postnatal day (P26)] and mature (P70) female and male Sprague-Dawley rats. METHOD: Dose response curves were established for the effects of intraperitoneal (ip) administration of the NMDA antagonist (+)MK-801 (0.75, 1.0, or 1.25 mg/kg), the GABAA agonist muscimol (0.75, 1.25, or 1.75 mg/kg), or saline on loss and regain of the righting reflex and trunk blood alcohol levels following 3.5 g/kg of ethanol. RESULTS: (+)MK-801 increased time to regain the righting reflex at both ages, maintaining the age-related increase in ethanol sensitivity typically observed, although a gender effect also emerged at P70, with females being more sensitive than males to (+)MK-801-induced increases in duration of loss of the righting reflex. In contrast to the across-age similarity in responsiveness to (+)MK-801, P26 animals were considerably more sensitive to muscimol's enhancing effect on ethanol sedation than mature animals. CONCLUSION: Although modulation of either the NMDA or GABA system enhances the sedative effects of ethanol at both ages, stimulation of the GABAA receptor is a more effective means of prolonging ethanol sedation in immature than mature animals.     

Prenatal ethanol exposure modifies [3H]MK-801 binding to NMDA receptors: spermidine and ifenprodil

BACKGROUND: It has been suggested that abnormalities seen in fetal alcohol syndrome are linked with NMDA receptor malfunction. Our laboratory has previously shown that prenatal ethanol treatment decreases [3H]MK-801 binding density at postnatal day 21, when NMDA receptor subunit protein levels were unaltered. Thus, the focus of the present study was to examine whether prenatal ethanol modifies native NMDA receptor levels. METHODS: Cerebral cortices were taken from offspring born to three treatment groups of pregnant Sprague Dawley(R) rats: an ethanol group given an ethanol liquid diet during the gestational period, a pair-fed control group that received a liquid diet without ethanol, and an ad libitum group fed rat chow and tap water. Western blot studies were carried out at postnatal days 1, 7, 14, and 21 to examine total protein expression of NR1 and NR1b splice variants. NR2 subunit levels were examined by [3H]MK-801 binding studies using spermidine, an endogenous polyamine, and ifenprodil, a selective NR2B antagonist. RESULTS: [3H]MK-801 binding density was significantly reduced in prenatal ethanol-treated groups compared with ad libitum and pair-fed control groups. Spermidine increased [3H]MK-801 binding, although potentiation by spermidine was not significantly different among all three experimental groups. Furthermore, no significant differences in total protein expression of NR1 or NR1b splice variants were observed in cortical membrane homogenates at postnatal days 1 through 21. [3H]MK-801 binding in the presence of ifenprodil showed that prenatal ethanol treatment significantly decreased low-affinity ifenprodil binding. High-affinity ifenprodil binding was reduced in both pair-fed and ethanol-treated groups. CONCLUSIONS: These results suggest that prenatal ethanol treatment reduces [3H]MK-801 binding and that this reduction may be due to a decrease in NR2A subunits.     

Effects of Early Postnatal Alcohol Exposure on Learning in the Developing Rat: Replication With Intubation Method of Delivery

Early postnatal exposure to alcohol during early development produces deficits in learned persistence, as reflected in the partial reinforcement extinction effect (PREE) in weanling rats, and deficits memory-based learning, as shown by patterned single alternation (PSA) discrimination learning in preweanling rats. We report a partial replication of these effects using the intubation method instead of artificial rearing. Rat pups were intubated once per day with 4.5 g/kg/day alcohol in a milk-based diet or control diet on postnatal days (PNDs) 4 to 9, and then assessed for the PREE on PNDs 20 and 21 or PSA learning on PNDs 17 and 18. Compared with previous artificial rearing reports, the intubation method produced healthier and heavier pups, and yielded a consistently lower and less variable blood alcohol levels. Even with the lower alcohol levels, intubation with alcohol eliminated the PREE. Intubation with alcohol had a weaker but still detrimental effect on PSA learning. These results suggest that alcohol exposure during development can produce behavioral deficits in the absence of the more severe effects on brain and body growth typically associated with fetal alcohol syndrome.     

Behavioral Sensitivity and Ethanol Potentiation of the N-Methyl-d-Aspartate Receptor Antagonist MK-801 in a Rat Line Selected for High Ethanol Sensitivity

The role of the N -methyl-d-aspartate (NMDA) receptors in differential ethanol sensitivity of the alcohol-insensitive [alcohol-tolerant (AT)] and alcohol-sensitive [alcohol-nontolerant (ANT)] rat lines selected for low and high sensitivity to ethanol-induced (2 g/kg) motor impairment was studied in behavioral and neurochemical experiments. A noncompetitive antagonist of the NMDA receptor, dirocilpine mal-eate (MK-801; 0.2 mg/kg), impaired motor function in ANT rats, but not in AT rats, in a tilting plane test. The impairment was further potentiated by a dose (0.75 glkg) of ethanol, which alone was inactive. This effect was apparently not associated with the locomotor stimulation produced by MK-801 (0.1 and 0.2 mg/kg), because stimulation did not differ between the rat lines. Locomotor stimulation was potentiated by the low ethanol dose in both rat lines. Ethanol treatment decreased the cerebellar and hippocampal cGMP concentrations both with and without MK-801 pretreatment in both rat lines. In situ hybridization using oligonucleotide probes specific for NMDA receptor subunit mRNAs NR1 and NR2A, B, C, and D revealed no clear differences in brain regional expression between ANT and AT rats. These results indicate that the alcohol-sensitive ANT rats are very sensitive to a low dose of ethanol in the presence of NMDA receptor antagonism, consistent with the hypothesis that this receptor system is involved in acute ethanol intoxication.     

Effects of Prenatal and Early Postnatal Ethanol Exposure on [3H]MK-801 Binding in Rat Cortex and Hippocampus

The effects of prenatal and/or early postnatal exposure to ethanol at high concentrations on N-methyl-D-aspartate (NMDA) receptor number and functioning in the weanling rat were examined. The bingelike exposure protocol was used in an animal model of acute ethanol effects at two critical periods of development. [³H]MK-801 binding parameters for the internal channel phencyclidine site were assessed in the presence of 10 μM glutamate and 10 μ M glycine activation. Four treatment groups were included: (1) animals exposed to ethanol both prenatal and postnatal; (2) animals exposed only prenatal; (3) animals exposed early postnatal only; and (4) control animals with no exposure to ethanol. The results of the [³H]MK-801 binding experiments showed that both prenatal and postnatal exposure to ethanol resulted in a significant decrease in the density of NMDA receptors. In addition, data indicated an apparent increase in the percentage of high-affinity state (open channel state) relative to low-affinity state (close channel state) receptors in the ethanoltreated groups. These results show that both prenatal and postnatal ethanol exposure decrease NMDA receptor density in the cortex and hippocampus. The findings are consistent with previous observations by our laboratory and others that NMDA-mediated calcium influx is reduced in these regions, as well as in whole brain by prenatal ethanol exposure. It is suggested that after ethanol exposure, the remaining functional NMDA receptors might have altered sensitiviity to coagonist activation with an increased probability of channel opening.     

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.     

The effects of NMDA and GABAA pharmacological manipulations on acute and rapid tolerance to ethanol during ontogeny

BACKGROUND: Sensitivity to several ethanol effects increases during ontogeny, perhaps in part because of a notable decline in acute tolerance. In contrast, rapid tolerance to ethanol-induced sedation emerges slowly during ontogeny. This study tested the hypothesis that ontogenetic differences in glutamate and/or gamma-aminobutyric acid systems influence tolerance expression. METHODS: Sprague-Dawley rats at postnatal day (P)26 or P70 received (+)MK-801, muscimol, or saline before ethanol (3.5 or 4.5 g/kg) or saline on day 1 and ethanol only on day 2. Loss of and time to regain the righting reflex and blood alcohol levels at recovery were recorded. The presence of acute tolerance was indicated as a positive slope of the linear regression of blood alcohol levels at recovery versus ethanol dose. Rapid tolerance was estimated on day 2 by comparing animals given ethanol only on day 2 with those given ethanol on both days. RESULTS: Acute tolerance on day 1 only was observed at P26; this was disrupted by (+)MK-801 but not muscimol. Evidence for acute tolerance also emerged in adults on day 2. Whereas both drugs increased ethanol sedation at both ages, they did not facilitate ontogenetic expression of rapid tolerance: rapid tolerance was not evident at P26 regardless of pretreatment when indexed in terms of recovery time. CONCLUSIONS: These data provide further evidence for an ontogenetic dissociation in the expression of acute and rapid tolerance to ethanol-induced sedation. Pharmacological attenuation of the expression of acute tolerance was sufficient but not necessary to delay recovery of righting after ethanol. The greater propensity of young animals to develop acute tolerance, seemingly modulated in part by NMDA receptors, may contribute to their relative resistance to ethanol, although other factors, including pharmacokinetic factors, also contribute to their more rapid recovery from ethanol sedation.     

Acamprosate,MK-801, and ifenprodil inhibit neurotoxicity and calcium entry induced by ethanol withdrawal in organotypic slice cultures from neonatal rat hippocampus

BACKGROUND: The antirelapse drug acamprosate has previously been reported to inhibit activating effects of polyamines on -methyl-D-aspartic acid receptor (NMDAR) function. Because increased synthesis of polyamines has been suggested as a mechanism for potentiation of NMDAR function during ethanol withdrawal, we evaluated the effects of acamprosate, MK-801, and ifenprodil in a cell culture model of ethanol withdrawal-induced neurotoxicity. METHODS: Organotypic hippocampal cultures from 8-day-old neonatal rats were maintained in vitro for 23 days before experimental use. The ethanol withdrawal model consisted of exposing cultures to ethanol (70-100 mM) for 4 days before being "withdrawn" into Calcium-Locke's buffer for 1 hr and then into minimal medium for 23 hr. Uptake of (45)CaCl(2) and propidium iodide by damaged cells was assessed 1 hr and 24 hr after the start of ethanol withdrawal, respectively. Additional studies examined effects of exposure to NMDA (50 microM) or spermidine (100 microM) on withdrawal-induced hippocampal damage. Last, these studies examined the ability of the sodium salt of acamprosate (Na-acamprosate, 200 microM), ifenprodil (50 microM), or MK-801 (30 microM) to inhibit neurotoxicity and (45)Ca(2+) entry produced by these insults. RESULTS: Ethanol withdrawal was associated with significantly greater toxicity and (45)Ca(2+) entry, relative to controls. Exposure to spermidine and NMDA during ethanol withdrawal further increased neurotoxicity and (45)Ca(2+) entry. Acamprosate, ifenprodil, and MK-801 almost completely prevented ethanol withdrawal-induced toxicity and (45)Ca(2+) entry. Acamprosate also reduced spermidine-induced neurotoxicity during ethanol withdrawal but was ineffective against NMDA-induced toxicity or (45)Ca(2+) entry at this time. CONCLUSIONS: The results support the contention that acamprosate, like ifenprodil, interacts with polyamines and that these compounds may be effective in reducing consequences of ethanol withdrawal. NMDAR activation is also strongly implicated in ethanol withdrawal neurotoxicity, but whether acamprosate causes any of these effects in this preparation directly via the NMDAR remains uncertain.     

Stability of [3H]MK-801 Binding Sites Following Chronic Ethanol Consumption

Previous work has demonstrated that short periods (1–2 weeks) of exposure to ethanol produce an upregulation of the N-methyl-D-aspartate (NMDA) receptor complex in hippocampus; an alteration that appears to be associated with the development of physical dependence, because a return to control levels occurs over a 24- to 48-hr abstinence period. Prolonged periods of chronic ethanol treatment (CET; 4–8 months of treatment) have been shown to produce severe and permanent alterations in the morphological and functional characteristics of hippocampal pyramidal neurons. Several lines of research have demonstrated that the NMDA receptor complex is involved in excitotoxic cell loss during certain pathological states. On the basis of this evidence, we hypothesized that prolonged ethanol exposure would be accompanied by an enduring increase in NMDA receptors and that NMDA receptor binding in cells surviving CET would be altered. To test this hypothesis, we measured the binding characteristics of the NMDA receptor complex in a variety of brain structures following CET. Animals were fed a nutritionally complete, ethanol-containing diet for 28 weeks and then allowed a 48-hr abstinence period. A control group was fed the same diet, except sucrose was isocalorically substituted for ethanol. We first examined the effect of CET on the binding properties of a noncompetitive antagonist to the NMDA receptor channel, [³H]diclozipene ([³H]MK-801). Next, as an indirect examination of NMDA receptor function, we measured the ability of glutamate to stimulate channel opening and thus [³H]MK-801 binding. In all brain structures examined, neither the K_d nor the B_max of [³H]MK-801 binding to the NMDA receptor was altered following CET. In addition, no effect of treatment was seen on the ability of glutamate to stimulate [³H]MK-801 binding.     

Acute ethanol inhibits extracellular signal-regulated kinase, protein kinase B, and adenosine 3':5'-cyclic monophosphate response element binding protein activity in an age- and brain region-specific manner

BACKGROUND: As little as a single episode of exposure of the developing brain to ethanol can result in developmental neuropathology and mental retardation. Extracellular signal-regulated kinases (ERKs), protein kinase B (PKB), and adenosine 3':5'-cyclic monophosphate response element binding protein (CREB) are messenger molecules that play important roles in neuronal plasticity and survival. This study was undertaken to examine the effects of acute ethanol on ERK, PKB, and CREB activation in the brain. METHODS: Immunoblot analysis was used to determine the effects of a 1-hr exposure of ethanol on levels of phospho-ERC in primary cortical cultures and in the cerebral cortex, hippocampus, and cerebellum of postnatal day 5 (PN5), postnatal day 21 (PN21), and adult rats. RESULTS: In cortical cultures, ethanol (100 mM) significantly reduced activity-dependent activation of phospho-ERK, phospho-PKB, and phospho-CREB by approximately 50%. In PN5 rats, ethanol (3.5 g/kg) inhibited both phospho-ERK and phospho-PKB in the cerebral cortex and hippocampus but was without effect in the cerebellum. A similar brain region-specific inhibition of phospho-ERK was observed in PN21 rats, whereas in adult rats, ethanol inhibited phospho-ERK in all three brain regions. In contrast, ethanol had no effect on phospho-PKB in either PN21 or adult rats. Without exception, ethanol inhibited phospho-CREB in an identical brain region- and age-dependent manner as was observed for phospho-ERK. Finally, administration of the NMDA antagonist MK-801 (0.5 mg/kg) to PN5 rats had no effect on phospho-ERK or phospho-PKB levels in any brain region. CONCLUSION: The results demonstrate that acute ethanol inhibits ERK/PKB/CREB signaling in brain. This inhibition occurs in an age- and brain region-specific manner, with inhibition of PKB restricted to a time during the brain growth-spurt period. Furthermore, the lack of effect of MK-801 suggests that inhibition of NMDA receptors is unlikely to play a major role in binge ethanol inhibition of ERK/PKB/CREB signaling in vivo.     

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.     

Effects of Ethanol and Temperature on NMDA Receptor Function in Different Mouse Genotypes

The present study investigated whether temperature-related changes in NMDA receptor sensitivity to ethanol might play a role in mediating the effects of body temperature on behavioral sensitivity to ethanol or in determining genotypic differences in sensitivity to ethanol. We accomplished this by determining the effects of ethanol on three different mouse genotypes (C57, LS, and SS) on two types of NMDA receptor-mediated responses at 30° and 35°C (i) extracellularly recorded synaptic potentials elicited in the CA1 region of the in vitro hippocampal slice preparation by stimulation of the Schaffer-commisural pathway in the presence of the cu-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blocker, 6,7-dinitroqui-noxaline-2,3-dione, and low magnesium concentration; and (ii) increase in [³H]MK-801 binding elicited by glutamate in telencephalic membrane preparations. Ethanol significantly decreased NMDA receptor-mediated excitatory postsynaptic potential (EPSP) amplitude and area in the three genotypes. In C57, the effect of ethanol on NMDA receptor-mediated EPSP amplitude and area was more pronounced at 30°C, compared with that at 35°C. In most cases, there was a good correlation between the effects of ethanol on EPSP amplitude and area. The order of sensitivity between the three genotypes was C57 = LS > SS at 35°C and C57 > LS = SS at 30°C. Similarly, ethanol significantly decreased glutamate-stimulated [³H]MK-801 binding in membrane fractions. The effect of ethanol was temperature-dependent, because ethanol produced more inhibition at 30°C than at 35°C in all genotypes. The effect of ethanol on MK-801 binding was concentration-dependent, and the sensitivity to 100 mM ethanol of the genotypes at 35°C was LS > SS = C57, whereas it was SS > LS = C57 at 30°C. Collectively, the results demonstrate that temperature is an important variable that can influence NMDA receptor sensitivity to ethanol measured via electro-physiological and binding techniques, and that temperature can influence relative sensitivity of NMDA receptors to ethanol between mouse genotypes. Furthermore, the findings indicate that temperature-induced changes in sensitivity of NMDA receptors to ethanol may play a role in mediating the effects of body temperature on behavioral sensitivity to ethanol in LS, but not C57 and SS mice.     

Corticosterone increases damage and cytosolic calcium accumulation associated with ethanol withdrawal in rat hippocampal slice cultures

BACKGROUND: Evidence suggests that stress hormones (i.e., glucocorticoids) may be increased during acute or chronic consumption of ethanol and during withdrawal from ethanol consumption, effects that may contribute to the development of cognitive impairment. The goal of the current studies was to examine the hypothesis that increased glucocorticoid levels in conjunction with ethanol exposure and withdrawal may cause hippocampal damage. METHODS: Organotypic hippocampal slice cultures were exposed to 50 mM ethanol for 10 days and withdrawn for 1 day. After withdrawal, cytotoxicity and cytosolic Ca2+ accumulation were measured using the nucleic acid stain propidium iodide and Calcium Orange, AM, respectively. Cultures were also treated with nontoxic concentrations of corticosterone (0.001-1 microM) during ethanol exposure and withdrawal or only during withdrawal. Additional cultures were coexposed to corticosterone and RU486 (0.1-10.0 microM), spironolactone (0.1-10.0 microM), or MK-801 (20 microM) during ethanol exposure and/or withdrawal. RESULTS: Ethanol withdrawal did not increase propidium iodide fluorescence and cytosolic Ca2+ levels. However, significant increases in propidium iodide fluorescence and in cytosolic Ca2+ accumulation were observed in cultures when corticosterone (> or = 100 nM) was exposed during ethanol treatment and/or withdrawal. These effects of corticosterone on ethanol withdrawal were attenuated by RU486 and MK-801 but not by spironolactone coexposure. CONCLUSIONS: This report demonstrated that corticosterone exposure during ethanol treatment and/or withdrawal resulted in significant hippocampal damage, possibly via activation of glucocorticoid receptors and enhancement of the glutamatergic cascade. The findings from these studies suggest that glucocorticoids contribute to the neuropathological consequences of alcohol dependence in humans.     

The Involvement of NMDA Receptors in Acute and Chronic Effects of Ethanol

Recent evidence indicates involvement of excitatory amino acid receptors sensitive to N-methyl-d-aspartate (NMDA) in the action of ethanol (EtOH). Pronounced inhibition of NMDA receptor function is seen in vitro with concentrations of EtOH corresponding to those present during alcohol intoxication in humans. The present study was devoted to investigate the role of NMDA receptors in the action of EtOH in rats. Acute experiments showed antagonism by EtOH of convulsions induced by intracerebroventricular injection of NMDA. A similar effect was seen with a high dose of diazepam. Convulsions induced by an agonist of another excitatory amino acid receptor subtype, kainate, were also inhibited by EtOH. An uncompetitive antagonist of NMDA receptors, 5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate (MK-801), potentiated EtOH-induced loss of righting, but attenuated the hypothermic action of EtOH. Moreover, MK-801 inhibited audiogenic convulsions in EtOH withdrawn rats. At the same time the effect of a proconvulsive dose of NMDA was not enhanced. Tolerance to the myorelaxant action of both EtOH and MK-801 upon repetitive administration was seen. Also some degree of cross-tolerance was observed. Moreover, MK-801 failed to modify EtOH preference in rats. The present results support involvement of NMDA receptors in expression of some acute and subchronic actions of EtOH and in expression of EtOH withdrawal.     

Alterations in Activity Following Alcohol Administration During the Third Trimester Equivalent in P and NP Rats

There is considerable variation in the consequences of alcohol abuse during pregnancy on infant outcome. Although it is clear that a number of factors contribute to this variability, one hypothesis that has received recent attention is the role of genetic differences in response to alcohol. This study examined activity levels in the alcohol-preferring (P) and alcohol-nonpreferring (NP) rats following neonatal alcohol exposure. Although these lines were selectively bred for differences in voluntary alcohol consumption, they also differ in their sensitivity and tolerance to alcohol. The P and NP offspring were artificially reared and administered ethanol (either 6 or 4 g/kg/day) from postnatal day 4 (PN 4) until PN 10 via intragastric cannula. An artificially reared isocaloric maltose group and a normally reared control group were also included. From PN 18 to PN 21, subjects were tested daily for 30 min in an automated activity monitor. Exposure to either the 4 or 6 g/kg dose of ethanol resulted in overactivity in P rats. However, only the 6 g/kg dose group displayed overactivity among the NP offspring. Furthermore, the level of overactivity displayed by the alcohol-exposed P rats was significantly greater than that displayed by the alcohol-exposed NP rats. These data suggest that genetic differences in response to alcohol may be a predictor for the behavioral teratogenic effects of alcohol.