Effects of Prenatal Ethanol Exposure on Brain Region NMDA-Mediated Increase in Intracellular Calcium and the NMDAR1 Subunit in Forebrain

The effects of prenatal ethanol exposure on N -methyl-d-aspartate (NMDA)-mediated calcium entry into neonatal dissociated neurons from hippocampus, forebrain, and cerebellum were investigated. Dissociated cells were isolated from less than 1-day-old pups of prenatally exposed, pair-fed control and ad libitum control groups and loaded with fura-2. Prenatal ethanol exposure significantly reduced the NMDA-stimulated increase in intracellular calcium in all three brain regions compared to the two control groups. These findings are very similar to those previously observed in neonatal dissociated whole brain neurons using the same ethanol exposure protocol. Studies were also conducted using forebrain to determine if prenatal ethanol exposure alters NMDAR1 subunit protein expression in this major brain area; however, the results indicated no significant differences between ethanol-exposed and control groups.     

N-Methyl-d-Aspartate-Stimulated Increases in Intracellular Calcium Exhibit Brain Regional Differences in Sensitivity to Inhibition by Ethanol

This study compared N -methyl- d -aspartate (NMDA)-stimulated increases in intracellular calcium in fura-2–loaded neurons dissociated from newborn rat brainstem (EC₅₀ in μM; 6.4), cerebellum (9.5), forebrain (6.3), and hippocampus (10.6). Ethanol inhibition of the response to 25 μM NMDA differed among the regions. The NMDA response in hippocampus was inhibited by 20 mM ethanol; cortex and cerebellum responses were inhibited by 80 mM ethanol, and no inhibition was seen in the brainstem. Addition of glycine (15 μM) failed to attenuate ethanol inhibition of the NMDA response. These results demonstrate that ethanol inhibition of NMDA-stimulated responses varies according to brain region. In contrast to previous findings from this laboratory using dissociated neurons from whole brain, the addition of glycine did not reverse the inhibitory effects of ethanol on NMDA-stimulated responses.     

Mechanism of Ethanol Inhibition of NMDA Receptor Function in Primary Cultures of Cerebral Cortical Cells

Ethanol is a potent inhibitor of the function of the N-methyl-o-aspar-tate (NMDA) subtype of glutamate receptor in various neuronal preparations. In primary cultures of cerebellar granule cells, ethanol was suggested to interact with the glycine co-agonist site of the receptor by a mechanism involving protein kinase C. In the present study, the interaction of ethanol with various sites on the NMDA receptor was examined in primary cultures of cerebral cortical cells from ernbryonic rats. NMDA receptor function was determined by measuring increases in intracellular Ca²⁺ with fura-2 fluorescence. Ethanol inhibited the function of the NMDA receptor in cerebral cortical cells, but in contrast to the results in cerebellar granule cells, phorbol ester treatment did not inhibit the NMDA response, and ethanol did not alter the effect of glycine on NMDA receptor function. Ethanol also did not affect inhibition of the NMDA response by Mg²⁺ or dizo-cilpine. The results support the hypothesis that the mechanism of ethanol inhibition of NMDA receptor function can vary in neurons from different brain regions.     

Hormonal Tolerance to Ethanol is Associated with Decreased Expression of the GTP-Binding Protein, Gsα, and Adenylyl Cyclase Activity in Ethanol-Treated LS Mice

Using the proopiomelanocortin (POMC) system as a marker, long sleep (LS) and short sleep (SS) lines of mice were investigated to explore the cellular events that occur during the acquisition of hormonal tolerance to ethanol. Four-day ethanol exposure (1.8 g/kg/24 hr) increased anterior pituitary POMC mRNA levels 4-fold in the LS line and 2-fold in the SS line. Following 7 days of ethanol exposure (1.8 g/kg/24 hr), anterior pituitary POMC levels returned to basal values in the LS line but remained elevated (2-fold) in the SS line. In this setting, the loss of ethanol's ability to sustain elevated POMC mRNA levels in the LS line is defined as hormonal tolerance. Since POMC biosynthesis is primarily regulated through adenylyl cyclase, ethanol-induced alterations in this signal transduction system were explored. Paralleling the effects of ethanol on POMC mRNA levels, ethanol exposure reduced GTP-gamma-S, AIF3-, and MnCl2-stimulated adenylyl cyclase activity by 35%, 21%, and 24%, respectively, in the LS line without effecting adenylyl cyclase activity in the SS line. To determine whether ethanol-induced changes in adenylyl cyclase activity in LS mice could result from alterations in G proteins, protein levels of G, alpha and Gi alpha were determined by western analysis before and after ethanol exposure. Paralleling the effect on POMC mRNA levels and adenylyl cyclase activity, ethanol induced a 35% reduction in Gs alpha protein levels in LS mice but did not alter Gi alpha levels. Neither Gs alpha nor Gi alpha levels were altered in the SS line.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct evidence for a cause-effect link between ethanol potentiation of GABA(A) receptor function and intoxication from hyperbaric studies in C57, LS, and SS mice

BACKGROUND: This article uses a direct ethanol antagonist, increased atmospheric pressure, to further test the causative link between ethanol potentiation of gamma-aminobutyric acid (GABA) type A receptor function and ethanol's behavioral effects. This was done by determining whether initial biochemical findings in long-sleep (LS) mice extended to other genotypes and whether the previously reported insensitivity of short-sleep (SS) mice to pressure antagonism of ethanol-induced loss of righting reflex extended to a nonselected ethanol-induced behavior. METHODS: The effects of 12 times normal atmospheric pressure of helium-oxygen gas (heliox) versus ethanol (25-200 mM) potentiation of GABA-activated Cl- uptake in brain membranes (microsacs) from C57, LS, and SS mice were tested by using a 36Cl- flux assay. The effects of pressure versus ethanol's (2 g/kg) anticonvulsant effect in SS mice were tested by using time to onset of isoniazid-induced myoclonic seizures. RESULTS: Exposure to 12 times normal atmospheric pressure heliox antagonized ethanol potentiation of GABA-activated Cl- uptake in all three genotypes across a range of ethanol concentrations that cause ethanol's behavioral and anesthetic effects. Pressure did not affect baseline receptor function. The threshold for initiating ethanol potentiation differed between genotypes in accordance with their behavioral sensitivities to ethanol (C57 and LS, < or =25 mM; SS, >50 mM). Pressure antagonized ethanol's anticonvulsant effect in SS mice. CONCLUSIONS: The results add important direct evidence supporting the hypothesis that ethanol potentiation of GABA(A) receptor function is an initial action of ethanol causing its behavioral effects. These findings also provide insight into possible effects of selective breeding on GABA(A) receptor function.     

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.     

Direct Antagonism of Ethanol's Effects On GABAA Receptors by Increased Atmospheric Pressure

Previous studies have shown that exposure to 12 times normal atmospheric pressure of helium-oxygen gas (heliox) directly antagonizes a range of ethanol's acute and chronic behavioral effects. The present study extends the investigation to the biochemical level by investigating the effects of pressure on ethanol-induced potentiation of GABA_A receptor function in mouse membrane vesicles (micro-sacs). Exposure to 12 atmospheric pressure heliox significantly antagonized ethanol (25 to 100 mM) potentiation of GABA-activated ³⁶Cl^- uptake, but did not significantly alter baseline GABA_A receptor function measured by the response of the system to GABA (10 to 100 μM), bicuculline (3 and 100 μM), or picrotoxin (100 μM). These findings add essential support for the hypothesis that hyperbaric exposure is a direct ethanol antagonist that can be used as a tool to help identify ethanol's initial cellular and molecular sites of action that cause its behavioral effects. Taken in context with previous behavioral studies, the present results also provide important new evidence for a cause-effect relationship between ethanol potentiation of GABA_A receptor function and ethanol's anesthetic and behavioral effects.     

Acamprosate is neuroprotective against glutamate-induced excitotoxicity when enhanced by ethanol withdrawal in neocortical cultures of fetal rat brain

BACKGROUND: Acamprosate reduces relapse, and the drug may interact with glutamate transmission and with glutamate/NMDA receptors. Because these systems are implicated in excess calcium entry leading to alcohol withdrawal-induced neurotoxicity, we evaluated the effects of acamprosate on these parameters in neuronal cultures. METHODS: Primary cultures of neocortical neurons from fetal Sprague-Dawley rats were maintained either in normal medium or in medium containing 100 mM ethanol for 4 days. After this time, cultures were challenged with glutamate for 10 min and then returned to medium (all in the absence of ethanol). 45Ca2+ uptake was measured during the challenge, and glutamate-induced toxicity was assessed after 20 hr. The effects of acamprosate present during the glutamate challenge were measured on both parameters. RESULTS: In controls, acamprosate did not significantly affect glutamate-induced neurotoxicity but produced a significant inhibition of calcium entry. The NMDA receptor antagonists dizocilpine and d-amino-phosphonovalerate (D-APV) inhibited both glutamate neurotoxicity and calcium entry. In cultures previously exposed to ethanol, glutamate-induced neurotoxicity and calcium entry were both significantly enhanced. Dizocilpine reduced both these parameters to unstimulated control values, and D-APV reduced both calcium entry and neurotoxicity with the same relation that we obtained in control cultures. In the ethanol-withdrawn cultures, acamprosate reduced both the enhanced glutamate-induced calcium entry and the enhanced neurotoxicity in a concentration-dependent manner. Acamprosate also significantly reduced calcium entry caused by 80 mM K+ in control and ethanol-exposed cultures. CONCLUSIONS: Acamprosate had protective effects against glutamate-induced neurotoxicity only in ethanol-withdrawn cultures. The neuroprotective effects of the drug did not correlate with its effects on calcium entry, making it unlikely that acamprosate directly affects NMDA receptors via the glutamate binding site or the receptor-operated calcium channel. The results are, however, compatible with other inhibitory effects on NMDA receptor function.     

Ethanol acceptance is high during early infancy and becomes still higher after previous ethanol ingestion

BACKGROUND: Infant (preweanling) rats readily accept ethanol without initiation procedures. During their second and third postnatal weeks, rats ingest large quantities of even high concentrations of ethanol. The present study tested the consequences of still earlier exposure to ethanol differing in concentration, mode of administration, and contextual circumstances. METHODS: Every 48 hours from postnatal day 2 (P2) to P10, pups were given access to 0, 5, 15, or 25% ethanol through an independent feeding procedure (consumption off the floor; COF) or by intragastric (i.g.) administration. The amount of ethanol consumed was matched for the 2 modes of ethanol delivery. On P12 pups were tested for intake of 15% ethanol through an intraoral infusion test or COF. RESULTS: Beginning on P6, pups ingested more ethanol solution than water, and by P8 and P10 they ingested large quantities of ethanol--1.5 to 2.0 g/kg ethanol from 15 or 25% ethanol solution within a 10-minute period. This early experience with ingestion of ethanol increased subsequent ethanol intake on P12, particularly when concentration and mode of ingestion were the same as before. CONCLUSIONS: Intake on P12 was increased by prior exposure to ethanol intragastrically as well as by the conventional oral route, suggesting pharmacological effects of prior ethanol exposure. Yet, the apparently greater influence of prior exposure by the oral route and the influence of prior ethanol concentration also implicate the importance of ethanol's chemosensory attributes for effects of prior ethanol exposure. The equivalent g/kg intake of ethanol in 15 or 25% solutions during the early part of the second postnatal week suggests that regulation of ethanol intake at P8 and P10 is similar to that observed previously in adults. Change in aspects of ethanol ingestion at about P6 may reflect the shift in function of the GABA system at about this age.     

Release and Accumulation of Neurotransmitters in the Rat Brain: Acute Effects of Ethanol In Vitro and Effects of Long-Term Voluntary Ethanol Intake

Release from and accumulation in tissue slices of some neurotransmitters under acute ethanol in naive rats and in long-term voluntarily ethanol drinking rats were investigated. Slices of the rat caudatoputamen were prelabeled with [³H]choline and release of [³H]acetylcholine was stimulated through either N -methyl- d -aspartate (NMDA) receptors or strychnine-sensitive glycine receptors. Ethanol in vitro at 2%, 4%, and 6% (34 mM, 68 mM, and 102 mM, respectively) concentration-dependently depressed the maximum effect of the concentration-response curve of NMDA in naive rats. In contrast, voluntary ethanol consumption over months led to a significantly enhanced NMDA receptor response characterized by an increase in the maximum effect of the concentration-response curve. The glycine receptor-mediated release of [³H]acetylcholine, which is inhibited by acute ethanol in a competitive-like fashion, was not changed in animals that ingested ethanol over months. Electrically evoked release of [³H]noradrenaline ([³H]NA) and its presynaptic modulation by morphine through μ-opioid receptors in neocortical slices of the rat, preloaded with [³H]NA, was nearly identical in both ethanol-naive rats and in ethanol drinking rats. The accumulation of [³H]γ-aminobutyric acid in rat cerebellum tissue was neither affected by acute ethanol in vitro nor after chronic ethanol consumption. In summary, long-term voluntary ethanol intake caused a significant increase in NMDA receptor function in the rat caudatoputamen, but did not result in changes in glycine-evoked [³H]acetylcholine release of electrically evoked [³H]NA release modulated by morphine or cerebellar [³H]γ-aminobutyric acid accumulation.     

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.     

Dose- and Age-Dependent Effects of Prenatal Ethanol Exposure on Hippocampal Metabotropic-Glutamate Receptor-Stimulated Phosphoinositide Hydrolysis

Prenatal ethanol exposure reduces the density of the N-methyl-D-aspartate (NMDA) receptor agonist binding sites and decreases the capacity to elicit long-term potentiation (LTP) in hippocampal tormation of 45-day-old rat offspring. We hypothesized that prenatal ethanol exposure would reduce metabotropic-glutamate receptor (mGluR)-activated phosphoinositide hydrolysis also. Sprague-Dawley rat dams were fed a liquid diet containing either 3.35% (v/v) ethanol or 5.0% ethanol throughout gestation. Control groups were pair-fed either isocalorically matched 0% ethanol liquid diets or lab chow ad libitum. (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) stimulated inositol-1-phosphate (IP₁) accumulation via activation of the mGluR in offspring whose mothers consumed the 3.35% ethanol liquid diet was not different compared with the control groups. Furthermore, trans-ACPD stimulated IP₁ accumulation in 10 to 13-day-old offspring of the 5.0% ethanol diet group was not different compared with the control groups. However, trans-ACPD stimulated IP₁ accumulation was reduced significantly in 56- to 82-day-old offspring of dams fed the 5.0% ethanol liquid diet compared with the control groups. In contrast, bethanechol stimulated IP₁ accumulation, mediated via activation of muscarinic cholinergic receptors, was not affected by maternal consumption of either ethanol liquid diet. These results suggest both dose- and age-dependent effects of prenatal ethanol exposure on hippocampal responsiveness to trans-ACPD-activated phosphoinositide hydrolysis. Furthermore, the ability of the 3.35% ethanol diet to alter hippocampal NMDA receptors without altering the mGluR response suggests a differential sensitivity to the effects of ethanol exposure in utero among hippocampal glutamate receptor subtypes. Recent studies indicate that activation of mGluRs facilitates NMDA receptor-dependent LTP. Thus, higher blood ethanol concentrations achieved by consumption of the 5.0% ethanol liquid diet adversely affects an additional glutamate receptor mechanism associated with LTP. This additional effect may lead to an even greater impact of prenatal ethanol exposure on LTP than occurs when NMDA receptor function alone is affected by maternal consumption of more moderate quantities of ethanol.     

Synapse-to-Neuron Ratio in the Lateral Geniculate Nucleus of Rats Exposed Chronically to Ethanol

Effects of chronic ethanol exposure on synapse-to-neuron ratio in the rat lateral geniculate nucleus were investigated. Male Sprague-Dawley rats were exposed to ethanol, using the Lieber-DeCarli liquid diets, for 4 months starting at the age of 5 weeks. Brains were per-fusion-fixed, and the region containing the dorsal lateral geniculate nucleus was cut into slabs (500 μm thick) that were epoxy resin-embedded. From each rat, three slabs containing the structure were serially sectioned for electron microscopy. Using the double disector method, the study shows an unaltered synapse-to-neuron ratio in ethanol-treated rats when compared with controls. The findings are in agreement with previous studies on the visual system using the seme exposure model. In contrast, a previous study has shown that the synapse-to-neuron ratio in locus ceruleus of ethanol-treated rats is reduced by 50%. Other studies have shown that, whereas the glutamatergic NMDA receptor is very sensitive to ethanol, the kainate/AMPA type of receptor is very much less so. Thus, the difference in ethanol-induced synapse elimination between the two regions may reflect this different sensitivity of the glutamatergic receptors, which are of the kainate/AMPA type in the lateral geniculate nucleus and of the NMDA type in the locus ceruleus.     

Effects of Ethanol on Recombinant Glycine Receptors Expressed in Mammalian Cell Lines

We examined the effects of acute ethanol exposure on recombinant human glycine receptors transiently transfected into HEK 293 cells and stably transfected into Ltk⁻ fibroblast-like cells. In our study of the effects of ethanol, we used the whole-cell patch-clamp configuration. Relatively low concentrations of ethanol (25 mM and 50 mM) did not affect glycine-gated currents in any of the cell lines studied. Higher concentrations of ethanol (100 mM and 200 mM) significantly potentiated glycine responses only in stably transfected Ltk⁻ cells expressing α₁ and α₂ subunits and in HEK 293 cells transiently expressing α₂ subunits. Cells stably expressing α₁ versus α₂ glycine receptors were modulated equally by ethanol. Both glycine α₁ and glycine α₁β receptors transiently expressed in HEK 293 cells were insensitive to all concentrations of ethanol tested; however, there was a trend toward potentiation at 100 and 200 mM ethanol concentrations. A population of cells (41–87%) that was sensitive to the potentiating effects of 100 and 200 mM ethanol (defined as more than 10% potentiation) was identified in both cell lines tested. In these sensitive cells, ethanol (100 and 200 mM) produced significant potentiation, independent of the cell line and the glycine receptor sub-unit tested. Together with published results from studies with Xenopus oocytes, these data indicate that the sensitivity of recombinant glycine receptors to ethanol depends upon the expression system.     

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.     

Reduced Sensitivity to Ethanol Reward, But Not Ethanol Aversion, in Mice Lacking 5=HT1B Receptors

Various serotonergic receptor systems are thought to influence the motivational effects of ethanol. This experiment characterized the acquisition of ethanol-induced conditioned taste aversion and ethanol-induced conditioned place preference in mutant knockout mice lacking 5-HT_1B receptors. In the taste conditioning procedure, adult homozygous knockout mice (-/-) and homozygous wild-type mice (+/+) received access to 0.2 M NaCl solution, followed immediately by intraperitoneal injection of 0 to 4 g/kg of ethanol. Ethanol produced dose-dependent conditioned taste aversion that was the same in both genotypes. In the place conditioning procedure, knockout and wild-type mice received six pairings of a tactile stimulus with ethanol (2 g/kg, ip). A different tactile stimulus was paired with saline. Ethanol produced increases in locomotor activity, with wild-type mice showing higher levels of ethanol-stimulated activity than knockout mice during conditioning trials 5 and 6. Wild-type mice demonstrated conditioned place preference for the ethanol-paired stimulus. In contrast, knockout mice showed no evidence of place conditioning. These results are generally consistent with an important role for serotonergic systems in ethanol reward and specifically indicate that 5-HT_1b receptors are important for ethanol's rewarding effects but not for ethanol's aversive effects.     

Ethanol enhances GABAA receptor function in short sleep and long sleep mouse brain membranes

BACKGROUND: SS and LS mice have been used to explore the genetic and neurochemical bases for differences in sensitivity to ethanol. The present study investigated the effects of ethanol on GABAA receptor function in microsacs from these genotypes. The purpose was to test a key element of the hypothesis that differences between these lines in sensitivity to ethanol-induced enhancement of GABAA receptor function underlie their selected differences in sensitivity to ethanol-induced loss of righting reflex (LORR). METHODS: The effects of ethanol on GABA-activated 36Cl- uptake in brain membranes (microsacs) isolated from male SS and LS mice were tested using a chloride flux filtration assay. RESULTS: Ethanol significantly enhanced GABA-activated 36Cl- uptake in SS microsacs at concentrations of 100-300 mM. Ethanol did not significantly affect GABA-activated chloride uptake in this preparation at concentrations of 25 and 50 mM. Ethanol significantly enhanced GABA-activated 36Cl- uptake in LS microsacs at concentrations of 25-100 mM, but not at 200 mM. CONCLUSION: The present studies are the first to show a statistically significant effect of ethanol on GABA-activated chloride uptake in both SS and LS mice with a clear difference between the genotypes in threshold. The relative threshold differences between SS and LS microsacs in sensitivity to ethanol indicate that selection for resistance to ethanol-induced LORR in SS mice has shifted the ethanol-GABAA receptor concentration-response curve to the right. The findings add key evidence that supports a cause-effect relationship between sensitivity to ethanol-induced potentiation of GABAA receptor function and genetically determined sensitivity to ethanol's behavioral effects.     

MK-801 Administration During Ethanol Withdrawal in Neonatal Rat Pups Attenuates Ethanol-Induced Behavioral Deficits

Alcohol exposure during development can produce central nervous system dysfunction, resulting in a wide range of behavioral alterations. The various mechanisms by which alcohol causes these behavioral changes, however, remain unknown. One mechanism that has been suggested is NMDA receptor-mediated excitotoxic cell death produced by ethanol withdrawal. The present study examined whether MK-801, an antagonist of the NMDA receptor that has been shown to protect against NMDA receptor-mediated excitotoxicity, could block alcohol's adverse effects on behavior. Sprague-Dawley rat pups were exposed to alcohol (6.0 g/kg) in a binge-like manner on postnatal day 6 using an artificial rearing procedure. Subjects then received an injection of MK-801 (0.1 mg/kg) or vehicle during withdrawal, 21 hr after ethanol exposure. At postnatal day 40, all subjects were tested on a serial spatial discrimination reversal task. Ethanolexposed subjects were impaired in both discrimination and reversal learning, and committed a significantly greater number of perseverative-type errors, compared with controls. MK-801 administration during ethanol withdrawal significantly attenuated ethanol-induced deficits in reversal learning and increases in perseverative-type errors, whereas MK-801 exposure by itself had no significant effect on performance. Thus, exposure to MK-801 during ethanol withdrawal partially protected against alcohol-related disruptions in spatial reversal learning. These results support the suggestion that NMDA receptor-mediated excitotoxicity may be one mechanism by which alcohol induces behavioral teratogenicity.