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.     

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.     

A review of the effects of prenatal or early postnatal ethanol exposure on brain ligand-gated ion channels

BACKGROUND: Ligand-gated ion channels mediate fast excitatory and inhibitory synaptic transmission in the developing central nervous system. These channels have been shown to have roles in neuronal proliferation, differentiation, and programmed cell death. Numerous studies over the past 10 years indicate that prenatal and/or early postnatal ethanol exposure affects neurotransmitter-gated ion channels. METHODS: We conducted a review of the relevant literature, identified by a computer-assisted literature search. This review presents an overview of studies performed with experimental preparations from the brains of rodents exposed to ethanol in utero and/or during the neonatal period and summarizes some of the salient issues that have developed in the course of these investigations. Differences in ethanol exposure paradigms and blood alcohol concentrations obtained in these studies are highlighted, and directions for future research are suggested. RESULTS: Most studies have focused on the effects of prenatal or early postnatal ethanol exposure on NMDA receptors. These studies show that ethanol exposure affects ligand binding, subunit expression, and function of this receptor. Fewer studies have examined ethanol's effects on ligand-gated ion channels other than NMDA receptors. For instance, a study reported changes in ligand binding to hippocampal kainate receptors. Another study found alterations in modulation of GABA(A) receptors by benzodiazepines and neurosteroids. CONCLUSIONS: These studies suggest that the effects of ethanol on brain ion channels may have a role in the pathophysiology of Alcohol-Related Neurodevelopmental Disorders and Fetal Alcohol Syndrome.     

Fetal alcohol exposure and temporal vulnerability: regional differences in cell loss as a function of the timing of binge-like alcohol exposure during brain development

This study was conducted to determine the temporal and regional vulnerability of the brain as a function of exposure to alcohol during brain development. Our goal was to manipulate the timing of alcohol exposure and assess the relative risk of cell loss in two different brain regions. Groups of timed pregnant Sprague-Dawley rats received binge-like alcohol exposure during either the first 10 days (first-trimester equivalent) or second 10 days of gestation (second-trimester equivalent), or the combination of first- and second-trimester equivalents for prenatal treatments. Offspring from some of the animals exposed to alcohol during the combined first- and second-trimester equivalent were reared artificially from postnatal days (P) 4 through 9 (part of the third-trimester equivalent) and also received binge-like alcohol during this period, producing animals that were exposed to alcohol during all three trimesters equivalent. Offspring from untreated dams were also reared artificially and received alcohol from only P4-9, thus creating animals that were exposed to alcohol only during part of the third-trimester equivalent. All pups were perfused on P10. Appropriate controls (nutritional and normally reared) were matched to every alcohol treatment combination. Peak blood alcohol concentrations were not different among the treatment groups for a given sampling time. Total cell numbers in the cerebellum (Purkinje and granule cells) and the olfactory bulb (mitral and granule cells) were estimated by the unbiased stereological technique, the optical disector. In terms of temporal vulnerability, alcohol exposure during the equivalent of all three trimesters resulted in a greater reduction in cerebellar Purkinje cell numbers compared with exposure to alcohol during the third-trimester equivalent, whereas both groups had a significant reduction in cell number compared with all other timing groups. Cerebellar granule cell number was reduced after alcohol exposure during all three trimesters equivalent, compared with all other timing groups. Alcohol exposure during the third-trimester equivalent resulted in a decrement in the number of olfactory bulb mitral cell numbers compared with all other groups, but there were no differences among the timing groups in numbers of olfactory bulb granule cells. When the cell loss in the two regions was compared within each alcohol treatment group to determine the relative regional vulnerability, the primary salient finding was that cerebellar Purkinje cells were more vulnerable to alcohol-induced loss subsequent to exposure during all three trimesters equivalent. No other regional differences were detected. These results extend earlier findings by showing that alcohol exposure during different periods of brain development results in regional differences in cell loss as a function of the timing of alcohol exposure during brain development and illustrate the variability of alcohol-induced neuronal loss.     

Fetal alcohol exposure and temporal vulnerability: effects of binge-like alcohol exposure on the ventrolateral nucleus of the thalamus

BACKGROUND: Prenatal alcohol exposure disrupts motor performance in affected offspring. The ventrolateral nucleus (VLN) of the thalamus functions to relay information between the cerebellum and motor cortex. Reductions in the size of the thalamus have been found in children with fetal alcohol syndrome, and therefore a rat model system was used to determine whether VLN size and neuronal number were altered by alcohol exposure during development. METHODS: Rat pups were exposed to alcohol in utero during the first 10 days of gestation (first trimester equivalent), the second 10 days of gestation (second trimester equivalent), or the first two trimesters equivalent combined. Some pups were exposed to alcohol in utero during the time of VLN neurogenesis. In addition, offspring from some of the dams treated during the first two trimesters equivalent were reared artificially from postnatal day (P) 4 through P9 (part of the third trimester equivalent) and received binge-like alcohol during this time, resulting in offspring exposed to alcohol during all three trimesters equivalent. Other offspring from untreated dams were reared in the same manner but received alcohol only during the third trimester equivalent. Control animals (nutritional and untreated) were reared for all treatment conditions. All pups were perfused on P10. RESULTS: A unique effect of alcohol treatment was not found for the VLN volume or the number of neural cells within the VLN. However, the period of VLN neurogenesis was found to be sensitive to both alcohol and nutritional control treatments, resulting in significant decreases in the VLN volume and neural cell number. CONCLUSIONS: Motor deficits seen in offspring exposed prenatally to alcohol do not seem to result from direct effects on the structure of the VLN of the thalamus.     

Role of major NMDA or AMPA receptor subunits in MK-801 potentiation of ethanol intoxication

Background: The glutamate system plays a major role in mediating EtOH’s effects on brain and behavior, and is implicated in the pathophysiology of alcohol‐related disorders. N‐methyl‐D‐aspartate receptor (NMDAR) antagonists such as MK‐801 (dizocilpine) interact with EtOH at the behavioral level, but the molecular basis of this interaction is unclear. Methods: We first characterized the effects of MK‐801 treatment on responses to the ataxic (accelerating rotarod), hypothermic and sedative/hypnotic effects of acute EtOH administration in C57BL/6J and 129/SvImJ inbred mice. Effects of another NMDAR antagonist, phencyclidine, on EtOH‐induced sedation/hypnosis were also assessed. Gene knockout of the NMDAR subunit NR2A or l ‐alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate GluR1 or pharmacological antagonism of the NMDAR subunit NR2B (via Ro 25‐6981) was employed to examine whether inactivating any one of these glutamate signaling molecules modified MK‐801’s effect on EtOH‐related behaviors. Results: MK‐801 markedly potentiated the ataxic effects of 1.75 g/kg EtOH and the sedative/hypnotic effects of 3.0 g/kg EtOH, but not the hypothermic effects of 3.0 g/kg EtOH, in C57BL/6J and 129/SvImJ mice. Phencyclidine potentiated EtOH‐induced sedation/hypnosis in both inbred strains. Neither NR2A nor GluR1 KO significantly altered basal EtOH‐induced ataxia, hypothermia, or sedation/hypnosis. Ro 25‐6981 modestly increased EtOH‐induced sedation/hypnosis. The ability of MK‐801 to potentiate EtOH‐induced ataxia and sedation/hypnosis was unaffected by GluR1 KO or NR2B antagonism. NR2A KO partially reduced MK‐801 + EtOH‐induced sedation/hypnosis, but not ataxia or hypothermia. Conclusions: Data confirm a robust and response‐specific potentiating effect of MK‐801 on sensitivity to EtOH’s intoxicating effects. Inactivation of three major components of glutamate signaling had no or only partial impact on the ability of MK‐801 to potentiate behavioral sensitivity to EtOH. Further work to elucidate the mechanisms underlying NMDAR × EtOH interactions could ultimately provide novel insight into the role of NMDARs in alcoholism and its treatment.     

Comparison of verbal learning and memory in children with heavy prenatal alcohol exposure or attention-deficit/hyperactivity disorder

Background: Children with fetal alcohol spectrum disorders (FASD) have deficits in verbal learning and recall. However, the specificity of these deficits has not been adequately tested. In the current study, verbal learning and memory performance of children with heavy prenatal alcohol exposure was compared to children with attention‐deficit/hyperactivity disorder (ADHD), a disorder commonly seen in alcohol‐exposed children. Methods: Performance on the California Verbal Learning Test—Children’s Version (CVLT‐C) was examined in 3 groups of children (N = 22/group): (i) heavy prenatal alcohol exposure and ADHD (ALC), (ii) nonexposed with ADHD (ADHD), and (iii) nonexposed typically developing (CON). Groups were matched on age, sex, race, ethnicity, handedness, and socioeconomic status (SES). Results: Group differences were noted on learning trials (CON > ADHD > ALC). On the delayed recall trial, CON children performed better than both clinical groups, who did not differ from each other. Children in the ALC group demonstrated poorer recognition than children in the CON and ADHD groups, who did not differ from each other. Marginally significant group differences were noted on retention of previously learned material. Post hoc analyses indicated that ADHD children showed worse retention relative to the CON group, whereas retention in the ALC children remained intact. Conclusions: These data suggest that children with heavy prenatal alcohol exposure and nonexposed children with ADHD show differential patterns of deficit on the CVLT‐C. Performance of alcohol‐exposed children reflects inefficient encoding of verbal material, whereas performance of the ADHD group may be better characterized by a deficit in retrieval of learned material. Differences noted between clinical groups add to a growing neurobehavioral profile of FASD that may aid in differential diagnosis.     

A review of social skills deficits in individuals with fetal alcohol spectrum disorders and prenatal alcohol exposure: profiles, mechanisms, and interventions

Background: Individuals gestationally exposed to alcohol experience a multitude of sociobehavioral impairments, including deficits in adaptive behaviors such as social skills. Methods: The goal of this report is to critically review research on social skills deficits in individuals with prenatal alcohol exposure, including individuals with and without fetal alcohol spectrum disorders (FASD). Results: Social deficits are found in alcohol‐exposed children, adults, and adolescents with and without a clinical presentation. These deficits tend to persist across the lifespan and may even worsen with age. Social deficits in this population appear to be independent of facial dysmorphology and IQ and are worse than can be predicted based on atypical behaviors alone. Abnormalities in neurobiology, executive function, sensory processing, and communication likely interact with contextual influences to produce the range of social deficits observed in FASD. Conclusions: Future investigations should strive to reconcile the relationship between social skills deficits in FASD and variables such as gender, age, cognitive profile, and structural and functional brain impairments to enable better characterization of the deficits observed in this population, which will enhance diagnosis and improve remediation.     

Effects of dose and period of neonatal alcohol exposure on the context preexposure facilitation effect

Background: Alcohol exposure in the rat on postnatal days (PD) 4 to 9 is known to partially damage the hippocampus and to impair hippocampus‐dependent behavioral tasks. We previously reported that PD4 to 9 alcohol exposure eliminated the context preexposure facilitation effect (CPFE) in juvenile rats, a hippocampus‐dependent variant of contextual fear conditioning. In the CPFE, context exposure and immediate shock occur on successive occasions and this produces conditioned freezing relative to a control group that is not preexposed to the training context. Here, we extend our earlier findings by examining the effects of neonatal alcohol administered at multiple doses or over different neonatal exposure periods. Method: Rat pups (male and female) were exposed to a single binge dose of alcohol at one of 3 doses (2.75, 4.00, or 5.25 g/kg/d) over PD4 to 9 (Experiment 1) or to 5.25 g over PD4 to 6 or PD7 to 9 (Experiment 2). Sham‐intubated (SI) and undisturbed (UD) rats served as controls. On PD31, rats were preexposed to either the training context (Pre) or an alternate context (No‐Pre). On PD32, rats received an immediate unsignaled footshock (1.5 mA, 2 seconds) in the training context. Finally, on PD33, all rats were returned to the training context and tested for contextual freezing over a 5‐minute period. Results: Undisturbed‐ and SI‐Pre rats showed the CPFE, i.e., context preexposure facilitated contextual conditioning, relative to their No‐Pre counterparts. The immediate shock deficit was present in all No‐Pre groups, regardless of previous alcohol exposure. In Experiment 1, blood alcohol level was negatively correlated with contextual freezing. Group 2.75 g‐Pre did not differ from controls. Group 4.00 g‐Pre froze significantly less than Groups UD‐ and SI‐Pre but more than Group 5.25‐Pre, which showed the immediate shock deficit. In Experiment 2, alcohol exposure limited to PD7 to 9, but not PD4 to 6, disrupted the CPFE. Conclusions: This is the first demonstration of dose‐related impairment on a hippocampus‐dependent task produced by neonatal alcohol exposure in the rat. Exposure period effects support previous studies of alcohol and spatial learning. The CPFE is a more sensitive behavioral task that can be used to elucidate developmental alcohol‐induced deficits over a range of alcohol doses that are more relevant to human exposure levels.     

Effect of duration of alcohol consumption on calcium and bone metabolism during pregnancy in the rat

BACKGROUND: Little is known about the consequences of drinking during pregnancy for the long-term health of the mother. Alcohol (ethanol) has been shown to disrupt calcium (Ca) homeostasis and is known to have deleterious effects on bone. During pregnancy, bone turnover is increased to maintain Ca homeostasis; therefore, pregnancy may be a time of life when maternal bone is particularly susceptible to the effects of ethanol. This study investigated the effect of duration of ethanol consumption on Ca homeostasis and bone during pregnancy in the rat. METHODS: Rats were fed ethanol (36% ethanol-derived calories) in liquid diets for 3 (21 days gestation only) or 6 (3 weeks before and throughout 21 days gestation) weeks. Maternal blood was analyzed for Ca (total and ionized Ca [iCa]), the Ca-regulating hormones (parathyroid hormone [PTH], 1,25(OH)2D, calcitonin), and osteocalcin (a marker for bone formation). Bone was analyzed for ash (mineral) content. RESULTS: Dams consuming ethanol (E dams) had decreased blood Ca levels (total and iCa) at both 3 and 6 weeks, but iCa was lower in E dams after 6 compared with 3 weeks. Importantly, ethanol seemed to interfere with the normal compensatory response to these decreased Ca levels. In contrast to pair-fed controls, serum PTH levels actually were decreased, 1,25(OH)2D levels failed to increase, and calcitonin levels were increased in ethanol-consuming dams, regardless of duration. Moreover, ethanol decreased bone formation, as indicated by serum osteocalcin levels, after both 3 and 6 weeks consumption, and after 6 weeks, the ash content of bone also was decreased. In addition, a relationship was found between the blood alcohol concentration (BAC) and some measures of Ca and bone metabolism. Serum 1,25(OH)2D and osteocalcin levels varied inversely, whereas serum calcitonin varied directly with BAC, suggesting that time of sampling after drinking may be an important variable for interpreting ethanol's effects on Ca and bone metabolism. In all rats, serum osteocalcin levels varied directly with PTH and 1,25(OH)2D levels. CONCLUSIONS: Ethanol consumption during pregnancy impaired Ca homeostasis in the dam, regardless of duration of consumption, and resulted in decreased bone formation and ash content of bone. Significant relationships among the Ca-regulating hormones, BAC, and osteocalcin support the hypothesis that ethanol's effects on the Ca-regulating hormones may mediate some of its effects on bone.     

Effect of duration of maternal alcohol consumption on calcium metabolism and bone in the fetal rat

BACKGROUND: Prenatal ethanol exposure can retard fetal growth and delay skeletal development. Ethanol also impairs maternal calcium (Ca) homeostasis and this impairment could mediate some of ethanol's effects on the fetal skeleton. Our previous studies suggest that the duration of maternal ethanol consumption may be an important factor for determining the severity of ethanol's effects on Ca homeostasis and fetal skeletal development. The purpose of this study was, therefore, to determine the effect of the duration of maternal ethanol consumption on fetal growth and skeletal development and to investigate the possibility that ethanol's effects may be related to perturbations in fetal/maternal Ca homeostasis. METHODS: Rats were fed ethanol (36% ethanol-derived calories) in liquid diets for 3 weeks (days 1-21 of gestation) or 6 weeks (for 3 weeks before and throughout gestation). Fetuses were collected on day 21 of gestation, and body weight and length were measured. Fetuses were stained to determine the degree of skeletal ossification, and fetal blood was analyzed for ethanol, Ca (total and ionic Ca), albumin, parathyroid hormone (PTH), and osteocalcin. RESULTS: Maternal ethanol consumption decreased fetal growth and delayed fetal skeletal development. Although there was a trend for fetal body length and serum osteocalcin levels to be more severely affected with an increased duration of maternal ethanol consumption, duration had no effect on fetal body weight or skeletal ossification. Fetal Ca homeostasis was also affected by ethanol exposure, with fetal hypocalcemia apparent after 6 weeks of maternal ethanol intake. A significant inverse relationship was found between fetal blood Ca levels and blood alcohol concentration (BAC), suggesting that the severity of the fetal hypocalcemia may have been related to differences in fetal BAC, rather than duration of maternal ethanol intake. Fetal serum PTH levels did not differ significantly among treatment groups indicating that the fetal hypocalcemia was not caused by a decrease in PTH levels. CONCLUSIONS: Prenatal ethanol exposure impaired Ca homeostasis and skeletal development in the fetal rat. The severity of ethanol's effects was only marginally dependent on the duration of maternal ethanol consumption per se and seemed to be more related to the relative exposure of the fetus to ethanol (fetal BAC). The relationship between the ethanol-induced fetal hypocalcemia and skeletal effects remains to be determined.     

Effects of Ethanol, Chlordiazepoxide, and MK-801 on Performance in the Elevated-Plus Maze and on Locomotor Activity

The effects of ethanol, chlordiazepoxide, and MK-801 on performance in the elevated-plus maze and on activity measured in a circular activity monitor were compared in Sprague-Dawley rats to determine whether these effects of ethanol could be explained by its action on either GABA_A or NMDA receptors. Both ethanol and chlordiarepoxide produced an increase in the time spent in the open arms of the elevated-plus maze and in the ratio of open arm to total arm entries, indicative of an anxiolytic action of these drugs. MK-801 did not alter either the time spent in the open arms or the ratio of open to total arm entries. Chlordiazepoxide and MK-801 produced an increase in total arm entries that suggested that these compounds were increasing locomotor activity. Ethanol also increased total arm entries, but the effect was not statistically reliable. Following habituation to an activity monitor, neither ethanol nor chlordiazepoxide increased activity in this task, whereas MK-801 produced a robust increase in locomotion. Additionally, neither ethanol nor chlordiazepoxide blocked the MK-801-induced locomotor stimulation. The latter finding suggests that the effects of ethanol on GABAA receptors was not Mocking an increased activity level produced by its antagonism of NMDA. Additionally, these results indicate that the anxiolytic and locomotor action of ethanol in rats parallel the effects of a benzodiazepine and not those of an NMDA antagonist. Finally, these results suggest that the consequence of ethanol's antagonism of NMDA receptor function is more restricted than that produced by MK-801.     

Fetal alcohol exposure alters GAP-43 phosphorylation and protein kinase C responses to contextual fear conditioning in the hippocampus of adult rat offspring

Abstract : Background: The growth‐ and plasticity‐associated protein GAP‐43 plays a significant role in the establishment and remodeling of neuronal connections. We have previously shown that GAP‐43 levels, protein kinase C (PKC) activity, and GAP‐43 phosphorylation increase during contextual fear conditioning and that fetal alcohol exposure (FAE) decreases PKC activity and GAP‐43 phosphorylation in the hippocampus of adult offspring. Drawing on these observations, we hypothesized that FAE manifests its cognitive impairment by disrupting PKC activation and membrane translocation, thereby decreasing GAP‐43 phosphorylation and function. Methods: Three groups of pregnant rat dams (FAE and two control diet groups) were placed on different diet regimens. Offspring from each of these groups were placed into each of four test groups, a contextual fear conditioned (CFC) group, a naïve unhandled group, and two nonlearning stress control groups. Hippocampi were dissected, homogenized, and used to prepare a cytosolic and a membrane fraction. These fractions were probed for total GAP‐43, PKC‐phosphorylated GAP‐43, and several PKC subtypes. PKC activity also was measured in total homogenates. Results: Compared with both control diet groups, FAE animals showed a deficit in the activation of PKC in the hippocampus at 24 hr but not at 1.5 hr after CFC. Likewise, we found that the amount of GAP‐43 and its phosphorylation were decreased 24 hr after CFC in FAE rats but not at early times after training. Analysis of the translocation of various PKC isoforms revealed that FAE animals had decreased levels of membrane‐bound PKC β₂ and PKC ε 24 hr after CFC. Conclusions: Considering the role of PKC activation and GAP‐43 phosphorylation in synaptic plasticity, our results suggest that deficient translocation of PKC β₂ and PKC ε in the hippocampus may mediate the electrophysiological and behavioral deficits observed in fetal alcohol exposed animals.     

Alteration of [3H]MK-801 Binding Associated with the JV-Methyl-d-Aspartate Receptor Complex by Acute Ethanol in Rat Cortex and Hippocampus In Vitro

We investigated the effect of ethanol on specific binding of [³H]MK-801 to the intrachannel phencyclidine (PCP) receptor site, as an index of change in the functional response of the N-methyl-d -Aspartate (NMDA)-associated ion channel. Saturation binding experiments were performed on synaptic membrane homogenates from adult rat cortex and hippocampus. [³H]MK-801 binding assays were conducted under conditions of basal, 10 μm glutamate, or 10 μm glutamate + 30 μm d -serine, with and without 50 or 100 mm ethanol. Association experiments of [³H]MK-801 binding (5 nm) were conducted under conditions of 0 or 10 μm glutamate, with varying concentrations of glycine (0.01, 0.10, and 10 μm) with and without 100 mm ethanol. Ethanol (50 and 100 mm ) significantly decreased the percentage of high-affinity (open-channel state) MK-801 receptors with a concomitant increase in percentage of low-affinity receptors, but did not change high- and low-affinity constants of the two binding states. An ethanol-induced increase in the closed-channel receptor density in basal and activated conditions was suggested by the saturation experiments. Association experiments further explained this finding, in that ethanol (100 mm ) significantly decreased fast component (open-channel) [³H]MK-801 binding in conditions of glycine (0.01–10 μm ) only and activated conditions of glutamate + glycine (0.01–0.10 μm ). However, the observed fast and slow kinetic rate constants of [³H]MK-801 binding, as well as total specific binding (fast + slow components), were not altered. Thus, ethanol seems to act as a noncompetitive antagonist upon the gating mechanism of, and ligand access to, the NMDA-coupled ion channel. These findings support previous observations of ethanol selectively reducing NMDA-activated calcium influx, and reducing the frequency and duration of ion channel opening in electrophysiological studies. Similar to previous reports on NMDA-stimulated calcium influx and [³H]MK-801 binding, glycine (at the maximal concentration of 10 μm ), in the presence of 10 μm glutamate, was found to reverse ethanol inhibition of fast component binding.     

Effects of acute ethanol exposure on regulatory mechanisms of Bcl-2-associated apoptosis promoter, bad, in neonatal rat cerebellum: differential effects during vulnerable and resistant developmental periods

BACKGROUND: Prenatal alcohol exposure produces anatomical and behavioral abnormalities associated with fetal alcohol syndrome (FAS). Animal FAS models have demonstrated temporal windows of vulnerability in the developing cerebellum, with substantial ethanol (EtOH)-mediated apoptotic activation during these periods. In rodents, the cerebellum is most sensitive to EtOH on postnatal days 4 to 6 (P4 to P6). At slightly later ages (P7 and later), this region is less vulnerable to EtOH. The present study investigated EtOH effects on mechanisms related to activities of Bad, a proapoptotic member of the Bcl-2 gene family, to further characterize processes underlying these disparate EtOH sensitivities. In healthy cells, Bad is retained in the cytosol by association with 14-3-3, a primarily cytosolic protein. Bad promotes apoptosis by disassociating from 14-3-3 and sequestering Bcl-xL through heterodimerization. This dimerization prevents the neutralizing association of Bcl-xL with Bax, freeing Bax to perform in a prodeath manner. Caspase-dependent cleavage of Bad to a 15-kDa fragment increases its proapoptogenic capacity. METHODS: Two hours following EtOH exposure of P4 and P7 animals via inhalation, we determined how exposure affects intracellular localization and proteolytic cleavage of Bad and expression of cerebellar 14-3-3, using subcellular fractionation and Western blot techniques. Ethanol effects on interactions between Bad and 14-3-3 or Bcl-xL at the more vulnerable and less vulnerable ages were determined using an enzyme-linked immunosorbent assay-based technique to detect native protein-protein interactions. RESULTS: At P4, EtOH increased mitochondrial localization of Bad, expression of a 15-kDa fragment recognized by Bad antibody, and formation of Bad:Bcl-xL complexes. At that more vulnerable age, EtOH also decreased formation of Bad:14-3-3 complexes. At P7, EtOH increased Bad:14-3-3 complexes and reduced Bad:Bcl-xL complexes. Cytosolic 14-3-3 remained unchanged by EtOH at P4 and P7. CONCLUSIONS: Ethanol-induced alterations of Bad-related mechanisms at P4 favor a prodeath response. EtOH does not influence these same mechanisms in a manner that promotes cell death at P7. Divergent Bad-related responses at these 2 developmental ages likely contribute to their differential EtOH vulnerability.     

Pretreatment with hydromorphone, a mu-opioid agonist, does not alter the acute behavioral and physiological effects of ethanol in humans

BACKGROUND: Endogenous opioid systems are thought to mediate at least some of the behavioral effects of ethanol. Opioid antagonists, like naloxone and naltrexone, decrease ethanol self-administration under a variety of conditions in different species of laboratory animals (e.g., rodents and nonhuman primates). Opioid agonists, like morphine, also alter ethanol consumption. However, the dose-response functions for opioid agonists are complex in that low doses increase ethanol self-administration, whereas moderate to high doses decrease ethanol self-administration. The results of prospective human laboratory studies that assessed the behavioral effects of ethanol after pretreatment with an opioid antagonist are mixed. The aim of the present study was to assess the acute subject-rated effects of ethanol (0, 0.5, and 1 g/kg) after pretreatment with hydromorphone, a mu-opioid agonist. METHODS: In the present experiment, the acute subject-rated, performance-impairing, and physiological effects of ethanol (0, 0.5, and 1 g/kg) were examined after pretreatment with hydromorphone (0, 1, and 2 mg), a mu-opioid agonist, in nine healthy volunteers. Volunteers received one of the nine possible ethanolhydromorphone combinations during each of nine experimental sessions. RESULTS: Ethanol produced prototypical subject-rated drug effects (e.g., dose dependently increased ratings of "high"), impaired performance, and increased heart rate. Hydromorphone pretreatment generally did not significantly alter the subject-rated, performance-impairing, or physiological effects of ethanol. CONCLUSIONS: The results of the present experiment suggest that hydromorphone pretreatment does not significantly affect the subject-rated effects of ethanol. Future human laboratory studies should test higher doses of hydromorphone. Future studies also might use more sophisticated behavioral procedures like self-administration, or perhaps drug discrimination, to determine if opioid agonists can modulate the behavioral effects of ethanol in humans.     

All three trimester binge alcohol exposure causes fetal cerebellar purkinje cell loss in the presence of maternal hypercapnea, acidemia, and normoxemia: ovine model

BACKGROUND: The third trimester equivalent has been identified, both in rat and sheep models, as a period of cerebellar vulnerability to alcohol-mediated injury. We wished to determine whether alcohol exposure throughout gestation results in greater injury compared with exposure limited to the third trimester equivalent. While this question has previously been addressed in the rat model, where the third trimester equivalent occurs postnatally, it has not yet been addressed in an animal model where all 3 trimester equivalents occur prenatally, as in the ovine. We also wished to correlate cerebellar Purkinje cell loss to alcohol-mediated alterations in maternal arterial pH and blood gases as these responses might be important mechanistically in mediating the damage. METHODS: Three groups of pregnant sheep were used: an untreated normal control group, a saline control group, and an alcohol group (1.75 g/kg of the body weight). The alcohol exposure regimen was designed to mimic a human binge pattern; alcohol was administered intravenously on 3 consecutive days, followed by 4 days without alcohol, beginning day 4 of gestation, continuing to the end of the third trimester equivalent of human brain growth, day 132 of gestation. RESULTS: All 3 trimester alcohol-exposed fetal brains exhibited significant deficits in cerebellar volume and Purkinje cell number compared with those of control subjects. We did not detect a difference in the reduction of Purkinje cell number when comparing between all 3 trimester and third trimester alcohol exposure studies. The neuronal loss was accompanied by maternal hypercapnea, acidemia, and normoxemia. CONCLUSIONS: These findings demonstrate in an ovine model where all 3 trimester equivalent of brain growth occur in utero that the fetal cerebellar Purkinje cells are more sensitive to the timing of alcohol exposure and less so to the duration of exposure. Decreases in maternal P(a)O(2) were not detected, suggesting that maternal hypoxia does not play a role in fetal Purkinje cell loss. And finally, we conclude that alcohol-induced changes in maternal arterial pH may play a role in alcohol-mediated developmental brain injury.     

Effect of acute alcohol treatment on the release of ACTH, corticosterone, and pro-inflammatory cytokines in response to endotoxin

This study investigated the effects of acute alcohol pretreatment on endotoxin lipopolysaccharide (LPS)-induced release of ACTH, corticosterone, and pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in plasma and at various tissues sites. Specifically, we wanted to determine whether alcohol pretreatment would alter the ACTH, corticosterone, and cytokine responses to LPS, and whether the alcohol-induced changes in ACTH/corticosterone secretory rates of endotoxemic rats were accompanied by similar changes in cytokine production. Alcohol, 3.0 g/kg, intragastric (i.g.), was administered 3 hr before LPS treatment [1.0 or 5.0 microg/kg, intravenous (i.v.)], and ACTH, corticosterone, and cytokines levels were measured over a 4 hr post LPS treatment. In intact rats, the alcohol-induced plasma ACTH and corticosterone responses had returned to basal levels by the time of LPS injection, and alcohol pretreatment increased the corticosterone but not the ACTH response after LPS treatment. In contrast, in adrenalectomized corticosterone-replaced animals, the alcohol-induced ACTH response was still elevated at the time of LPS injection. However, the overall ACTH response of rats pretreated with the vehicle or alcohol was statistically similar. As expected, LPS also significantly stimulated both TNF-alpha and IL-6 release into the general circulation. The IL-6, but not the TNF-alpha, response was inhibited by alcohol pretreatment in intact rats, a phenomenon that was not present in adrenalectomized animals. Finally, we showed that LPS also augmented the TNF-alpha and/or IL-6 content of the pituitary, adrenal glands, and spleen, and that these responses were not altered by alcohol pretreatment. On the basis of these results, we concluded that acute alcohol treatment increased LPS-induced corticosterone response, while it blunted the IL-6 response. LPS also significantly elevated pituitary, adrenal, and splenic contents of TNF-alpha and IL-6, and alcohol did not influence these changes.