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Brain regional gamma-aminobutyric acid type A (GABAA) receptor subunit mRNA expression was studied in ethanol-preferring AA (Alko, Alcohol) rats after moderate ethanol drinking for up to 2 years of age. In situ hybridization with oligonucleotide probes specific for 13 different subunits was used with coronal cryostat sections of the brains. Selective alterations were observed by ethanol exposure and/or aging in signals for several subunits. Most interestingly, the putative highly ethanol-sensitive alpha4 and beta3 subunit mRNAs were significantly decreased in several brain regions. The age-related alterations in alpha4 subunit expression were parallel to those caused by lifelong ethanol drinking, whereas aging had no significant effect on beta3 subunit expression. The results suggest that prolonged ethanol consumption leading to blood concentrations of about 10 mM may downregulate the mRNA expression of selected GABAA receptor subunits and that aging might have partly similar effects.

In vitro ligand binding studies were used to compare GABA/benzodiazepine receptor/chloride ionophore complexes in various brain regions of ethanol-sensitive ANT and ethanol-insensitive AT rats. In naive rats, there were several, but fairly small line differences in the binding parameters of [3H]muscimol and [3H]flunitrazepam to cerebral cortical, cerebellar or hippocampal membranes washed with or without a detergent. GABA-stimulation of flunitrazepam binding in the cerebral cortex membranes was slightly greater in the AT than ANT rats. In detergent solubilized receptors, the GABA-stimulation of flunitrazepam binding emerged only in the presence of ethanol in most AT samples, whereas the GABA-stimulation was always observed in ANT samples and ethanol had no further effect. Pharmacological characteristics of [3H]t-butylbicycloorthobenzoate binding displaceable by picrotoxin were similar in both lines. Chronic ethanol administration tended to increase the number of these binding sites in the cerebral cortex of AT rats and to decrease them in the ANT rats. Although many differences between the lines were observed, our results indicate that the ethanol-sensitivity difference between the AT and ANT rat lines cannot be explained by enhanced function of the GABA/benzodiazepine receptor/chloride ionophore complex as far as this is revealed by in vitro binding studies. It remains to be studied whether these lines differ in presynaptic GABAergic mechanisms or in the actual function of the postsynaptic chloride channels before the role of GABA can be more accurately assessed in this genetic model for ethanol-induced motor impairment.

The Collaborative Initiative on Fetal Alcohol Spectrum Disorders (CIFASD) was created in 2003 to further understanding of fetal alcohol spectrum disorders. Clinical and basic science projects collect data across multiple sites using standardized methodology. This article describes the methodology being used by the clinical projects that pertain to assessment of children and adolescents. Domains being addressed are dysmorphology, neurobehavior, 3-D facial imaging, and brain imaging.

A microdialysis system for measuring the ethanol concentration curve in the nucleus accumbens of the rat brain was developed and tested in three different rat lines (AA, ANA, and Wistar) after an intraperitoneal (IP) and an intragastric (IG) dose of 1.0 g/kg ethanol. The flow rate of the modified Ringer solution was set at 5 microliters/min; samples were taken every minute after ethanol administration and analyzed with headspace gas chromatography. After IP administration, the brain ethanol levels rose much more rapidly than tail blood ethanol levels in the same animals. The maximum brain ethanol level after IG administration was lower and occurred later, and were similar to the tail blood levels in AA and ANA rats. No clear difference between the lines was found after IP administration but there was some indication that ANA rats may absorb alcohol after IG intubation faster than AA or Wistar rats.

We examined whether an early-life event - ethanol exposure in the initial stages of pregnancy - affected offspring brain structure, energy metabolism, and body composition in later life. Consumption of 10% (v/v) ethanol by inbred C57BL/6J female mice from 0.5 to 8.5 days post coitum was used to model alcohol exposure during the first 3-4 weeks of gestation in humans, when pregnancy is not typically recognized. At adolescence (postnatal day [P] 28) and adulthood (P64), the brains of male offspring were scanned ex vivo using ultra-high field (16.4 T) magnetic resonance imaging and diffusion tensor imaging. Energy metabolism and body composition were measured in adulthood by indirect calorimetry and dual-energy X-ray absorptiometry (DXA), respectively. Ethanol exposure had no substantial impact on white matter organization in the anterior commissure, corpus callosum, hippocampal commissure, internal capsule, optic tract, or thalamus. Whole brain volume and the volumes of the neocortex, cerebellum, and caudate putamen were also unaffected. Subtle, but non-significant, effects were observed on the hippocampus and the hypothalamus in adult ethanol-exposed male offspring. Ethanol exposure was additionally associated with a trend toward decreased oxygen consumption, carbon dioxide production, and reduced daily energy expenditure, as well as significantly increased adiposity, albeit with normal body weight and food intake, in adult male offspring. In summary, ethanol exposure restricted to early gestation had subtle long-term effects on the structure of specific brain regions in male offspring. The sensitivity of the hippocampus to ethanol-induced damage is reminiscent of that reported by other studies - despite differences in the level, timing, and duration of exposure - and likely contributes to the cognitive impairment that characteristically results from prenatal ethanol exposure. The hypothalamus plays an important role in regulating metabolism and energy homeostasis. Our finding of altered daily energy expenditure and adiposity in adult ethanol-exposed males is consistent with the idea that central nervous system abnormalities also underpin some of the metabolic phenotypes associated with ethanol exposure in pregnancy.

The effects of lorazepam and sodium barbital on GABAA receptor function were evaluated in rat lines selected for differential sensitivity to the motor-impairing effects of ethanol [alcohol-insensitive (AT) and alcohol-sensitive (ANT) lines]. The effect of GABA on [3H]flunitrazepam and [3H]Ro 15-4513 binding and the effects of lorazepam and sodium barbital on [3H]muscimol binding were measured in cerebellar, cerebrocortical, and hippocampal membrane preparations. The effects of lorazepam and sodium barbital on muscimol-stimulated 36Cl- influx were measured using membrane vesicle suspensions from the same brain areas. No differences were found between the rat lines in the GABA-induced stimulation of [3H]flunitrazepam binding or in the lorazepam and sodium barbital-induced enhancement of either [3]muscimol binding or muscimol-stimulated 36Cl- flux. Neither was desensitization of the 36Cl- flux affected differently by ethanol, lorazepam, and barbital in vitro between the lines. The affinity of cerebellar diazepam-insensitive (DZ-IS) [3H]Ro 15-4513-binding sites for benzodiazepine agonists has been shown to be much greater in the ANT than the AT rats. In the present study, at 0 degrees C, GABA decreased [3H]Ro 15-4513 binding in the presence of diazepam only in ANT rats. Similarly, GABA decreased this binding at 37 degrees C in ANT rats having a high affinity for diazepam, whereas it enhanced the binding in all AT samples in those ANT samples where diazepam had a poor AT-like affinity. The decrease in binding in ANT samples is apparently caused by the enhancing effect of GABA on diazepam binding to DZ-IS [3H]Ro 15-4513-binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

N-Methyl-D-aspartate (NMDA) receptors are sensitive to ethanol at concentrations relevant to intoxication. To ascertain possible involvement of NMDA receptors in differential ethanol sensitivity between alcohol-sensitive ANT (alcohol-nontolerant) and alcohol-insensitive AT (alcohol-tolerant) rat lines, characterization of a noncompetitive NMDA antagonist [3H]MK-801 binding to brain membranes was carried out. Saturation analyses of [3H]MK-801 binding to cerebrocortical, hippocampal, and cerebellar synaptosomal membranes revealed no statistically significant differences in either the affinity constant (Kd) or binding site density (Bmax) between the rat lines. Autoradiographic analysis of [3H]MK-801 binding to ANT and AT brain sections revealed a regionally heterogenous distribution of binding, without any detectable differences between the AT and ANT sections whether these were prepared from the brains of acutely ethanol-treated or nontreated animals. Glutamate, glycine, or the two in combination greatly increased [3H]MK-801 binding to brain membranes. In extensively washed crude cerebrocortical membranes, the maximal effect (Emax), but not potency (EC50) of glycine to increase [3H]MK-801 was slightly greater (p < 0.01) in the ANT than AT rats. The effects of glutamate or glutamate in the presence of saturating concentration of glycine (30 microM) were not significantly different between the two lines. Association parameters (t1/2 and Beq values) of [3H]MK-801 to its cortical binding sites were also similar. These results do not indicate any clear qualitative difference in [3H]MK-801 binding to NMDA receptors or in its modulation by glutamate and glycine between the ANT and AT rat lines.

Magnetoencephalography (MEG) is a noninvasive method of studying magnetic fields from outside the skull that are generated by at least partially synchronized neuronal populations in the brain. The advantage of MEG over electroencephalography (EEG) is the transparency of the skull, scalp, and brain tissue to the magnetic fields, which facilitates easy localization of the cortical activity. In MEG, alcohol increased the relative power of the alpha rhythm and reduced the relative power of beta activity in parieto-occipital regions. In contrast, no changes were observed in EEG, indicating that these methods differently detect alcohol's action on the cortex. Furthermore, MEG and EEG also differently detected the effects of alcohol on cognition. Alcohol reduced magnetic and electric auditory N1 and mismatch negativity amplitudes. P3a amplitudes were also reduced in EEG but not in MEG, suggesting that different cortical areas are responsible for alcohol's action on involuntary attention. Transcranial magnetic stimulation (TMS) provides new possibilities for studying localized changes in the electrical properties of the human cortex, especially when combined with EEG. Different cortical areas can be stimulated and the subsequent brain activity can be measured, yielding information about cortical excitability and connectivity. Alcohol modulates EEG responses evoked by motor-cortex TMS, the effects being largest at the right prefrontal cortex (assessed by minimum-norm estimation), meaning that alcohol changed the functional connectivity between motor and prefrontal cortices. Furthermore, alcohol decreases amplitudes of EEG responses after the left prefrontal stimulation of anterior parts of the cortex, which may be associated with the decrease of prefrontal cortical excitability. Taken together, MEG and TMS combined with EEG provide new insight into the focal actions of alcohol on the cortex with a temporal resolution of milliseconds, giving information different from that given by other brain imaging modalities.