Postnatal ethanol exposure blunts upregulation of GABAA receptor currents in Purkinje neurons.

Recently, we found that early postnatal ethanol exposure inhibits the maturation of GABAA receptors (GABAARs) in developing medial septum/diagonal band (MS/DB) neurons, suggesting that these receptors may represent a target for ethanol related to fetal alcohol syndrome (FAS). To determine whether GABAARs on other neurons are also sensitive to a postnatal ethanol insult, postnatal day (PD) 4-9, rat pups were artificially reared and exposed to ethanol (4.5 g kg-1 day-1, 10.2% v/v). The pharmacological profile of acutely dissociated cerebellar Purkinje cell GABAARs from untreated, artificially reared controls and ethanol-treated animals was examined with conventional whole-cell patch clamp recordings during PD 12-16 (juveniles) and PD 25-35 (young adults). For untreated animals, GABA (0.3-100 microM) consistently induced inward Cl- currents in a concentration-dependent manner showing an age-related increase in maximum response without change in EC50 or slope value. Acute ethanol (100 mM) consistently inhibited 3 microM GABA currents (10-20%); positive modulators, pentobarbital (10 microM), midazolam (1 microM) and loreclezole (10 microM), consistently potentiated; the negative modulator, Zn2+ (30 microM), inhibited GABA currents across both juvenile and young adult groups. Loreclezole potentiation increased while Zn2+ inhibition decreased with age in untreated Purkinje neurons. Postnatal ethanol exposure (PD 4-9) decreased GABAAR maximum current density in young adult Purkinje cells but not in juvenile neurons. However, sensitivity to allosteric modulators did not change after ethanol. These data are consistent with the hypothesis that postnatal ethanol exposure during the brain growth spurt can disturb GABAAR development across the brain, although the mechanism(s) underlying this action remains to be determined.

DNA fragmentation during exposure of rat cerebella to ethanol under hypoxia imposed in vitro.

To gain a better understanding into the mechanisms of damage incurred by neurons in periods following heavy alcohol exposure during development, we used an in vitro system to monitor the effects of alcohol and hypoxia on cell survival and DNA integrity. Samples representing the first few hours of exposure to alcohol and hypoxia were compared to those resulting from hypoxia alone. Measurements were taken from cell counts using Trypan blue exclusion and TUNEL assays as well as digital scans of the ethidium bromide fluorescence of genomic DNA isolated from the treated tissue. We found that DNA degradation from hypoxia was accelerated by several hours in the presence of 100 mM ethanol. This result depended on age, with adult animals (>8 months) having a similar response to 4-day postnatal animals, while the effect on 10-day postnatal animals and those of intermediate age (45 days postnatal) was increasingly delayed. Different methods of inducing the processive degradation of DNA produced laddering typical of apoptosis, a biphasic degradative process, or patterns usually associated with necrosis.

Development of GABAA receptors on medial septum/diagonal band (MS/DB) neurons after postnatal ethanol exposure.

The impact of 'binge-like' ethanol exposure on postnatal days (PD) 4-9 was examined on development of gamma-aminobutyric acid type A receptors (GABAAR) during the first month of life in the rat. Whole-cell patch-clamp recordings in acutely isolated medial septum/diagonal band (MS/DB) neurons were used to define effects of rapidly applied ethanol and other allosteric modulators on bicuculline-sensitive GABA currents. Three age groups were examined including 'pups' (PD 4-10), 'juveniles' (PD 11-16) and 'young adults' (PD 25-35). In untreated neurons, maximum responses to GABA and the apparent GABA EC50 increased approximately 2-fold during the first month of life. Potentiation of GABA responses by pentobarbital, midazolam, and loreclezole all increased with age, while Zn2+ inhibition declined. Initial inhibition by ethanol switched to potentiation of GABA responses during this time. In vivo, binge-like ethanol treatment (4.5 g kg-1 day-1 divided into two doses, 2 h apart on PD 4-9) reduced both the GABA maximal response and GABA EC50 measured on PD 11-16. These measures returned to control levels by PD 25-35. After binge-like postnatal ethanol exposure, age-dependent loss of Zn2+ inhibition of GABA responses was increased, while potentiating actions of in vitro ethanol were blocked. GABAAR modulation by other drugs was unaffected. These data suggest that early postnatal ethanol exposure disrupts the expected developmental pattern of GABAAR function in MS/DB neurons, an action that could contribute to neurobehavioral deficits associated with the fetal alcohol syndrome. Whether these changes are due to cellular damage, delayed gene expression or post-translational modification needs to be determined.

Electrophysiological characterization of cerebellar neurons from adult rats exposed to ethanol during development.

The purpose of this study was to investigate the spontaneous activity of mature rat cerebellar neurons that had been exposed to ethanol (EtOH) during postnatal days 4 to 10, which corresponds to the third trimester in humans. Newborn Sprague-Dawley rats were implanted with gastric feeding tubes and were artificially reared from postnatal days 4 to 10 with two different diets. The experimental group received 4.5 g/kg/day of EtOH delivered in a milk solution. Controls received similar feeding with an isocaloric supplement replacing the EtOH. Electrophysiological evaluations were performed after an EtOH-free rearing period. Although lobules IX and X of the cerebellar vermis appeared morphologically smaller in the animals neonatally exposed to EtOH, compared with controls, extracellular recordings from both Purkinje cells and Golgi interneurons in adult rats showed no differences in spontaneous activity or firing pattern between the control and EtOH-exposed animals. Similarly, excitations and inhibitions of Purkinje neuron activity evoked by parallel pathway stimulation appeared unaffected by the developmental EtOH exposure. However, we did observe a significant decrease in the proportion of Purkinje neurons generating complex spike bursts in the group exposed to EtOH neonatally. These data suggest that, although fewer Purkinje neurons may survive the brain growth spurt if exposed to EtOH during this critical period of development, those that do survive appear to function normally. The observed abnormality in complex spike production may result from EtOH effects on developing neurons in the inferior olive that give rise to the climbing fibers that cause this bursting pattern in Purkinje neurons.

Developing rat Purkinje cells are more vulnerable to alcohol-induced depletion during differentiation than during neurogenesis.

This study compared the extent of cerebellar Purkinje cell depletion induced by administering alcohol to rats during two temporally distinct periods of Purkinje cell development--neurogenesis and early differentiation. One group received alcohol (5 g/kg/day) during and shortly after Purkinje cell neurogenesis (gestational days 13-18) via oral intubation of pregnant dams. A second group received alcohol (2.5 g/kg/day) during early Purkinje cell differentiation (postnatal days 4-9) via artificial rearing of pups. The two alcohol treatment protocols were designed to match the cyclic daily blood alcohol profiles of the two groups as closely as possible. Pair-fed intubated controls, artificially reared gastrostomy controls, and normally reared ad lib/suckle controls were also evaluated. Mean peak blood alcohol concentrations (BACs) were 266 mg/dl for the intubated pregnant dams and 205 mg/dl for the pups exposed postnatally. Purkinje cell profiles were counted from single, 2-microns-thick midsaggital sections on postnatal day 10. Alcohol exposure during neurogenesis resulted in no significant change in Purkinje cell profile densities. Exposure during differentiation produced significant reductions in Purkinje cell profile densities, predominantly in the early maturing regions of the vermis (lobules I-IV and IX-X). These results indicate that Purkinje cells are more vulnerable to alcohol-induced population depletion during differentiation than during neurogenesis.

Transient cortical astrogliosis induced by alcohol exposure during the neonatal brain growth spurt in rats.

The astrocyte response to central nervous system injury induced by neonatal alcohol exposure was evaluated using radioimmunoassay and immunocytochemistry of glial fibrillary acidic protein (GFAP). Rat pups were exposed to alcohol on postnatal days 4 through 9 via artificial rearing. Alcohol solutions were administered as one of the following treatments: 10.2% (v/v) in two feedings (4.5 g/kg/day), 5.1% (v/v) in four feedings (4.5 g/kg/day), or 2.5% (v/v) in 12 feedings (6.6 g/kg/day), producing mean blood alcohol concentrations (BACs) of approximately 300, 180, and 50 mg/dl, respectively. Littermates were included as gastrostomy controls (GC) and suckle controls (SC). On postnatal day 10, GFAP concentration increased as a function of BAC, and the 10.2% alcohol treatment significantly and dramatically increased GFAP in the cortex (325% of SC). GFAP immunocytochemistry revealed frequent loci of heavily labeled reactive astrocytes surrounding small cortical blood vessels in the 10.2% group. In addition, a generalized increase in GFAP immunoreactivity was present in the deep layers of the cortex in all alcohol groups, marked by astrocytic fibrillary hypertrophy and increased density. Three-dimensional counts in layer V of parietal cortex using confocal microscopy indicated that the density of GFAP-labeled astrocytes of the 10.2% group was twice that of controls. The layer V gliosis was observable even at low BACs, while gliosis around the vasculature occurred only with high BACs. By postnatal day 15, the astroglial effects were no longer evident. These transient astroglial reactions likely constitute an important aspect of cortical pathophysiology resulting from binge alcohol exposure during the brain growth spurt of the third trimester equivalent.

Prenatal ethanol exposure during the last third of gestation in rat reduces hippocampal NMDA agonist binding site density in 45-day-old offspring.

The effect of ethanol exposure during different periods of prenatal or postnatal development on hippocampal N-methyl-D-aspartate (NMDA) receptor binding was studied in rat. Fetal rat pups were exposed to ethanol for different periods of time during gestation via maternal consumption of a 3.35% ethanol liquid diet. In a separate experiment, neonatal pups were fed 2.51 g ethanol/kg body weight/day from Postnatal Day (PD) 4 to PD 10 via intragastric feeding tube. These two ethanol administration paradigms produced average peak maternal and pup blood ethanol concentrations of 39 mg/dl and 57 mg/dl, respectively. At 45 days of age, offspring from each treatment group were sacrificed for measurements of hippocampal NMDA-sensitive [3H]-glutamate binding site density using in vitro radiohistochemical techniques. As observed previously, prenatal ethanol exposure throughout gestation resulted in NMDA-sensitive [3H]-glutamate binding site reductions in the apical dendritic field regions of dentate gyrus, hippocampal CA1 and subiculum of dorsal hippocampal formation compared to the ad lib or pair-fed control groups. NMDA-sensitive [3H]-glutamate binding was not different than control in rats exposed to ethanol during the first half of gestation only. Prenatal ethanol exposure during the last half or the last third of gestation resulted in NMDA-sensitive [3H]-glutamate binding site reductions comparable to the binding site reductions observed in rats exposed to ethanol throughout gestation. Hippocampal NMDA-sensitive [3H]-glutamate binding site density in postnatal ethanol-exposed rats was not different than the suckling or gastrostomy control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Indices of stress in rats: effects of sex, perinatal alcohol and artificial rearing.

Three groups of rats were normally reared by dams, artificially reared but not exposed to alcohol or artificially reared and exposed to 6.6 g/kg/day of ethanol condensed into 8 h of each 24-h period. Alcohol exposure was given from postnatal day 4 through 9 and artificial rearing was conducted from postnatal day 4 to 12. In adulthood, approximately half of the rats were fitted with a chronic jugular catheter and were exposed to swim stress for 1 min in 21 degrees C water. Alcohol exposure augmented the corticosteroid stress response to the swim stress in female rats but not in male rats. The other half of the animals were tested for acquisition and extinction of a conditioned emotional response (CER). While alcohol exposure had no effects on the CER, extinction of CER was greatly accelerated by artificial rearing alone in female rats but not in male rats. Taken together, these findings suggest that some aspects of the stress response in female rats are more vulnerable to insults during the early postnatal period than those in male rats and that the insult-induced alterations can affect hormonal and behavioral measures differentially.

Changes in brain microvasculature resulting from early postnatal alcohol exposure.

The microvasculature in selected brain regions in rats was examined on postnatal day 10 following exposure to alcohol on postnatal days 4 to 10. The alcohol-exposed rats were artificially reared and given 6.6 g/kg of ethanol condensed into 8 hr of each 24-hr period. A gastrostomy-control group was reared in the same manner but maltose-dextrin was substituted for ethanol in the formula. A suckle-control group was reared normally by dams. Measurements were taken from midsagittal sections of the cerebellum and sections of the hippocampal formation at a midtemporal level. Although the overall area of the vermal cerebellum was decreased as a consequence of the alcohol exposure, the capillary density was unchanged. However, cerebellar capillary diameters were increased in some lobules in the alcohol-exposed rats. In the dentate gyrus, there was a trend toward a decrease in capillary numerical density but no change in regional area or capillary diameter in the alcohol-exposed rats. In the hippocampus proper, there was a decrease in regional area, no change in capillary density, and an increase in capillary diameter due to alcohol. These results indicate that alcohol exposure during the early postnatal period affects the microvasculature differentially in the cerebellum, hippocampus proper and dentate gyrus.

Brain growth deficits following a single day of alcohol exposure in the neonatal rat.

Alcohol (7.5 g/kg) was administered to neonatal rats in a single day via artificial rearing procedures, being given in four consecutive feedings spaced two hours apart on either postnatal day 4, 5, or 6. These single days of alcohol exposure resulted in high peak blood alcohol concentrations (mean peak BACs of 380, 439 and 460 mg/dl, respectively) and significantly restricted brain weight when measured on postnatal day 10. The cerebellum was affected more than total brain, forebrain or brain stem. Additionally, growth of the cerebellum was more stunted by alcohol exposure on either day 4 or 5 than when the alcohol exposure occurred on day 6. Small but significant delays in body growth occurred 1-2 days after the alcohol exposure for each group. The interference with brain growth following high BACs for a short period of time has important clinical and experimental implications related to binge drinking and the mechanisms underlying alcohol-induced effects on the developing brain.