Cell Cycle Kinetics in Fetal Rat Cerebral Cortex: Effects of Prenatal Treatment with Ethanol Assessed by a Cumulative Labeling Technique with Flow Cytometry

The effects of ethanol on the cell cycle kinetics of cortical precursor cells during the period of cortical neuronogenesis [between gestational day (G) 12 and G21] was systematically examined. Samples of dissociated cortical cells were harvested from the cerebral cortices of 13-, 15-, 17-, 19-, and 21-day-old fetuses. The fetuses were obtained from pregnant rats: (a) fed a liquid diet containing 6.7% (v/v) ethanol (Et) ad libitum, (b) pair-fed an Isocaloric liquid control diet (Ct), or (c) fed chow and water (Ch) ad libitum. Before harvesting the cells, the fetuses were administered a series of 1–5 injections of bromodeoxyuridine (BrdU). The proportion of cells that incorporated the BrdU was assessed. Using these raw data, the S-phase length (T_B, total cell cycle length (T_c), and the growth fraction (GF; the fraction of the total population that was actively cycling) were determined with a cumulative labeling procedure. The T₃ was –8–9 hr, regardless of either the date of the injection or the dietary treatment of the dam. On the other hand, the T_c for the Ct- and Ch-treated rats increased over the gestational period. That is, the T_c was shortest on G13 and longest on G21. The T_c for Et-treated rats, however, did not change between G13 and G21. For the Ch- and Ct-treated groups, the GF decreased > 15-fold between G13 and G21. The decline (5-fold) for the Et-treated group over the same period, however, was not as great as it was for the Ct-treated fetuses. Thus, by G17 (and thereafter), the GF for Et-treated fetuses was significantly greater than it was for the Ct-treated group. Ethanol treatment has opposite effects on the two cortical germinal zones; it stimulates the proliferation of subventricular cells, whereas it inhibits the proliferation of ventricular cells). Thus, the ethanol-induced changes in the T_c and the GF reflect the combined effects of ethanol on the two cortical proliferative zones.     

Effects of Prenatal Ethanol Exposure on Parvalbumin-Expressing GABAergic Neurons in the Adult Rat Medial Septum

Exposure of human fetuses to ethanol often results in the fetal alcohol syndrome. Animal models of fetal alcohol syndrome have been developed and used to examine the consequences of prenatal ethanol exposure on the central nervous system. The objective of this study was to determine the long-term effects of prenatal ethanol exposure on parvalbumin-expressing (PA+) GABAergic neurons of the rat medial septum. Pregnant Long-Evans rats were maintained on 1 of 3 diets from gestational day 0 to 21: an ethanol-containing liquid diet in which ethanol accounted for 35% of the total calories, a similar diet with the isocaloric substitution of sucrose for ethanol, or a lab chow control diet. Offspring were killed on postnatal day 60, and their brains were prepared for parvalbumin immunocy to chemistry. Female rats exposed to the ethanol-containing diet during gestation had 42% fewer PA+ neurons in the medial septum and reduced PA+ cell density when compared with female rats exposed to the sucrose diet. Ethanol females also had fewer PA+ neurons per unit volume than sucrose females. Male rats exposed to ethanol did not display a similar reduction in PA+ neurons or density. No effect of prenatal diet was found on the area or volume of the medial septum, nor were cell diameters affected. As such, prenatal exposure to ethanol seems to reduce permanently the number of PA+ neurons in the female rat medial septum without affecting area, volume, or neuronal size. Functional implications and possible relations to the fetal alcohol syndrome are discussed.     

In Vitro Comparison of the Effects of Ethanol and Acetaldehyde on Dorsal Root Ganglion Neurons

Results of previous experiments designed to investigate the role of acetaldehyde, the primary metabolite of ethanol, have been contradictory. Experiments have provided evidence that supports and refutes the idea that acetaldehyde is responsible for the teratogenic effects observed in fetal alcohol syndrome. In the present study, cell culture techniques were used to examine the effects of acetaldehyde, both independently and in conjunction with ethanol. The purpose was to determine whether acetaldehyde had any effect on survival and process outgrowth of dorsal root ganglion (DRG) neurons cultured in vitro. This study revealed that acetaldehyde was as toxic to DRG survival as is ethanol, but had a lesser effect on neurite outgrowth than ethanol. Also, acetaldehyde and ethanol do not act synergistically to damage neurons in culture. The results indicate that, although acetaldehyde is probably not solely responsible for ethanol neurotoxicity, it does exhibit a secondary toxicity that could be the subject of future studies.     

Effects of Prenatal Ethanol Exposure on Phospholipase C-β1 and Phospholipase A2 in Hippocampus and Medial Frontal Cortex of Adult Rat Offspring

Previous studies in our laboratory using a rat model of fetal alcohol exposure (FAE) suggest that FAE-induced behavioral deficits are, in part, linked to neurochemical and electrophysiological deficits in long-term potentiation (LTP) in the entorhinal cortical perforant path projection to the hippocampal formation. Several findings suggest that signal-activated phospholipase C (PLC) and phospholipase A₂ (PLA₂) are critical to the induction and maintenance of LTP. Thus, alterations in phospholipid metabolism may play a significant role in the LTP deficits observed in FAE offspring. To test this hypothesis, we measured PLC-β1 and PLA₂ activities in the hippocampus and medial frontal cortex of adult rats prenatally exposed to ethanol. PLC-β1 activities were significantly decreased by 20 to 30% in both the hippocampus and medial frontal cortex of FAE rats, compared with ad libitum and pair-fed controls. Total Ca²⁺-dependent PLA₂ activity was 25% lower in the medial frontal cortex of FAE rats, but did not significantly differ from controls in the hippocampal formation. Approximately 30% of the measured activity in both the medial frontal cortex and hippocampal formation of ad libitum and pair-fed animals was associated with an 85 kDa cytosolic PLA₂ form. Cytosolic PLA₂ activities were significantly reduced in both the medial frontal cortex and hippocampal formation of FAE rats, compared with controls. These changes in Ca²⁺-dependent PLA₂ and PLC-β1 activities, coupled with reports of FAE-induced deficits in protein kinase C activity, indicate that prenatal exposure to moderate quantities of ethanol causes profound and long-lasting deficits in the cellular signaling mechanisms associated with activity-dependent synaptic plasticity and memory formation.     

Effects of Prenatal Ethanol Exposure on Later Sensitivity to the Low-Dose Stimulant Actions of Ethanol in Mouse Offspring: Possible Role of Catecholamines

The purpose of this study was to examine whether prenatal ethanol (EtOH) exposure alters later sensitivity to the low-dose stimulant effects of EtOH. Because the locomotor stimulant effects of EtOH are thought to be mediated, at least in part, by activation of brain monoamine systems, and because prenatal EtOH exposure has been shown to alter brain monoamine activity, it was hypothesized that prenatal EtOH exposure may alter sensitivity to the stimulant actions of EtOH. To test this hypothesis, sensitivity to the locomotor stimulant effects of various challenge doses of EtOH was examined in male and female offspring from prenatal alcohol (A), pair-fed (PF), and lab chow (LC) groups at different ages. In addition, to address the hypothesis further, sensitivity to the catecholamine synthesis inhibitor α-methyl-p-tyrosine (AMPT) was examined in these offspring, as well. Results indicated that male offspring prenatally exposed to EtOH exhibited reduced baseline activity and a blunted stimulant response to all challenge doses of EtOH (0.75–1.5 g/kg) in comparison with control offspring at 30 days of age, but these effects appeared to “normalize” at 70 days of age. Female EtOH-exposed offspring also exhibited a reduced baseline level of activity relative to control offspring, as well as a blunted stimulant response to the lowest challenge dose of EtOH (0.75 g/kg) at 30 days of age, and these effects persisted into adulthood. The stimulant response to higher doses of EtOH did not significantly differ among prenatal treatment groups in young or adult female offspring. However, because baseline activity was significantly lower in female EtOH-exposed offspring than control offspring, the stimulant response to these doses of EtOH (1.125 and 1.5 g/kg) was relatively greater than that for PF and LC offspring. Importantly, none of the differences in performance among the prenatal treatment groups could be attributed to an alteration in EtOH pharmacokinetics, because blood EtOH levels measured immediately following the 10-min test session were similar for all prenatal treatment groups across all of the EtOH test doses. Further, a similar response profile as that observed following EtOH challenge at 70 days of age was obtained following phenobar-bital challenge (10–40 mg/kg). Finally, whereas AMPT (50–400 mg/ kg) dose-dependently antagonized the stimulant effects of EtOH in all prenatal treatment groups, this effect of AMPT was significantly greater in mice prenatally exposed to EtOH in comparison with control offspring. Thus, sensitivity to the stimulant effects of EtOH was significantly altered in adult male mice when these animals were additionally challenged with a drug that further reduced central catecholamine activity. Taken together, these results provide support for the hypothesis that prenatal EtOH exposure alters later sensitivity to the low-dose stimulant properties of the drug, and that this effect may be the result of an alteration in brain monoamine activity in these offspring.     

Effects of Prenatal Exposure to Ethanol on the Number of Axons in the Pyramidal Tract of the Rat

We examined the effect of gestational ethanol exposure on the number of axons in the caudal pyramidal tract. Between gestational day (G)6 and G21, inclusive, pregnant rats were fed a liquid ethanol-containing diet (Et), an isocaloric liquid control diet (Ct), or a diet of chow and water (Ch). On postnatal day 30, the offspring of these rats were killed and their caudal medullas were processed for electron microscopy. The overall size of the pyramidal tract and the space occupied by the axons was smaller in the Et-treated rats than in the Ct-treated rats. The myelinated axons were smaller and the myelin was thinner in the Et-treated rats than in the Ct-treated rats. These decreases produced an ethanol-induced increase in the density of axons in the pyramidal tract. In particular, the density of myelinated axons (but not nonmyelinated axons) was greater in Et-treated rats. The net result was that the estimated number of axons in the pyramidal tracts of the Et-treated rats was not significantly different than the number in the Ch-and Ct-treated rats. The present data demonstrate that ethanol does not affect the absolute number of axons in the pyramidal tract. As a result of the ethanol-induced microencephaly, however, the data translate into a relative increase in the number of pyramidal tract axons. This relative increase matches the ethanol-induced increase in the density of corticospinal projection neurons that may result from the retention of a developmentally exuberant projection.     

Changes in Blood Alcohol Levels as a Function of Alcohol Concentration and Repeated Alcohol Exposure in Adult Female Rats: Potential Risk Factors for Alcohol-Induced Fetal Brain Injury

Fetal alcohol syndrome and alcohol-related birth defects are the result of heavy maternal alcohol consumption during gestation. The magnitude of deficit manifested by the offspring is invariably a consequence of several risk factors that may result in high peak blood alcohol concentrations (BACs), such as the duration, timing, or pattern of alcohol consumption. In addition, the alcohol content of the consumed beverage may play a role in determining offspring developmental consequences. Because higher BACs are positively correlated with risk and severity of brain injury early in postnatal lie, initially it was important to determine how BAC is influenced by alcohol concentration and whether that influence is constant over repeated alcohol treatments. Groups of female Sprague-Dawley rats received daily intragastric intubations of 5 g/kg alcohol in one of several concentrations: 45% (v/v), 30% (v/v), 22.5% (v/v), or 15% (v/v) for a duration of 18 consecutive days. Blood samples were taken at various times postintubation on days 3,8,13, and 18 of treatment, and analyzed by headspace gas chromatography. Multivariate analyses of peak BAC, average BAC, and time to reach peak BAC revealed some noteworthy results. First, peak BAC and average BAC were significantly lower in the 45% group, compared with the other concentration groups, whereas this group also took a longer time to reach peak BAC than the other three groups. Second, peak BAC and averege BAC were higher on the last day of treatment than any of the other treatment days. These results suggest that alcohol concentration and repeated alcohol exposure can influence BAC and, as such, are important risk factors to be considered in the appraisal of alcohol-induced fetal brain injuries.     

Differential Sensitivity of Human Neuroblastoma Cell Lines to Ethanol: Correlations with Their Proliferative Responses to Mitogenic Growth Factors and Expression of Growth Factor Receptors

Early ethanol exposure depletes neurons in the developing nervous system, however the effects on neuronal precursors are not homogeneous. Some cells are more susceptible to ethanol toxicity than others. Growth factors are important mitogens for neuronal precursors. We tested the hypothesis that the differential sensitivity of neuronal precursors to ethanol is determined by their responses to growth factors using an in vitro model (SH-SY5Y, SK-N-SH, and IMR32 neuroblastoma cells) of neuronal precursors. The three cell lines were raised in a medium containing 10% or 0% fetal calf serum. Cells were exposed to ethanol and/or a growth factor. These factors included basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-l, nerve growth factor, and platelet-derived growth factors AA and BB. The numbers of cells per culture were counted both before and after 3 days of ethanol and/or growth factor treatment. In addition, the effect of ethanol exposure on the expression of receptors for these growth factors was examined. Neuroblastoma cells displayed differential sensitivity to ethanol. The growth of SH-SY5Y and SK-N-SH cells was inhibited by ethanol in a concentration-dependent manner. Ethanol did not affect cell viability. Thus, this inhibition resulted from a reduction of cell proliferation. In contrast, IMR32 cells were not affected by ethanol (even at concentrations as high as 800 mg/dl). The response to growth factors was also heterogeneous. In serum-supplemented medium, SH-SY5Y and SK-N-SH cells were stimulated by all of the tested growth factors. For cells raised in a serum-free medium, only the nerve growth factor was ineffective. IMR32 cells, however, were unaffected by most of these growth factors, regardless of the medium conditions. Ethanol blocked the action of all growth factors tested. In general, all cells expressed the specific receptors for the six growth factors. Only the expression of the basic fibroblast growth factor, insulin-like growth factor-l, and nerve growth factor receptors were reduced by ethanol exposure. In summary, neuroblastoma cells exhibit differential susceptibility to ethanol, and this correlates with their response to mitogenic growth factors. Some growth factors are a target of ethanol toxicity. These heterogeneous effects seem to parallel ethanol-induced changes of proliferating neuronal precursors in vivo.     

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.     

Prenatal Ethanol Exposure: Susceptibility to Convulsions and Ethanol's Anticonvulsant Effect in Amygdala-Kindled Rats

The present experiments assessed the effects of prenatal ethanol exposure on the susceptibility to convulsions and on the anticonvulsant effect of ethanol using the electrical kindling model of epilepsy in rats. Adult male Sprague-Dawley rats from prenatal ethanol (E), pair-fed (PF), and ad libitum-fed control (C) treatment groups were tested following the implantation of a stimulation electrode in the left amygdala complex. The same rate viete tested in tout consecutive experiments.
Both E and PF rats showed a slightly slower rate of kindling than C rats, as measured by convulsion class but not as measured by forelimb clonus duration (experiment 1). However, the groups did not differ significantly in the electrical stimulation threshold for kindled convulsions (experiment 2). Furthermore, prenatal ethanol exposure had no significant effect on the dose-response curve for ethanol's (0, 0.9, 1.1, 1.3, and 1.5 g/kg, ip) anticonvulsant effect (experiment 3), or on the rate of tolerance development to ethanol's (1.5 g/kg, ip) anticonvulsant effect (experiment 4) on kindled convulsions. Thus, prenatal exposure to ethanol does not appear to have long-term effects on the susceptibility to convulsions or on the anticonvulsant effect of ethanol in adult male rats in the kindling model as used in the present experiments.     

Ethanol Reduces Expression of the Nerve Growth Factor Receptor, But Not Nerve Growth Factor Protein Levels in the Neonatal Rat Cerebellum

The cerebellum is especially vulnerable to ethanol's neurotoxic effects during development, and ethanol exposure during the brain growth spurt will deplete cerebellar neurons. The mechanisms underlying this neuronal cell loss remain elusive. Nerve growth factor (NGF) is a neurotrophin that promotes cell survival in various brain areas, and there is evidence that NGF may play a role in the developing cerebellum. This study examined whether ethanol exposure of the neonatal rat cerebellum altered the levels of either NGF or the expression of p75 and trkA, which are two components of the NGF receptor. Ethanol exposure had no effect on NGF levels in the neonatal cerebellum, as determined by an NGF-specific ELISA. Immunohistochemical labeling techniques indicated that both the p75 and trkA NGF receptors were expressed on Purkinje cell dendrites in the developing cerebellum, with posterior lobules expressing higher levels of p75 and trkA NGF receptor, compared with anterior lobules. Ethanol exposure of neonatal rats reduced the expression of both p75 and trkA NGF receptors on the Purkinje cell dendrites. These results suggest that ethanol could interfere with neurotrophic support of Purkinje cells by reducing the levels of available NGF receptor.     

Treatment of Pregnant Alcohol-Consuming Rats with Buspirone: Effects on Serotonin and 5-Hydroxyindoleacetic Acid Content in Offspring

This laboratory previously demonstrated that in utero ethanol exposure markedly impairs the development of the serotonergic system in rat brain. Developmental abnormalities could be detected as early as G15 in the brainstem and G19 in the cortex. Because of the importance of fetal serotonin (5-HT) and 5-HT_1A receptors for the normal development of 5-HT containing neurons, we initiated studies to determine whether administration of a 5-HT_1A agonist, buspirone, to pregnant rats could overcome the adverse effects of in utero ethanol exposure on the developing serotonergic system in offspring. Female, Sprague-Dawley rats were given daily subcutaneous injections of buspirone (4.5 mg/kg) from gestational day 13 (G13) to G20. 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) content were determined in the cortex and cortical regions. These experiments demonstrated that the ethanol-associated abnormalities in the development of the serotonergic system can be partially overcome by in utero exposure to buspirone. Specifically, whereas untreated ethanol rats had a deficiency of 5-HT and/or 5-HIAA in whole cortex on PN5, and in the motor cortex on PN19 and 35, no significant differences were detected in these regions of the age-matched offspring of buspirone-treated, ethanol-fed rats. In contrast, the 5-HT and 5-HIAA deficiency in the somatosensory cortex of 19-dayold offspring of ethanol-fed rats was not corrected by in utero buspirone treatment. These results suggest that the abnormal development of cortical projections of serotonergic neurons may be due in part to the low fetal 5-HT content in ethanol-exposed rats and may potentially be overcome by in utero treatment with a 5-HT_1A agonist. However, additional studies are needed to elucidate the importance of dose and timing of buspirone administration, and the effects of in utero buspirone exposure on other components of serotonergic and nonserotonergic neurons.     

Pharmacokinetics of the Ethanol Bioavailability in the Regenerating Rat Liver Induced by Partial Hepatectomy

It is well known that a single ethanol administration is capable of inhibiting the two-thirds partial hepatectomy (PH)-induced liver regeneration (LR); nonetheless, it has not been elucidated how ethanol metabolism by the remnant liver is exerting the deleterious ethanol actions on LR. Indeed, pharmacokinetics analysis of ethanol elimination is lacking in rats subjected to PH, which might extend our understanding in the mechanisms that account for the ethanol-induced inhibition on LR after PH in the rat. Therefore, the present study is a pharmacokinetics analysis comparing intragastric and in-traperitoneal administrations of ethanol to rats under PH, at several times after surgery (0 to 96 hr postsurgery). Our results show that PH rats had a much lower blood ethanol peak than sham-operated, when intragastrically administered during the first 4 hr after surgery that was transient and normalized at 6 hr post-PH. The area under the curve for blood ethanol was higher in PH animals, starting after 6 hr postsurgery and extended to the all replicative period, and returned within the control values thereafter. The quantity of ethanol absorbed after its intraperitoneal injection was essentially the same as the administered dose for all of the groups tested. Hence, ethanol bioavailability diminished due to an enhanced rate of the first-pass metabolism for ethanol in PH rats at the very early times post-PH. At later times of PH, ethanol bioavailability was practically normalized, and these effects were accompanied by a drastic increase in the liver capacity to metabolize ethanol, mainly at 48 to 96 hr after surgery, as calculated as ethanol elimination per gram of liver, as well as by total body weight. The very early changes in ethanol bioavailability in PH rats were not accounted for gastric ethanol retention in these animals. In conclusion, first-pass metabolism importantly participates in the modified ethanol bioavailability at very early times after PH, an event presumably attained to gastric catabolism of ethanol. However, the very enhanced metabolism of ethanol showed by the regenerating liver, particularly after the first 24 hr postsurgery, seems to be the main factor affecting ethanol pharmacokinetics in rats subjected to PH. The underlying mechanisms in this liver enhancement of ethanol oxidation by PH rats remains to be elucidated.     

Effects of Ethanol on Hexose Uptake by Cultured Rat Brain Cells

The effects of ethanol on hexose uptake by glial cells was investigated using primary cultures prepared from term rat fetuses. Specific 3H 2-deoxy-D-glucose (2DG) uptake was significantly reduced by a 4-hr exposure to ethanol at concentrations of 25, 50, and 100 mM, but not 200 or 300 mM. The inhibitory effect of 50 mM ethanol increased with the duration of exposure, with 2DG uptake inhibited by 36% after 18 hr. Astrocytes cultured from the brains of term fetuses of rats fed ethanol during pregnancy showed essentially the same 2DG uptake response to in vitro ethanol treatment. Kinetics of 2DG uptake showed a significant decrease of Vmax in the presence of ethanol. No interaction was found between ethanol and insulin, which stimulated 2DG uptake and protein content of the cultures. The data suggest that ethanol can modulate hexose uptake by astrocytes cultured from fetal rat brain. However, insulin actions on glial cells were not affected by ethanol.