The enduring impact of neurulation stage alcohol exposure: A combined behavioral and structural neuroimaging study in adult male and female C57BL/6J mice.

Prenatal alcohol exposure (PAE) can cause behavioral and brain alterations over the lifespan. In animal models, these effects can occur following PAE confined to critical developmental periods, equivalent to the third and fourth weeks of human gestation, before pregnancy is usually recognized. The current study focuses on PAE during early neurulation and examines the behavioral and brain structural consequences that appear in adulthood. On gestational day 8 C57BL/6J dams received two alcohol (2.8g/kg, i.p), or vehicle, administrations, four hours apart. Male and female offspring were reared to adulthood and examined for performance on the elevated plus maze, rotarod, open field, Morris water maze, acoustic startle, social preference (i.e. three-chambered social approach test), and the hot plate. A subset of these mice was later evaluated using magnetic resonance imaging to detect changes in regional brain volumes and shapes. In males, PAE increased exploratory behaviors on the elevated plus maze and in the open field; these changes were associated with increased fractional anisotropy in the anterior commissure. In females, PAE reduced social preference and the startle response, and decreased cerebral cortex and brain stem volumes. Vehicle-treated females had larger pituitaries than did vehicle-treated males, but PAE attenuated this sex difference. In males, pituitary size correlated with open field activity, while in females, pituitary size correlated with social activity. These findings indicate that early neurulation PAE causes sex specific behavioral and brain changes in adulthood. Changes in the pituitary suggest that this structure is especially vulnerable to neurulation stage PAE.

Acute alcohol exposure during neurulation: Behavioral and brain structural consequences in adolescent C57BL/6J mice.

Prenatal alcohol exposure (PAE) can induce physical malformations and behavioral abnormalities that depend in part on thedevelopmental timing of alcohol exposure. The current studies employed a mouse FASD model to characterize the long-term behavioral and brain structural consequences of a binge-like alcohol exposure during neurulation; a first-trimester stage when women are typically unaware that they are pregnant. Time-mated C57BL/6J female mice were administered two alcohol doses (2.8g/kg, four hours apart) or vehicle starting at gestational day 8.0. Male and female adolescent offspring (postnatal day 28-45) were then examined for motor activity (open field and elevated plus maze), coordination (rotarod), spatial learning and memory (Morris water maze), sensory motor gating (acoustic startle and prepulse inhibition), sociability (three-chambered social test), and nociceptive responses (hot plate). Regional brain volumes and shapes were determined using magnetic resonance imaging. In males, PAE increased activity on the elevated plus maze and reduced social novelty preference, while in females PAE increased exploratory behavior in the open field and transiently impaired rotarod performance. In both males and females, PAE modestly impaired Morris water maze performance and decreased the latency to respond on the hot plate. There were no brain volume differences; however, significant shape differences were found in the cerebellum, hypothalamus, striatum, and corpus callosum. These results demonstrate that alcohol exposure during neurulation can have functional consequences into adolescence, even in the absence of significant brain regional volumetric changes. However, PAE-induced regional shape changes provide evidence for persistent brain alterations and suggest alternative clinical diagnostic markers.

Alcohol-mediated Purkinje cell loss in the absence of hypoxemia during the third trimester in an ovine model system.

BACKGROUND: Although the mechanisms that underlie fetal alcohol-induced neuronal loss have not been determined, hypoxia/hypoxemia has been considered a leading candidate. This study was designed to test the hypothesis that neuronal loss could occur in the developing brain in the absence of fetal hypoxemia. METHODS: Three groups of pregnant sheep were used: a control group, a binge-drinking group, and a pair-fed group. The alcohol and pair-fed animals were anesthetized on day 113 of pregnancy, and the mothers and fetuses were instrumented with arterial and venous catheters. All animals were killed on day 133. Stereological cell counting techniques were used to estimate the total number of Purkinje cells in the fetal cerebellum. RESULTS: Peak maternal and fetal blood alcohol concentrations did not produce fetal hypoxemia. Nevertheless, there was a 25% loss of Purkinje cells of the cerebellum in the alcohol-exposed fetuses compared with that in the pair-fed controls. The loss of neurons was not accompanied by microencephaly or a concomitant decrease in either cerebellar weight or volume of the fetal cerebellum. CONCLUSIONS: Neuronal loss can be observed after alcohol exposure during the third trimester equivalent in fetal sheep in the absence of alcohol-induced hypoxemia. Furthermore, cell loss in the absence of deficits in gross brain weight or regional brain volume indicates that the lack of gross brain volume deficits from magnetic resonance imaging techniques is not a reliable indication that the brain is unaffected by the alcohol exposure.

Third trimester binge ethanol exposure results in fetal hypercapnea and acidemia but not hypoxemia in pregnant sheep.

BACKGROUND: The mechanisms by which maternal ethanol abuse during pregnancy causes neurodevelopmental injury in the fetus are not well understood. The purpose of this study was to use a chronically instrumented fetal sheep model system to determine if a binge pattern of ethanol exposure administered throughout the third trimester reduced fetal arterial partial pressure of oxygen (PaO2); a positive finding would support the hypothesis that fetal hypoxemia may play a role in mediating ethanol-related birth defects. METHODS: Pregnant ewes received saline or 0.75, 1.25, 1.5, or 1.75 g/kg of ethanol intravenously over 1 hr beginning on day 109 of gestation (term = 145 days) for 3 consecutive days per week followed by 4 days without exposure. The fetuses were surgically instrumented on day 113, and experiments were performed on days 118 or 132, the 6th and the 12th ethanol exposure, respectively. RESULTS: Ethanol infusions resulted in peak blood ethanol concentrations of 80.8 +/- 6.5, 182.5 +/- 13.5, 224.4 +/- 13.9, and 260.6 +/- 20.0 mg/dl +/- SEM (maternal) and 70.0 +/- 5.9, 149.7 +/- 9.0, 216.9 +/- 14.0, and 233.3 +/- 19.8 mg/dl +/- SEM (fetal) in response to the 0.75, 1.25, 1.5, and 1.75 g/kg doses, respectively. Maternal and fetal heart rate and maternal blood pressure increased whereas fetal blood pressure decreased in a dose-dependent manner in response to ethanol infusions. Maternal and fetal arterial pH decreased and arterial partial pressures of carbon dioxide increased in response to ethanol infusions. Maternal PaO2 decreased whereas fetal PaO2 did not change in response to ethanol infusions. CONCLUSIONS: A binge ethanol exposure paradigm, three consecutive days per week throughout the third trimester at ethanol doses that created blood ethanol concentrations commonly achieved by human ethanol abusers, resulted in changes in maternal and fetal heart rate, changes in blood pressure, hypercapnea, acidemia, and maternal, but not fetal, hypoxemia. We conclude that in an ovine model system, ethanol doses that create blood ethanol concentrations as high as 260 mg/dl do not result in fetal hypoxemia. Remaining issues to address with this model system are whether neurodevelopmental injuries that are associated with maternal ethanol abuse are mediated by a reduction in fetal cerebral blood flow, fetal hypercapnea, or acidemia.

Early postnatal alcohol exposure produced long-term deficits in brain weight, but not the number of neurons in the locus coeruleus.

The locus coeruleus (LC), a small nucleus in the pontine tegmentum with clusters of norepinephrine (NE) containing neurons, projects to more brain regions than any other nucleus in the central nervous system. Therefore, any neuroanatomical deficits in the LC may have wide-ranging functional implications. Previous studies have shown that heavy alcohol exposure during development can damage several brain regions receiving extensive innervation from the LC (e.g., cerebellum and olfactory bulb). In this study, we examined the effects of early alcohol exposure during development on neuronal numbers in the LC of the adult rat. Sprague-Dawley rat pups were reared using artificial rearing techniques during the brain growth spurt period (part of the third trimester equivalent). The pups in the alcohol group received 4.5 g/kg per day in two of their 12 daily feedings from postnatal days (PDs) 4-9. Gastrostomy and suckle control groups were also included. On PD 90, the animals were sacrificed and their brains were processed for stereological cell counting. Whole brain weight, the number of neurons in the LC, the volume and neuronal density of the LC were assessed. There was a significant reduction in whole brain weight of the alcohol group compared with that of the gastrostomy control group, demonstrating a long lasting effect of alcohol on overall brain growth. Nevertheless, there were no differences in neuronal number, density and volume of the LC between alcohol and gastrostomy groups. Results were discussed in relation to the issues of temporal and regional vulnerabilities.

Effects of alcohol and nicotine on developing olfactory bulb: loss of mitral cells and alterations in neurotransmitter levels.

Previous research from our laboratory has shown that [ethanol (EtOH)] exposure during the brain growth spurt is detrimental to olfactory bulb development. This study extends those findings by examining the effects of EtOH, nicotine (NIC), and the combination of these drugs (EtOH/NIC) on olfactory bulb mitral cell numbers, as well as on various major neurotransmitter levels in neonatal rats. An artificial rearing paradigm was used in the present studies. These artificially reared pups were given 4 g/kg/day of EtOH and/or 6 mg/kg/day of NIC on postnatal day (PD) 4 to PD 9, except in the case of the acute neurochemistry study, in which the pups received treatment on PD 9 only. An artificially reared gastrostomy control group (GC) and a suckle control group were included. The mean total numbers of mitral cells in the EtOH and NIC groups were significantly reduced from that of the GC, as well as the volume of the left main olfactory bulb. There was no difference among any of the groups in mitral cell density. As for neurochemistry data, there was no difference in neurotransmitter levels among any of the groups in the repeat exposure regimen. There were, however, changes after the acute exposure (exposure on PD 9 only). Both serotonin and GABA levels were significantly increased only after NIC exposure. However, norepinephrine levels were significantly decreased after acute exposure in all three drug treatment groups, compared with that of the control group. Except for the GC control group, dopamine levels were not detected consistently after acute exposure to EtOH, NIC, or EtOH/NIC. Collectively, these findings demonstrate that exposure to EtOH or NIC individually during the brain growth spurt results in developmental deficits in the olfactory bulb, suggesting that both EtOH and NIC are neuroteratogens. Furthermore, this study demonstrated the capability of NIC to antagonize (protect) EtOH-induced mitral cell loss in the developing olfactory bulb.

Neonatal alcohol and nicotine exposure limits brain growth and depletes cerebellar Purkinje cells.

The present study examined the effects of coexposure of alcohol and nicotine during the brain growth spurt period on brain weights and cerebellar Purkinje cell numbers. Sprague-Dawley rat pups were randomly assigned into five groups (four artificially reared groups and one suckle control). Artificially reared pups were given alcohol (0 or 4 g/kg/day) and/or nicotine (0 or 6 mg/kg/day) daily from postnatal days (PDs) 4 to 9, and the suckle controls received no experimental treatments. The results are summarized below. (1) Surprisingly, nicotine reduced the peak blood alcohol concentration from about 300 to 230 mg/dl, but alcohol did not affect urine cotinine levels (approximately 12,000-13,000 ng/ml). (2) Alcohol significantly reduced the weights of forebrain, cerebellum, and brain stem, but nicotine limited only the growth of the forebrain. (3) Purkinje cell numbers in the cerebellar vermis were significantly reduced in response to alcohol, nicotine, and the combination of both drugs. (4) No statistically significant interactive effect was found following the cotreatment of alcohol and nicotine. Collectively, the present study replicated our previous findings demonstrating alcohol's detrimental effects on brain development; it also presented new evidence documenting nicotine's neuroteratogenic effects on restricting brain growth and depleting cerebellar Purkinje cells during the brain growth spurt.