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.     

Long-Term Behavioral Changes in Response to Early Developmental Exposure to Ethanol in Zebrafish

Background:  Zebrafish is becoming an important research tool for the analysis of brain function and behavior. It has been proposed to model human alcoholism as well as fetal alcohol syndrome. Previous studies investigating the consequences of exposure to ethanol during early development of zebrafish employed robust dosing regimens (high ethanol concentration and long exposure) that may model a rare situation in the human clinic. These studies found major structural abnormalities developing in the exposed fish.
Methods:  Here we hope to avoid such gross changes and administer only low doses of ethanol (0.00, 0.25, 0.50, 0.75, 1.00 vol/vol %) at 24-hour postfertilization and for only a short period of time (for 2 hours). We analyze the behavior of exposed fish at adult stage using computerized stimulus presentation and automated videotracking response quantification.
Results:  Despite the short ethanol exposure period and the modest concentrations, significant behavioral alterations were found: fish exposed to higher doses of ethanol swam at an increased distance from a computer-animated zebrafish shoal while their activity levels did not change.
Conclusions:  Although the interpretation of and the mechanisms underlying this finding will require further investigation, the results suggest that zebrafish will be an appropriate model organism for the analysis of the effects of moderate to mild prenatal ethanol exposure.     

Prenatal Ethanol Exposure Induces a Sexually Dimorphic Effect on Daily Water Consumption in Prepubertal and Adult Rats

Previous studies have shown that female rats consume significantly more water than males on a weight basis. Because exposure to alcohol during the last week of gestation is associated with incomplete behavioral defeminization in male rats, we examined daily water intake in fetal alcohol-exposed (FAE) males and females. Time-pregnant multiparous Sprague-Dawley dams were administered an ethanol liquid diet containing 35% ethanol-derived calories from day 14 through parturition. At 80 days of age, daily water consumption of FAE males and female litter representatives was measured for 7 days. FAE males, but not females, consumed significantly more water than their pair-fed counterparts. Subsequent experiments determined that the increased water consumption in FAE males is present prepubertally, persists into mature adulthood, and is not influenced by prenatal or postnatal castration. Chronic estrogen treatment induced large increases in water consumption, but consumption of FAE males remained elevated over elevated pair-fed male consumption, indicating that pituitary sensitivity to estrogen was not increased in FAE males. Morphometric studies of hypothalamic nuclei containing vasopressin cells revealed no long-term effects of prenatal ethanol exposure on the volume of the supraoptic nucleus or paraventricular nucleus in males, nor was an effect observed in the ventromedial nucleus measured as a control. In FAE females, the volume of the paraventricular nucleus was significantly smaller than chow-fed controls. Whereas baseline plasma and pituitary arginine vasopressin (AVP) levels of FAE animals and pair-fed controls were not significantly different, AVP content was significantly reduced in the septal/bed nucleus region in brains of FAE animals of both sexes. Overall, these data indicate that prenatal ethanol exposure increases male water consumption in the absence of alterations in basal plasma AVP.     

Neuromuscular Responses to Disturbance of Balance in Children with Prenatal Exposure to Alcohol

Alcohol-exposed children display delayed motor development and impaired fine- and gross-motor skills, including deficits in the maintenance of balance. In a recent study, we assessed the contribution of visual, somatosensory, and vestibular information to the ability to maintain balance. Our findings suggested that alcohol-exposed children were overly reliant on somatosensory information and were unable to compensate by using the visual and/or vestibular systems. To understand the nature of these observed balance deficits, corrective postural reactions were examined by exposing standing subjects to rapid toe-up movements of the support surface. Subjects for this study were alcohol-exposed (ALC) and normal control (NC) children matched for age and sex. Postural reactions were quantified by measuring electromyographic activity of the triceps surae and anterior tibialis muscles. Analyses revealed no differences between the ALC and NC groups on short- and medium-latency electromyographic responses, which are thought to be involuntary mono- and polysynaptic spinal reflexes, respectively. However, when compared with the NC group, the ALC group displayed increased long-latency responses, which are thought to involve a transcortical pathway. Although we are not able to rule out the possibility of additional peripheral (e.g., vestibular) disturbance as a contributing factor to postural instability, our findings suggest that the balance deficits seen in alcohol-exposed children are, at least in part, central in nature.     

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 Exposure to Alcohol Affects the Ability to Maintain Postural Balance

Prenatal exposure to alcohol is known to affect gross motor functioning. Animal studies have shown that balance is particularly affected, and there is some evidence that similar deficits exist in alcohol-exposed children. In the current study, postural balance, or the ability to maintain equilibrium, was assessed in a group of alcohol-exposed children (ALC group; n =11) and controls (NC group; n = 11) individually matched for age and sex. Balance was measured across six conditions designed to systematically manipulate or eliminate visual or somatosensory information. Equilibrium and strategy scores for each condition and a derived composite balance score were analyzed. Although the ALC group had a lower mean composite balance score, their performance was similar to that of the NC group on all conditions where somatosensory input was reliable. However, when somatosensory input was manipulated, and when both somatosensory and visual input were inaccurate, the ALC group performed more poorly than controls. Interestingly, there were no differences between the ALC group and NC group in the type of control strategy used to maintain balance. These results suggest that alcohol-exposed children are overly reliant on somatosensory input. When this input is atypical, alcohol-exposed children display significantly greater anterior-posterior body sway and are unable to compensate using available visual or vestibular information. These deficits may be related to cerebellar anomalies previously reported in fetal alcohol syndrome children.     

Applying a Developmental Framework to the Self‐Regulatory Difficulties of Young Children with Prenatal Alcohol Exposure: A Review

Prenatal alcohol exposure (PAE) can be associated with significant difficulties in self‐regulatory abilities. As such, interventions have been developed that focus on improving varying aspects of self‐regulation for this population. The application of a multilevel theoretical framework that describes the development of self‐regulation during early childhood could further advance the field. First, this framework could assist in elucidating mechanisms in the trajectories of early adjustment problems in this population and, second, informing the development of more precise assessment and interventions for those affected by PAE. The aims of the current review were to provide an overview of the self‐regulatory framework proposed by Calkins and colleagues (e.g., Calkins, 2007; Calkins and Fox, 2002); examine the self‐regulatory difficulties that are commonly experienced during infancy (i.e., 0 to 2 years) and early childhood (i.e., 3 to 8 years) in children with PAE in the context of the developmental framework; and describe how the framework can inform the development of future assessment and intervention provision for young children with PAE. The application of a developmental framework, such as proposed by Calkins and colleagues, allows for a systematic and theoretically driven approach to assessment and intervention programs for young children with PAE.     

Effects of Prenatal Ethanol Exposure and Stress in Adulthood on Lymphocyte Populations in Rats

The present study was undertaken to assess the possible interactive effects of prenatal ethanol exposure and stress in adulthood on lymphocyte populations in rat offspring, and to examine differential vulnerability of males and females to these challenges. Male and female offspring from prenatal ethanol-exposed (E), pair-fed, and ad libitum-fed control conditions were exposed to a 3-week chronic intermittent stress regimen in adulthood. Animals were exposed to two of six different stressors daily, one each at random times in the morning and afternoon, with the same pair of stressors being repeated every 4 days. Following the 3-week stress period, lymphocytes from four compartments (peripheral blood, spleen, thymus, and cervical lymph nodes) were analyzed for expression of differentiation antigens. Data demonstrate that, whereas a number of the effects of prenatal ethanol on lymphocyte populations appeared to be nutritionally mediated, the additional challenge of exposure to stressors differentially affected animals exposed to ethanol prenatally and appeared to have effects primarily in male offspring. Stressed E males had a greater reduction in the number of pan T-cells in the thymus and peripheral blood, compared with nonstressed E males, but showed an increased peripheral blood pan T-antigen expression. Stressed E males also had reduced numbers of peripheral blood CD4⁺ T-cells and thymic CD4⁺CD8⁺ T-cells, compared with controls. In addition, several effects of stress were observed in animals in all three prenatal treatment groups, including decreased numbers of lymph node pan T- and CD4⁺ T-cells and decreased numbers of total peripheral blood lymphocytes in males, increased numbers of total splenic and thymic lymphocytes in females, and increased numbers of splenic CD8⁺ T-cells, as well as a decreased ratio of CD4⁺:CD8⁺ T-cells in the lymph node and spleen in both males and females. These findings indicate that, although exposure to chronic intermittent stress in adulthood may have marked effects on lymphocyte populations across all treatment groups, specific deficits in the immune system of fetal E animals may become apparent only when these animals are subjected to the additional challenge of stress. Moreover, male and female offspring may be differentially affected by the two challenges of ethanol and stress.     

Effect of Prenatal Ethanol Exposure on Fetal Calcium Metabolism

Alcohol consumption has adverse effects on both adult and developing bone. The mechanisms by which alcohol affects bone, however, are unknown. This study examined the possibility that maternal alcohol consumption may affect fetal bone development by altering fetal levels of parathyroid hormone (PTH), 1,25(OH)₂D, or calcitonin (hormones that regulate calcium (Ca) and bone metabolism in the adult animal). Female Sprague-Dawley rats were bred and divided into three groups: 1 group was fed lab chow ad libitum (Control; C) and the other 2 groups received a liquid diet with (Ethanol; E) or without (Pair-fed; PF) ethanol. Blood from dams and fetuses was collected on day 21 of gestation, and selected fetuses were stained for determination of the degree of bone ossification. Mean fetal body weight and fetal skeletal ossification were reduced in the E compared with PF and C groups. Total Ca levels in fetal serum, however, showed a trend to be increased in E compared with PF and C fetuses, and no significant group differences were found in fetal serum levels of albumin, PTH, or calcitonin. Serum levels of 25-OH-D and 1,25(OH)₂D were significantly decreased in E and PF, compared with C fetuses. Total Ca levels in maternal serum did not vary with the group; however, serum albumin levels were higher in E, compared with PF and C dams, suggesting that serum ionic Ca levels may have been reduced in the E dams. Serum levels of 25-OH-D were reduced in the E, compared with PF and C dams, whereas levels of 1,25(OH)₂D were elevated. PTH levels did not vary among groups. Interestingly, serum calcitonin levels were elevated in the E, compared with PF and C, dams. These results indicate that the effects of ethanol on fetal bone development do not appear to be related to alterations in fetal serum levels of PTH, 1,25(OH)₂D, or calcitonin. Maternal ethanol consumption, however, results in reduced appetite and a decrease in dietary Ca intake. Despite the reduced Ca intake, the ability of the dam to maintain Ca homeostasis appeared intact, although this may be dependent on the duration of ethanol consumption.     

Effects of Prenatal Ethanol Exposure on Hippocampal Ionotropic-Quisqualate and Kainate Receptors

Previous studies from our laboratories have shown that the consumption of moderate quantities of ethanol by rat dams during pregnancy reduces N-methyl-D-aspartate (NMDA) agonist receptor binding and NMDA-mediated electrophysiological responses in the hippocampal formation of adult offspring. We hypothesized that prenatal ethanol exposure would produce similar effects on receptor number and agonist-mediated responses of two so-called "non-NMDA" subtypes of glutamate receptors, the ionotropic-quisqualate (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-sensitive and the kainate-sensitive receptors. Sprague-Dawley rats were fed either a liquid diet containing 3.35% ethanol, an isocalorically matched liquid diet, or lab chow ad libitum throughout gestation. No significant differences between offspring from these three groups in the agonist concentration-response curves for either AMPA-induced or kainate-induced depolarization of hippocampal CA1 pyramidal neurons were observed. Furthermore, no significant differences in the density of [3H]-AMPA or [3H]-vinylidene kainic acid binding sites in any of the apical dendritic field regions of dorsal or ventral hippocampal formation were observed between the groups. These results indicate that the ionotropic quisqualate and kainate receptors, located in the apical dendritic field regions of the principal hippocampal neurons, are not affected by the same degree of prenatal ethanol exposure, which is known to reduce NMDA receptor binding and function in these same regions.     

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.     

Generation of Neurons in the Rat Dentate Gyrus and Hippocampus: Effects of Prenatal and Postnatal Treatment with Ethanol

Neurons in the rat hippocampal formation (the dentate gyrus and the hippocampus) are born over a protracted period, from gestational day (G) 15 into adulthood. Dentate gyral neurons born prenatally are generated from the ventricular zone, whereas those born postnatally are derived from a secondary proliferative zone, the intrahilar zone. In contrast, hippocampal pyramidal neurons are generated only prenatally from the ventricular zone. In the neocortex, ethanol depresses the proliferation of cells in the ventricular zone and stimulates the proliferation of cells in the secondary proliferative zone. The present study tests the hypotheses that prenatal treatment with ethanol has a different effect on the generation of dentate gyral neurons than does postnatal ethanol treatment, and that these differences are determined by the timing of the ethanol exposure relative to the period and site of neuronal generation. Rats were treated with ethanol between G6 and G21 or between postnatal day (P) 4 and P12. They were given an injection of [³H]thymidine on G15, G18, G21, P6, P9, or P12. Rats were killed on P30–P35. The tissue was processed by standard autoradiographic methods and assessed using rigorous stereological procedures. The total number of neurons and the density of radiolabeled neurons in both the dentate gyrus and the CA1 region of the hippocampus were determined. Prenatal ethanol treatment decreased the total number of neurons in the CA1 segment of the hippocampus and had little impact on neuronal number in the dentate gyrus. Likewise, the number of hippocampal and dentate gyral neurons generated daily was significantly lower in ethanol-treated rats than in controls. Postnatal treatment to ethanol, however, significantly increased the total number of dentate gyral neurons and the density of neurons generated postnatally. These postnatal changes depended on the blood ethanol concentration (BEC). At moderate BECs, the total number of neurons in the dentate gyrus and the number of neurons generated was increased. At high BECs, however, neuronal number and neuronal generation were decreased. Postnatal ethanol treatment had no effect on the number of (total or radiolabeled) CA1 neurons. Thus, pre- and postnatal exposure to ethanol have opposite effects both on the number of neurons in the dentate gyrus and on the generation of neurons. These paradoxical effects likely result from three causes: the differential effects of ethanol on the two proliferative zones, the critical period of neuronal development, and the potentially opposite effects of moderate and high BEC.     

Neonatal Ethanol Exposure Impairs Eyeblink Conditioning in Weanling Rats

Eyeblink conditioning depends on an identified brainstem-cerebellar circuit and may be useful in functional studies of early cerebellar damage produced by neurotoxicants. The present study asked whether binge-like neonatal ethanol exposure that damages the cerebellum would also result in eyeblink conditioning deficits. On postnatal day (PND) 23 to PND24, three groups of Long-Evans rat pups were tested for eyeblink conditioning: (1) ETOH, a group that received intragastric administration of 5.25 g/kg/day of ethanol on PND4 through PND9 via artificial rearing; (2) GC, a gastrostomy control group that received calorically matched milk formula on those days; and (3) SC, suckle controls that were reared normally with their dams. Eyeblink conditioning was severely impaired in the ethanol-treated group relative to the GC and SC groups, which did not differ. This impairment did not reflect sensory, motor, or motivational effects of ethanol treatment, because startle responses to the auditory conditioned stimulus and reflexive eyeblink responses to the unconditioned stimulus did not differ across the three treatment groups. These results suggest that neonatal binge ethanol exposure disrupted brain development in a manner that selectively impaired associative processes involved in eyeblink conditioning, consistent with alcohol-induced damage to the brainstem-cerebellar circuit necessary for this form of learning.