Jennifer D. Thomas

Neonatal choline supplementation ameliorates the effects of prenatal alcohol exposure on a discrimination learning task in rats

Prenatal alcohol exposure can disrupt brain development and lead to a myriad of behavioral alterations, including motor coordination deficits, hyperactivity, and learning deficits. There remains a need, however, to identify treatments and interventions for reducing the severity of alcohol-related neurodevelopmental disorders. Some of the alcohol-induced deficits in learning may be related to alterations in cholinergic functioning. Interestingly, there is a growing literature demonstrating that pre- and/or early postnatal choline supplementation can lead to long-term enhancement in learning and memory and cholinergic activity in rats. The present study examined whether such early choline supplementation might counter the effects of prenatal alcohol treatment on a visuospatial discrimination task. Pregnant Sprague-Dawley rats were randomly assigned to one of three prenatal treatment groups. One group received a liquid diet containing 35% ethanol-derived calories (EDC) from gestational day (GD) 6-20. A second group served as a pair-fed (PF) control group and the third group served as an ad lib lab chow (LC) control. On postnatal day (PD) 2, pups were assigned within-litter to one of three postnatal treatments: choline, saline vehicle, or no treatment. Choline and vehicle pups were intubated with a choline chloride solution or vehicle daily from PD 2 to 21, whereas the non-treated pups were handled daily but not intubated. On PD 45, subjects were tested on a visuospatial discrimination task. Ethanol-exposed subjects who were not treated neonatally with choline committed a significantly greater number of errors both during acquisition and during delayed discrimination training compared to both PF and LC controls. Neonatal choline treatment significantly improved performance on the discrimination task in all groups; however, the beneficial effects of choline were significantly larger in ethanol-exposed subjects. Indeed, the performance of ethanol-exposed pups treated with neonatal choline did not differ from any of the PF or LC groups on any measure. Thus, early postnatal choline supplementation significantly attenuated the effects of prenatal alcohol on this learning task. Importantly, these effects were not due to the acute effects of choline, but rather to long-term changes in brain and behavioral development. These data suggest that early dietary interventions may reduce the severity of fetal alcohol effects.

Prenatal choline supplementation mitigates the adverse effects of prenatal alcohol exposure on development in rats

Prenatal alcohol exposure can lead to a range of physical, neurological, and behavioral alterations referred to as fetal alcohol spectrum disorders (FASD). Variability in outcome observed among children with FASD is likely related to various pre- and postnatal factors, including nutritional variables. Choline is an essential nutrient that influences brain and behavioral development. Recent animal research indicates that prenatal choline supplementation leads to long-lasting cognitive enhancement, as well as changes in brain morphology, electrophysiology and neurochemistry. The present study examined whether choline supplementation during ethanol exposure effectively reduces fetal alcohol effects. Pregnant dams were exposed to 6.0g/kg/day ethanol via intubation from gestational days (GD) 5-20; pair-fed and lab chow controls were included. During treatment, subjects from each group received choline chloride (250mg/kg/day) or vehicle. Physical development and behavioral development (righting reflex, geotactic reflex, cliff avoidance, reflex suspension and hindlimb coordination) were examined. Subjects prenatally exposed to alcohol exhibited reduced birth weight and brain weight, delays in eye opening and incisor emergence, and alterations in the development of all behaviors. Choline supplementation significantly attenuated ethanols effects on birth and brain weight, incisor emergence, and most behavioral measures. In fact, behavioral performance of ethanol-exposed subjects treated with choline did not differ from that of controls. Importantly, choline supplementation did not influence peak blood alcohol level or metabolism, indicating that cholines effects were not due to differential alcohol exposure. These data indicate early dietary supplements may reduce the severity of some fetal alcohol effects, findings with important implications for children of women who drink alcohol during pregnancy.

Forebrain ischemia induces selective behavioral impairments associated with hippocampal injury in rats.

Two groups of rats were tested on a variety of motor and cognitive tasks after either 10 minutes of two-vessel occlusion forebrain ischemia (n = 8) or sham operative procedures (n = 6). Histological injury was absent in the sham-operated group. In the ischemic group, hippocampal injury was restricted to field CA1, while damage in the neocortex and caudoputamen was sparse. Motor tests performed on postoperative days 18 and 28 revealed no significant differences between the ischemic and sham-operated groups. Retention performance of a radial maze discrimination task was impaired, with a significant but transient increase in both working and reference memory errors. Passive avoidance acquisition and retention were not significantly affected, although conclusions concerning the utility of this task must be reserved because of variability in the behavior of the sham-operated rats. Morris maze spatial navigation (place learning) and open-field activity were insensitive to treatment group. These functional results are consistent with the observed histological injury and what is known about hippocampal injury and behavior, and they provide further guidance for the development of neurological assays appropriate for discriminating outcome from forebrain ischemia in rats.

Long-term deficits in cerebellar growth and rotarod performance of rats following “binge-like” alcohol exposure during the neonatal brain growth spurt

The cerebellum is vulnerable to growth restriction and neuronal depletion induced by alcohol exposure during the brain growth spurt of neonatal rats. This study examined whether neonatal alcohol exposure permanently restricted brain growth and induced motor performance deficits in adults. Two groups of rats were given 4.5 g/kg of alcohol per day during postnatal days 4 through 9, using artificial-rearing procedures. One group was given the alcohol as a 10.2% (v/v) solution in two of the 12 daily feedings, producing peak BACs of 361 mg/dl. The second group was given the alcohol as a 5.1% (v/v) solution in four of the feedings, producing peak BACs of 187 mg/dl. Controls included an artificially reared group and a normally reared group. All rats were tested on a rotarod at approximately 405 days of age, then perfused 1-2 weeks later. The 10.2% group was significantly impaired in acquiring the task and had significant reductions in whole brain and cerebellar weight, compared to controls. The 5.1% treatment also significantly restricted whole brain and cerebellar growth, and rotarod performance of that group was intermediate between the control groups and the 10.2% group. The cerebellar reductions and deficits in motor performance in adulthood demonstrate permanent structural and functional consequences of binge-like alcohol exposure during the brain growth spurt.

Prenatal choline supplementation mitigates behavioral alterations associated with prenatal alcohol exposure in rats.

BACKGROUND Prenatal alcohol exposure can alter physical and behavioral development, leading to a range of fetal alcohol spectrum disorders. Despite warning labels, pregnant women continue to drink alcohol, creating a need to identify effective interventions to reduce the severity of alcohols teratogenic effects. Choline is an essential nutrient that influences brain and behavioral development. Recent studies indicate that choline supplementation can reduce the teratogenic effects of developmental alcohol exposure. The present study examined whether choline supplementation during prenatal ethanol treatment could mitigate the adverse effects of ethanol on behavioral development. METHODS Pregnant Sprague-Dawley rats were intubated with 6 g/kg/day ethanol in a binge-like manner from gestational days 5-20; pair-fed and ad libitum chow controls were included. During treatment, subjects from each group were intubated with either 250 mg/kg/day choline chloride or vehicle. Spontaneous alternation, parallel bar motor coordination, Morris water maze, and spatial working memory were assessed in male and female offspring. RESULTS Subjects prenatally exposed to alcohol exhibited delayed development of spontaneous alternation behavior and deficits on the working memory version of the Morris water maze during adulthood, effects that were mitigated with prenatal choline supplementation. Neither alcohol nor choline influenced performance on the motor coordination task. CONCLUSIONS These data indicate that choline supplementation during prenatal alcohol exposure may reduce the severity of fetal alcohol effects, particularly on alterations in tasks that require behavioral flexibility. These findings have important implications for children of women who drink alcohol during pregnancy.

Choline supplementation attenuates learning deficits associated with neonatal alcohol exposure in the rat: Effects of varying the timing of choline administration

Despite the harmful effects of fetal alcohol exposure, some pregnant women continue to drink alcohol. Thus, it is imperative to pursue safe, effective treatments for children with fetal alcohol spectrum disorders. Using an animal model, our laboratory has demonstrated that choline, an essential nutrient, effectively reduces the severity of some fetal alcohol effects, even when administered after the ethanol insult is complete. The present study investigated whether there is a critical developmental period when choline is most effective in attenuating ethanols teratogenic effects. Sprague-Dawley rats were exposed to 5.25 g/kg/day ethanol during the third trimester equivalent brain growth spurt (postnatal days (PD) 4-9) via intubation. A non-intubation control group and a sham intubation control group were included. Following ethanol exposure, pups received subcutaneous injections of saline vehicle or choline chloride (100 mg/kg/day) from PD 11-20, PD 21-30, or PD 11-30. Beginning on PD 45, subjects were tested on a Morris water maze spatial learning task. Performance of both the ethanol-exposed group that did not receive choline and the ethanol-exposed group treated with choline from PD 21-30 was significantly impaired compared to controls during acquisition of the Morris water maze task. Performance of ethanol-exposed groups treated with choline from PD 11-20 or PD 11-30 was intermediate, not differing significantly from any other groups. However, during the probe trial, ethanol exposure produced significant deficits in spatial memory which were mitigated by all choline treatments, regardless of the timing of administration. These findings suggest that cholines therapeutic window may be very large, or spans across the two developmental periods examined in this study. Importantly, these findings indicate that choline supplementation may effectively reduce some alcohol-related learning impairments, even when administered in later childhood.

BEHAVIORAL DEFICITS INDUCED BY BINGELIKE EXPOSURE TO ALCOHOL IN NEONATAL RATS : IMPORTANCE OF DEVELOPMENTAL TIMING AND NUMBER OF EPISODES

The importance of the timing and number of episodes of bingelike alcohol exposure in neonatal rats on subsequent behavioral outcomes was evaluated with a parallel bar task and a spatial conditional alternation task. Different groups of Sprague-Dawley rat pups were exposed to alcohol delivered via artificial rearing procedures either on postnatal Days (PD) 4 and 5, on PD 8 and 9, or on both PD 4/5 and 8/9 (Combined), producing daily peak blood alcohol concentrations around 400 mg/dl. Controls included an artificially reared group and a normally reared group. Exposure during PD 4/5 produced significantly more severe motor deficits and significantly more severe reductions in cerebellar and brainstem weights than did exposure on PD 8/9. Combined exposure produced greater deficits on these measures than either of the limited exposures. Significant deficits in the acquisition rates for conditional alternation were found only with the Combined exposure, although both the PD 8/9 and Combined groups committed significantly more within-trial errors. All three alcohol treatments produced significant and comparable reductions in forebrain weight. The type and severity of behavioral and neural deficits induced by neonatal bingelike alcohol exposure depend on the timing and number of exposures.

Choline Supplementation and DNA Methylation in the Hippocampus and Prefrontal Cortex of Rats Exposed to Alcohol During Development

BACKGROUND Some of the most frequent deficits seen in children with fetal alcohol spectrum disorders (FASD) and in animal models of FASD are spatial memory impairments and impaired executive functioning, which are likely related to alcohol-induced alterations of the hippocampus and prefrontal cortex (PFC), respectively. Choline, a nutrient supplement, has been shown in a rat model to ameliorate some of alcohols teratogenic effects, and this effect may be mediated through cholines effects on DNA methylation. METHODS Alcohol was given by intragastric intubation to rat pups during the neonatal period (postnatal days 2 to 10) (ET group), which is equivalent to the third trimester in humans and a period of heightened vulnerability of the brain to alcohol exposure. Control groups included an intubated control group given the intubation procedure without alcohol (IC) and a nontreated control group (NC). Choline or saline was administered subcutaneously to each subject from postnatal days 2 to 20. On postnatal day 21, the brains of the subjects were removed and assayed for global DNA methylation patterning as measured by chemiluminescence using the cpGlobal assay in both the hippocampal region and PFC. RESULTS Alcohol exposure caused hypermethylation in the hippocampus and PFC, which was significantly reduced after choline supplementation. In contrast, control animals showed increases in DNA methylation in both regions after choline supplementation, suggesting that choline supplementation has different effects depending upon the initial state of the brain. CONCLUSIONS This study is the first to show changes in global DNA methylation of the hippocampal region and PFC after neonatal alcohol exposure. Choline supplementation impacts global DNA methylation in these 2 brain regions in alcohol-exposed and control animals in a differential manner. The current findings suggest that both alcohol and choline have substantial impact on the epigenome in the PFC and hippocampus, and future studies will be needed to describe which gene families are impacted in such a way that function of the nervous system is changed.

Choline Supplementation Mitigates Trace, but not Delay, Eyeblink Conditioning Deficits in Rats Exposed to Alcohol During Development

Children exposed to alcohol prenatally suffer from a range of physical, neuropathological, and behavioral alterations, referred to as fetal alcohol spectrum disorders (FASD). Both the cerebellum and hippocampus are affected by alcohol exposure during development, which may contribute to behavioral and cognitive deficits observed in children with FASD. Despite the known neuropathology associated with prenatal alcohol exposure, many pregnant women continue to drink (heavy drinkers, in particular), creating a need to identify effective treatments for their children who are adversely affected by alcohol. We previously reported that choline supplementation can mitigate alcohols effects on cognitive development, specifically on tasks which depend on the functional integrity of the hippocampus. The present study examined whether choline supplementation could differentially mitigate alcohols effects on trace eyeblink classical conditioning (ECC, a hippocampal‐dependent task) and delay ECC (a cerebellar‐dependent task). Long‐Evans rats were exposed to 5.25 g/kg/day alcohol via gastric intubation from postnatal days (PD) 4–9, a period of brain development equivalent to late gestation in humans. A sham‐intubated control group was included. From PD 10–30, subjects received subcutaneous injections of 100 mg/kg choline chloride or vehicle. Beginning on PD 32–34, subjects were trained on either delay or trace eyeblink conditioning. Performance of subjects exposed to alcohol was significantly impaired on both tasks, as indicated by significant reductions in percentage and amplitude of conditioned eyeblink responses, an effect that was attenuated by choline supplementation on the trace, but not delay conditioning task. Indeed, alcohol‐exposed subjects treated with choline performed at control levels on the trace eyeblink conditioning task. There were no significant main or interactive effects of sex. These data indicate that choline supplementation can significantly reduce the severity of trace eyeblink conditioning deficits associated with early alcohol exposure, even when administered after the alcohol insult is complete. These findings have important implications for the treatment of fetal alcohol spectrum disorders.

Voluntary exercise influences behavioral development in rats exposed to alcohol during the neonatal brain growth spurt

Children exposed to alcohol prenatally may suffer from severe brain damage, expressed as a variety of behavioral problems, including hyperactivity and learning deficits. There is a critical need to identify effective treatments for fetal alcohol effects. Physical exercise enhances cognitive ability and increases neurogenesis in the hippocampus, a brain area important for learning and memory. Thus, the present study examined whether physical exercise might reduce the severity of alcohol-induced behavioral alterations. Sprague-Dawley rats were intubated with 5.25 g/kg/day ethanol during the third trimester equivalent (postnatal days [PDs] 4-9). Intubated sham control and nontreated controls were included. From PD 21 to PD 51, half of the subjects were given access to running wheels. On PD 52, subjects were tested on the Morris water maze, and on PD 60, open field activity levels were measured. Morris maze performance was significantly impaired among ethanol-exposed subjects; exercise significantly improved performance of all groups. Similarly, ethanol-exposed subjects were overactive in the open field, an effect attenuated with exercise. In sum, these data suggest that exercise may increase neuronal plasticity not only in controls, but also in subjects exposed to alcohol during development.