Tuan D. Tran

Critical periods for ethanol-induced cell loss in the hippocampal formation

Rodent models of fetal alcohol syndrome (FAS) have revealed discrepant findings in ethanols (EtOH) ability to alter the survival of principal neurons within hippocampal areas CA1, CA3 and the dentate gyrus (DG). One issue is the lack of systematic examination of the timing of EtOH exposure over key periods of hippocampal cell development. The present study examined whether systematic developmental EtOH exposure produces long-term hippocampal cell loss that is related to a specific time course in which either generation, migration or synaptogenic events in this neural region occurs. EtOH treatment occurred during the periods equivalent to the first, second, third and all three trimesters in humans using similar administration procedures for both mothers and pups. Unbiased stereological estimates of the total number of pyramidal and granule cells within hippocampal regions CA1, CA3 and DG were performed when rats reached adulthood. The findings confirm previous reports that area CA1 is highly susceptible to EtOH exposure that occurs during either the early neonatal period or all three trimesters equivalent, while areas CA3 and DG are more resistant to EtOH-induced insult during all periods of hippocampal development examined.

Critical periods for the effects of alcohol exposure on brain weight, body weight, activity and investigation.

Using an animal model of fetal alcohol syndrome - which equates peak blood alcohol concentrations across different developmental periods - critical periods for the effect of alcohol on brain weight, activity and investigative behavior were examined. The periods of alcohol exposure were from gestational day (GD) 1 through 10, GD 11 through 22, postnatal day (PD) 2 through 10, or all three periods combined. The critical period of alcohol exposure for an increase in activity in juveniles was GD 11 through 22. This pattern was not seen in the same animals in adulthood; instead, increases in both activity and investigation were seen in animals exposed from PD 2 through 10 and not seen in animals exposed during all three periods combined. Brain weight was reduced by alcohol exposure from GD 11 through 22, PD 2 through 10 and all three periods combined. The period from PD 2 through 10 was the only period when the brain weight to body weight ratio was reduced. In conclusion, exposure to alcohol during the periods in the latter half of gestation or early postnatal period seem to have the most deleterious effects on the brain, activity and investigation in the rat. In addition, the effects of alcohol exposure over both the prenatal and postnatal period cannot be easily predicted from the effects of shorter periods of exposure.

Vitamin E does not protect against neonatal ethanol-induced cerebellar damage or deficits in eyeblink classical conditioning in rats

BACKGROUND Rodent studies have shown that heavy binge-like ethanol (EtOH) exposure during the brain growth spurt [postnatal days (PD) 4-9] causes cerebellar neuronal loss and deficits in cerebellar-mediated eyeblink classical conditioning (ECC). Oxidative stress has been implicated in EtOH-mediated brain damage, and studies using vitamin E have reported amelioration of EtOH-induced tissue damage, including protection in rats against EtOH-induced cerebellar Purkinje cell (PC) loss on PD 4 to 5. The purpose of this study was to determine whether dietary supplementation with vitamin E concurrent with binge EtOH exposure on PD 4 to 9 in rats would attenuate the cerebellar cell death and ECC deficits. METHODS Rat pups were given one of five different neonatal treatments: (1) intubation with EtOH in milk formula (twice daily, total dose 5.25 g/kg/day), (2) intubation with EtOH in milk formula supplemented with vitamin E (12.26 mg/kg/feeding), (3) intubation with milk formula that contained vitamin E only, (4) sham intubations, or (5) normally reared unintubated controls. Between PD 26 and 33, subjects received short-delay ECC for 3 consecutive days. Unbiased stereological cell counts were performed on cerebellar PCs of left cerebellar lobules I to VI and neurons of the interpositus nucleus. In a separate study with PD 4 pups, the effects of vitamin E on EtOH-induced expression of caspase-3 active subunits were assessed using Western blot analysis. RESULTS EtOH-treated groups showed significant deficits in acquisition of conditioned eyeblink responses and reductions in cerebellar PCs and interpositus nucleus neurons compared with controls. Vitamin E supplementation failed to protect against these deficits. Vitamin E also failed to protect against increases in caspase-3 active subunit expression induced by acute binge EtOH exposure on PD 4. CONCLUSIONS In contrast to the previously reported neuroprotective potential of antioxidants on EtOH-mediated cerebellar damage, vitamin E supplementation did not diminish EtOH-induced structural and functional damage to the cerebellum in this model of binge EtOH exposure during the brain growth spurt in rats.

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.

Neonatal ethanol produces cerebellar deep nuclear cell loss and correlated disruption of eyeblink conditioning in adult rats

Binge-like neonatal exposure to ethanol (EtOH) in rats, during the period of brain development comparable to that of the human third trimester, produces significant, dose-dependent Purkinje cell loss in the cerebellum and deficits in eyeblink classical conditioning. There are currently no published reports of whether neuronal loss in the cerebellar deep nuclei also results from binge-like neonatal exposure to EtOH and what the functional consequences of any cell loss might be. Since eyeblink conditioning requires cerebellar deep nuclear cells for normal learning to occur, we examined the effects of binge-like neonatal EtOH exposure on the total number of deep nuclear cells and eyeblink conditioning in adult rats. Group Ethanol (n=11) received EtOH doses of 5.25 g/kg/day on postnatal days 4-9, producing average peak blood alcohol concentrations of 363 mg/dl. Group Sham Intubated (n=11) underwent acute intragastric intubation on postnatal days 4-9 but did not receive any EtOH infusions. Group Unintubated Control (n=10) did not receive any intubations. When rats were at least 3 months old, they received either paired eyeblink conditioning or unpaired training. Following training, estimates of the total number of cerebellar deep nuclear cells were obtained using the optical fractionator, an unbiased stereological counting procedure. Rats in Group Ethanol had approximately 50% fewer deep nuclear cells compared to rats in Groups Sham Intubated and Unintubated Control, which did not differ. For 21 rats that received paired eyeblink conditioning, a highly significant correlation (+0.80) was found between the number of deep nuclear cells and learning rate in eyeblink conditioning.

Critical periods for the effects of alcohol exposure on learning in rats.

Critical periods for alcohol-induced deficits in spatial navigation and passive avoidance learning were investigated with a rat model of fetal alcohol syndrome. Rats were exposed to alcohol prenatally (Gestational Days 1-10 or 11-22) or postnatally (Postnatal Days 2-10) or throughout all 3 periods. Offspring were tested in either a spatial navigation or an avoidance task as juveniles or adults. As juveniles, the combined exposure group took longer to learn the spatial navigation task compared with all other groups. This effect was not seen in adults. Passive avoidance performance was not affected. These results suggest that long-term exposure to alcohol during development has adverse effects on spatial learning. The lack of differences in the short-term exposure groups implies that there may not be 1 critical period of alcohol exposure, but that the adverse effects of alcohol during development may be cumulative on some behaviors.

Alcohol Exposure during Development Alters Social Recognition and Social Communication in Rats

The present study examined the effects of postnatal alcohol exposure via gastric intubation on social communication of diet preference and social recognition. Rats were placed in one of three treatment groups. All treatments occurred from postnatal day (PD) 2 through 10 and Experiments 1 and 2 were conducted when the rats reached 60 and 100 days of age, respectively. Alcohol-exposed pups received a 3.0 g/kg dose of ethanol in milk solution that was delivered by insertion of PE-10 tubing down the esophagus daily from PD 2 through 10. Intubated control animals underwent intubation without ethanol or milk. Nontreated control pups were weighted daily. In Experiment 1, a nonexperimental rat was initially given access to lab chow mixed with a spice and then housed with an experimental rat for 30 min. The experimental rat was subsequently given access to two diets--one that the nonexperimental rat had consumed and a novel diet. It was found that the alcohol-exposed females consumed a greater percent of the communicated diet than the control females. In Experiment 2, the experimental rats were first exposed to a juvenile for 5 min and then exposed to the same juvenile after a delay of 30 or 90 min. Investigation time was recorded in both sessions and a reduction of investigation time in the second session is an indicator of social recognition memory. Alcohol-exposed rats of both sexes had poorer memory of a juvenile than both control groups after a 90-min delay. Together, these data indicate that basic components of social behavior may be altered by alcohol exposure during development.

Adequacy of maternal iron status protects against behavioral, neuroanatomical, and growth deficits in fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorders (FASD) are the leading non-genetic cause of neurodevelopmental disability in children. Although alcohol is clearly teratogenic, environmental factors such as gravidity and socioeconomic status significantly modify individual FASD risk despite equivalent alcohol intake. An explanation for this variability could inform FASD prevention. Here we show that the most common nutritional deficiency of pregnancy, iron deficiency without anemia (ID), is a potent and synergistic modifier of FASD risk. Using an established rat model of third trimester-equivalent binge drinking, we show that ID significantly interacts with alcohol to impair postnatal somatic growth, associative learning, and white matter formation, as compared with either insult separately. For the associative learning and myelination deficits, the ID-alcohol interaction was synergistic and the deficits persisted even after the offsprings’ iron status had normalized. Importantly, the observed deficits in the ID-alcohol animals comprise key diagnostic criteria of FASD. Other neurobehaviors were normal, showing the ID-alcohol interaction was selective and did not reflect a generalized malnutrition. Importantly ID worsened FASD outcome even though the mothers lacked overt anemia; thus diagnostics that emphasize hematological markers will not identify pregnancies at-risk. This is the first direct demonstration that, as suggested by clinical studies, maternal iron status has a unique influence upon FASD outcome. While alcohol is unquestionably teratogenic, this ID-alcohol interaction likely represents a significant portion of FASD diagnoses because ID is more common in alcohol-abusing pregnancies than generally appreciated. Iron status may also underlie the associations between FASD and parity or socioeconomic status. We propose that increased attention to normalizing maternal iron status will substantially improve FASD outcome, even if maternal alcohol abuse continues. These findings offer novel insights into how alcohol damages the developing brain.

Alterations in Hippocampal and Hypothalamic Monoaminergic Neurotransmitter Systems after Alcohol Exposure During All Three Trimester Equivalents in Adult Rats

Summary. Animal models of Fetal Alcohol Syndrome (FAS) often rely on exposing the developing organism to alcohol during either the prenatal or postnatal period only. Very few studies have examined brain changes resulting from alcohol exposure during both the prenatal and postnatal period, a period which is equivalent to all three trimesters in humans. In this study, we examined the effects of alcohol exposure during this prolonged period of neural development on hippocampal and hypothalamic neurotransmitters in the rat. Pregnant dams were intubated with alcohol from gestational day (GD) 1 to GD 22 and then their pups were intubated with alcohol from postnatal day (PD) 2 to PD 10. Alcohol-exposed rats of both sexes exhibited increased hippocampal noradrenaline (NE) concentration compared to intubated and nontreated control animals. Within the hypothalamus, alcohol-exposed females but not males exhibited increased NE concentration. Hypothalamic serotonin (5-HT) concentration was reduced in both alcohol-exposed and intubated-control rats compared to nontreated controls. The results suggest that both the hippocampal and hypothalamic NE systems are especially vulnerable to alcohol insult that occurs during a period of neural development that corresponds to the full human prenatal period.

Comparison of compound and cross-modal training on postoperative visual relearning of visual decorticate rats

The effects of postoperative bimodal compound conditioning and cross-modal transfer of learning on behavior were compared by training rats prior to visual decortication to avoid shock with visual intensity cues. On Postop Day 6, rats were given avoidance training in one of three cue conditions: auditory intensity cues (cross-modal), paired auditory and visual cues (compound conditioning), or no cues (no-training control). On Postop Day 7 rats in the no-training control and the cross-modal transfer conditions were retrained with the visual discrimination while rats in the compound conditioning group were either retrained with the visual intensity cue or trained with the auditory intensity cue. Postoperative cross-modal transfer training enhanced visual relearning whereas bimodal compound conditioning interfered with relearning. However, compound conditioning facilitated subsequent auditory discrimination learning. These results support the notion of an injury-induced neurological bias that is increased after bimodal compound conditioning and reduced after cross-modal training. Potential implications for neurological rehabilitation are also discussed.