James R. West - US grants

It has been postulated that axon sprouting may be a mechanism which underlies some recovery of function following brain damage. Ethanol exposure may have deleterious effects on sprouting and recovery regardless of whether the damage is ethanol-induced or induced by trauma. However, almost no information is available concerning whether CNS neurons exposed chronically to ethanol are even capable of sprouting. The purpose of this proposal is to evaluate the effects of ethanol on sprouting in the adult mammalian brain. For several reasons, monitoring the reorganization of the rat dentate gyrus after partial deafferentation (by the unilateral removal of the entorhinal cortex) will be the method used to assess the effects of ethanol on sprouting: 1) unlike ethanol-induced damage, the entorhinal lesion leaves the contralateral region virtually intact, allowing an intraanimal control which increases the power of quantitative analysis; 2) it will provide data directly related to whether trauma-induced sprouting is retarded or inhibited by ethanol; and 3) it permits focusing on the behavioral recovery of a specific deficit that has been linked previously to sprouting. Quantitative methods including computerized image processing of acetyl-cholinesterase (AChE) histochemical and anterograde autoradiographic pathway tracing techniques will be used to evaluate the stereotypic sprouting responses of the septal, commissural, and crossed temperodentate projections to the dentate gyrus. Recovery of function will be evaluated by monitoring changes in spontaneous alternation correlated with neuroanatomical measures of sprouting within the same animals. By determining if long-term ethanol exposure inhibits sprouting and recovery. it will then be possible to assess whether abstinence has a positive (recoupable) affect on sprouting and recovery. These data will be important for developing and modifying theories to account for the reversibility and recovery from ethanol-induced and lesion-induced brain damage in alcoholics.

Alcohol consumption during pregnancy puts the fetus at risk for mental retardation and severe damage to the central nervous system (CNS). However, the severity of effects in the fetal alcohol syndrome are quite variable across individuals. Clinical observations and limited animal studies indicate that genetic differences among individuals can influence the severity of fetal alcohol effects. The proposed research will investigate the contributions of genetic differences to individual differences in susceptibility to alcohol-induced CNS damage during development. The long range objective is to identify factors influenced by genetic variation which can lead to differences in susceptibility to fetal alcohol effects on brain growth and structure and on behavior. The experimental approach will compare the susceptibility of rats from eight different inbred strains (those used in forming the NIH heterogeneous stock of rats for pharmacogenetic research) to effects of exposure to alcohol during the brain growth spurt, the period of most rapid brain growth. This period of brain development, which occurs postnatally in the first two weeks of life in rats, is roughly equivalent to that of the human third trimester. Exposure to alcohol during postnatal days 4-10 in randomly bred Sprague- Dawley rats has severe and enduring effects on brain growth and behavior. We will use artificial rearing procedures during this period to deliver controlled doses of alcohol in a nutritionally adequate diet to pups of the eight strains to characterize their susceptibility to fetal alcohol effects. There are four specific aims: 1) to identify those inbred strains which are most (and least) susceptible to restriction of brain growth and to determine the role of differences in blood alcohol concentrations in accounting for the strain differences in brain growth; 2) to determine whether strains differ both in terms of risks related to pharmacokinetic differences and in susceptibility of the brain independent of factors controlling blood alcohol concentration; 3) to examine the two most susceptible and most resistant strains identified on the basis of brain growth restriction for differences in neuron loss and neuromorphological changes in the hippocampal formation; 4) to test the identified strains for differences in behavioral impairments on tasks sensitive to hippocampal dysfunction. The proposed research will establish the contribution of genetic differences to variation in brain growth restriction, structural damage to the hippocampus and related behavioral impairments following alcohol exposure during the brain growth spurt.

The overall objective of this proposal is to establish an animal model system which will be useful for investigating the effects of cocaine exposure on the developing brain. Specifically, a technique for artificially rearing neonatal ras will be used to characterize neuromorphological changes resulting from cocaine exposure during the brain growth spurt, the period of most rapid brain growth. That period, which is roughly equivalent to that of the human third trimester, is a time when the developing brain is especially vulnerable to the effects of other insults. The first objective will be to determine basic developmental pharmacokinetic properties of cocaine in the neonatal rats using the artificial-rearing procedure. The first hypothesis to be tested is that cocaine exposure during the brain growth spurt can produce stunted brain growth (microencephaly) either on a temporary or permanent basis. The second hypothesis is that cocaine exposure during the brain growth spurt causes reductions in neuronal populations, and that neurons in various brain regions exhibit differential vulnerability to cocaine. State-of-the-art stereological counting techniques will be used to provide accurate quantitative estimates of entire populations of pyramidal and granule cells in the hippocampal formation, and Purkinje and granule cells in the cerebellum. The third hypothesis to be tested is that cocaine exposure during the third trimester equivalent results in vascular disruption and reactive astrogliosis especially in the neocortex. Glial fibrillary acidic protein (GFAP) immunocytochemistry will be used to identify affected regions throughout the brain. The elucidation of the types and loci of structural brain damage resulting from cocaine exposure during the brain growth spurt will be helpful in identifying stages of development when the brain is especially vulnerable to cocaine and in determining whether the pattern and timing of the cocaine exposure influences the type or extent of damage. These studies will also contribute to establishing a comprehensive picture of the spectrum of structural damage to the mammalian brain that can occur following cocaine exposure during the period of its most rapid development.

DESCRIPTION: (Adapted from the Investigator's Abstract) Although it has been 25 years since fetal alcohol syndrome (FAS) was first defined, it is still not clear whether or not the embryo suffers long-term brain deficits after alcohol exposure early in gestation. Since many women are binge drinkers, including those who can stop or significantly reduce their alcohol consumption once they are aware they are pregnant, and since many of them will not know they are pregnant for at least several weeks after conception, binge drinking during this early period may represent a very dangerous practice. Alarmingly, recent studies have provided evidence that contradicts the commonly held view that early pregnancy is not a vulnerable period for inducing birth defects. Therefore, until we understand the consequences of such exposure, misconceptions about the vulnerability of the conceptus during early development increases the likelihood of alcohol related birth defects, and delays the chances that the cause of those birth defects will be identified. This competitive renewal application represents the first comprehensive attempt to evaluate the effects of binge-like alcohol exposure during different discrete developmental events early in pregnancy on long-term structure and function of the brain. First, it utilizes the C57BL/6J mouse, which is known to be susceptible to alcohol-induced craniofacial and brain dysmorphology associated with FAS. Second, the timing of the alcohol exposure will focus on discrete embryonic events. Third by using a standard series of peak blood alcohol concentrations, it will be possible to compare the "relative vulnerability" of different early prenatal periods. The studies will determine whether embryos exposed to alcohol during early development, even at blood alcohol concentrations (BACs) below that resulting in facial dysmorphology, nevertheless display lasting central nervous system dysfunction. Specific Aim #1 will evaluate the effects of oral alcohol exposure occurring during gastrulation (gestation day [GD] 7:0) on fetal (GD14:0) somatic growth, brain morphology and craniofacial anomalies, to determine the threshold at which brain deficits may be induced in the absence of craniofacial dysmorphology. Specific Aim #2 will evaluate the effects of alcohol exposure during specific developmental events from fertilization to early gestation. Specific Aim #3 will test the hypothesis that binge-like alcohol exposure that is time-locked to specific developmental events early in development will have more deleterious effects than will similar duration of alcohol exposure that specifically misses the critical events identified in Specific Aim #2. The results will be beneficial for counseling women about the risks of drinking during the initial weeks of pregnancy, and may help to explain the variation in brain damage observed in children damaged by in utero alcohol exposure. The studies also will provide the foundation for mechanistic studies to identify how alcohol exposure damages the developing conceptus.