Kim E. Light - US grants

This proposal will test the hypothesis that acetylcholine synapses in various brain regions will show specific neurochemical alterations in their developmental ontogeny when exposed to alcohol. Furthermore, these alterations will be dependent upon 1) the dose and duration of alcohol exposure; 2) the specific developmental stage of a particular brain region at the time of alcohol exposure, and (3) the developmental stage of the particular brain region during which analysis for specific alterations is conducted. Rat pups will be administered ethanol or isocaloric sucrose via twice daily intragastric intubation with a fine diameter Silastic tube. The pups will be kept with lactating dams in litters of ten. Exposure periods will inlcude post-natal day one (PN1) to PN5; PN1 to PN10; PN1 to PN15 and PN1 to PN20. Animals will be sacrificed and various brain regions dissected for analysis. For each period of exposure, dates of sacrifice will include the day following the last dose, five days following the last dose, PN20, PN60 and PN120. Brain regions to be dissected and analyzed include cortex, striatum, hippocampus, midbrain-thalamus, hypothalamus, medulla-pons and cerebellum. Regional neurochemical analyses will involve measurements of acetylcholinesterase activity, choline acetyltransferase activity and determinations of muscarinie selective receptor affinity (Kd), and density (Bmax) via saturation analysis using the radioligands (3H)-QNB and (3H)-pirenzepine. Further analysis of muscarine selective receptors will include competition studies to analyze receptor selectivity and ethanol-induced alterations in receptor specificity. The functionality of alcohol-induced alterations of receptor binding parameters and/or enzyme alterations will be assessed by studies on the muscarinic receptor stimulated release of inositol phosphates. Postnatally, the rat brain undergoes the same type of growth and development as does the human brain during the third trimester of pregancy. Accordingly, these studies in rats are designed to look for possible neurochemical outcomes of human fetal ethanol exposure.

One of the most extensively studied aspects of ethanol-induced neural toxicity is the specific loss of Purkinje ce s the cerebellum. The consequences of Purkinje cell loss are thought to be involved in the deficiencies of motor coordination and gait exhibited by children diagnosed with fetal alcohol syndrome. Although loss of Purkinje cells has been repeatedly demonstrated to be a consistent and reliable consequence of early postnatal ethanol exposure in the rat, the manner and time course of this cell loss has not been clearly identified. We hypothesize that Purkinje cell loss in the cerebellum results from the induction of apaptosis in a manner similar to an acute toxic response (time course related to peak blood ethanol concentration). Thus, a linear relationship will exist between ethanol concentrations and the extent of apoptosis. Evaluation of this hypothesis will include in vivo exposures to ethanol using the intra gastric intubation technique and in vitro experiments using the organotypic slice culture model. Throughout these studies we will use ethanol exposures on PN4 (or the in vitro equivalent) in the rat, since the strongest relationship between peak BEC and percent reduction of Purkinje cells has been demonstrated to occur on this day. This exposure paradigm is a model of third trimester ethanol exposure in the human. Two specific aims will guide this research: (1) The first aim involves the identification of the relationship between peak blood ethanol concentration (BEC) and Purkinje cell death following in vivo ethanol administration. These experiments will identify the timing and manner of Purkinje cell death as well as the linear relationship between the magnitude of cell death to peak BEC. Apoptotic cell death will be identified by the presence of fragmented DNA, apoptotic cell morphology, increased expression of pro-apoptotic associated antigens (Bax, caspase-3) and decreased expression of anti-apoptotic associated antigens (p53). (2) The second specific aim will use the in vitro organotypic slice culture technique to further explore the nature and mechanisms of ethanol-induced Purkinje cell apoptosis. Initially, these studies will parallel those described under the first specific aim in order to confirm the ability of ethanol to produce Purkinie cell apoptosis. These studies will also establish the direct linear relationship between ethanol concentration and the magnitude of Purkinje cell death. This second specific aim provides the foundation for the use of this technique as an experimental paradigm to determine the mechanism and specific pathway(s) involved in ethanol-induced apoptosis as well as to dissect the regulation of these pathway(s) with standard biochemical and pharmacological techniques.