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.
