The objectives of this research are to ascertain the gangliosides and their molecular structures that modulate central nervous system (CNS) ethanol sensitivity and to reveal their mechanisms of action. One approach to alcoholism therapy or avoidance strategies is to discover biochemical components of the disorder, devise methods for altering those components, and testing their efficacy in altering alcohol abuse. Clinical testing is far removed from this research, but accomplishment of the specific aims will provide information on a clinically safe class of compounds, gangliosides, that can modify CNS ethanol sensitivity. LS and SS mice are lines derived from the heterogeneous HS stock in a bidirectional genetic selection experiment in which the selection was made only for differential CNS sensitivity to ethanol hypnosis. The HS strain, LS and SS lines and LS x SS recombinant inbred strains (RIS) will be used in this research.
The specific aims and methods of this research are: 1. To determine which gangliosides modulate ethanol sensitivity in vivo. Exogenous gangliosides administered intracerebroventricularly with micro-osmotic pumps in LS and SS mice will be used. The functional groups required for ethanol action will also be ascertained. 2. To determine which gangliosides modulate ethanol sensitivity in vitro. Primary cultures of neo-natal cerebellar granule cells and pre-natal Purkinje cell enriched cultures from HS mice will be used to determine the effect of exogenous gangliosides on ethanol inhibition of NMDA receptor-gated calcium flux. Synaptosomes from brains of LS and SS mice will be used to determine the effect of the B subunit of cholera toxin (which binds GM1) or exogenous gangliosides, on the genetic differences in ethanol enhancement of GABA-gated chloride flux. 3. To determine that GM1 increases the interaction of ethanol at the surface of synaptosomal plasma membranes (SPM) and to ascertain the correlation of GM1 concentration with CNS sensitivity to ethanol. These studies will use SS SPM with and without exogenously added GM1 and Fourier Transform NMR to determine the effects of GM1 on membrane-ethanol interactions, and LS and SS RIS to determine the correlation of ethanol sensitivity with GM1 concentration. 4. To determine ethanol-induced changes in ganglioside concentration and composition in specific cell types in vitro. These studies will use HS neo-natal cerebellar granule cells and pre-natal Purkinje cell enriched cultures to ascertain the effects of ethanol on ganglioside concentration and composition. The effects of ethanol on these parameters will be characterized for differential responses in the same cell cultures from LS and SS mice. 5. To determine calcium-induced changes in the surface exposure of gangliosides in vitro in the absence and presence of ethanol. These studies will use LS and SS synaptosomes, cerebellar granule cells and Purkinje cell enriched cultures which have been exposed to galactose oxidase as an enzyme probe of plasma membrane ganglioside surface exposure.
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