The mechanism by which ethanol exerts its effect on brain morphology is not well understood. Therefore, this proposal is designed to study the mechanism underlying morphological changes after chronic ethanol exposure and withdrawal from this exposure. Our experiments and those reported in the literature suggest that morphological changes occur prevailingly in dendrites, dendritic spines, and synaptic contacts. Since microtubules are responsible for maintaining dendritic and axonal arborization and actin filaments are responsible for maintaining dendritic spines and synaptic contacts, it is possible that alcohol-induced alteration of these cytoskeletal structures constitutes the mechanism of morphological changes observed following chronic ethanol administration. In order to establish the nature of these changes, we shall determine possible modifications in the composition of microtubules and actin filaments in individual neuronal compartments. We shall also determine whether mutual interactions between microtubules and actin filaments and their respective associated proteins are affected. To this end, we have designed the following experiments. Experiment I. With quantitative immunoblotting, we shall establish the relative levels of polymerized and nonpolymerized tubulin and actin and the relative levels of posttranslationally modified alpha-tubulin (acetylated and tyrosinated) in the whole brain. Experiment II. With immunoelectron microscopy, we shall study: a) relative levels of posttranslational modifications: acetylation and tyrosination of (alpha-tubulin subunit in axons and dendrites, respectively; b) distribution of microtubule associated proteins (MAP 2 and MAP tau); c) colocalization of MAP 2 and MAP tau with tyrosinated and acetylated microtubules in dendrites and axons, respectively; d) distribution of actin filaments in dendritic spines and their colocalization with actin regulatory proteins (myosin and MAP 2); e) colocalization of actin and MAP 2 in the postsynaptic density (PSD) of axosomatic and axospinous synapses. Experiment III. With conventional electron microscopy, we shall determine in selected brain regions the: a) density of microtubules in dendrites and axons; b) microtubule spacing in dendrites. Alcohol-sensitive (LS[IBG]) mice will be used in these experiments.
