The increased abuse of cocaine in the United States has resulted in a significant number of babies, up to 20% in urban hospitals, being exposed to cocaine in utero. These infants have a number of congenital problems, including delayed in neural development leading to long-term changes in cognitive functioning. The mechanisms through which cocaine alters the developing nervous system are not well understood. Cocaine has been shown to block the development of neuritic processes in cultured cells. The initiation and outgrowth of axons and dendrites is highly controlled. When the developing neuron is activated through the proper signaling cascade specific kinases in the cytoplasm phosphorylate cytoskeletal proteins which are required for axon initiation and maturation. It is known that prenatal cocaine exposure inhibits G-proteins that activate key kinases required for cytoskeletal phosphorylation. Aberrant neuronal development could result from altered axon growth if in utero cocaine exposure caused even transient blocks in G-protein function. The purpose of this application is to study the hypothesis that cocaine interferes with axonal initiation and outgrowth through a direct interaction with G-proteins in the signal transduction cascade leading to a down regulation of cytoskeletal proteins and aberrant axonal outgrowth. The immediate goals of this research are to determine if cocaine inhibits neurite initiation and outgrowth in cultured cells through alterations in cytoskeletal proteins, and to determine whether cocaine causes transient alterations in G-proteins leading to changes in the differentiation or maturation of developing neurons. The effects of cocaine will be studied in cultured cells which are induced into a neuronal phenotype through different signaling cascades. Levels of cytoskeletal proteins will be assessed with immunofluorescence microscopy and quantitated using western blot analysis. In addition Gi alpha and p21ras activation with be examined using western blot analysis and northern analysis. Immediate early genes c-fos and jun, which are induced through G-protein signaling, will also be studied to determine whether cocaine-induced alterations in these proto-oncogenes are regulated through alteration in G-protein signaling. Once the actions of cocaine have been resolved in the cell culture model, it is the long-term objective of this study to look at these changes in a whole animal model.
