During the last funding period, a core facility was established for the production of monoclonal antibodies against phosphoproteins of the basal ganglia. In addition, a second facility was established for the study of intact embryonic basal ganglia (striatal) cells in vitro, using a rotation-mediated reaggregation culture system (originally part of Project IV). Because these two facilities subserve common goals and utilize similar materials, the projects have been combined into a common core facility. We propose to study the regulation of protein phosphorylation in intact striatal neurons, employing the reaggregate culture system. The long-range goal of such studies will be to determine the role of protein phosphorylation in the development and function of striatal neurons. Specifically, this proposal will focus on three lines of investigation. First, we will study the regulation of the developmental expression of striatal-specific phosphoproteins. In particular, we will consider the roles of cell-cell interaction (e.g. neuron-glia, striatal neuron-cortical neuron, etc.) and diffusible factors (eg. neuronal growth factors). Second, we will study the regulation of the cyclic AMP-dependent protein phosphorylation pathway. These studies will concentrate on the role of dopamine and the potential role of a convergent pathway involving casein kinase II. Third, we will study the action of the excitatory amino acid glutamate, the major excitatory neurotransmitter in the cortico-striatal pathway, in the modulation of dopamine-regulated protein phosphorylation in the striatum. Because many of the experiments will involve pharmacological regulation, it is anticipated that our studies will furnish new insights into the response of striatal neurons to neuroactive and psychoactive drugs. We propose to generate monoclonal antibodies against striatal phosphoproteins, particularly those that have been difficult to purify as well as those that are membrane-associated, in order to allow their convenient purification, their structural and functional characterization, and their immunocytochemical localization. One strategy will be to raise monoclonal antibodies against specific amino acid sequences of these phosphoproteins by use of synthetic peptides or proteolytic fragments. In the case of membrane proteins, we will use peptides encompassing sequences that most likely have a cytoplasmic localization and are therefore more highly antigenic. In addition, antibodies that distinguish between phosphorylated and dephosphorylated forms of these proteins will be generated; again, peptides comprising specific amino acid sequences of a given protein will be most useful. The selectivity and titer of any resultant antibodies will be determined using a variety of strictly defined assays. The reaggregate striatal cell culture system and a battery of highly specific monoclonal antibodies will be essential tools, useful to the entire program project, as well as by a substantial number of other investigators being supported by grants from the NIMH, in the elucidation of the role of protein phosphorylation in basal ganglia function.
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