Brief sensory stimulation can produce very prolonged or even permanent changes in the behavior of an animal. The mechanisms that neurons use to generate such prolonged changes are not yet understood. This project will investigate the role of two protein kinases in a long-lasting change in neuronal excitability that occurs in a simple system of invertebrate neurons, the bag cell neurons of Aplysia. In response to brief synaptic stimulation, these neurons generate a 30 minute afterdischarge that is followed by a prolonged period of relative inexcitability. In vivo, this sequence triggers a series of reproductive behaviors lasting several hours. At the onset of afterdischarge, there is an elevation of cyclic AMP levels, and the stimulation of phosphoinositide hydrolysis in bag cell neurons. The first series of experiments win use patch clamp recordings, video-enhanced microscopy and digital imaging of intracellular calcium to test the hypothesis that the activation of protein kinase C in isolated neurons leads to an extension of plasma membrane at the distal tips of neurites and that a new species of voltage-dependent calcium channels is specifically activated in this newly-extended membrane. The second series of experiments will analyse the action of autoactive peptides that alter cyclic AMP levels. The effects of these peptides on potassium currents and on the movement of secretory granules into the tips of neurites will be analysed and the role of the cyclic AMPdependent protein kinase in these events will be tested using specific protein kinase inhibitors. Studies of the isolated neurons will then be coupled with measurements of neuropeptide release from intact clusters of bag cell neurons to determine the effect of simultaneous activation of the cyclic AMP system and of protein kinase C. In particular, these experiments will test the possibility that such co-activation leads to a rapid alteration in the distribution of active ion channels that is coupled to the formation of new morphological sites for the release of the neuropeptides. Finally, the molecular identity of the potassium and calcium channels that are regulated by these protein kinases will be determined using molecular cloning. Antibodies against these channel proteins will then be used to determine directly whether the changes in the electrical properties of the bag cell neurons that occur at the onset of afterdischarge are associated with changes in the phosphorylation state of the channel proteins and/or in their cellular localization.
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