Derangements in synaptic transmission are an important part of the pathology of several neurological and mental diseases including epilepsy, schizophrenia, depression, and perhaps Alzheimer's disease. Despite the medical significance of synaptic transmission and the important roles of synapses in information processing and storage in the brain, relatively little is known about the molecular composition of the key synaptic organelles involved in transmission or about the mechanisms by which the functions of these organelles are regulated. The proposed research involves a study of the regulation by protein phosphorylation of identified proteins at synapses in the central nervous system. The goal of this project is to measure the time course of regulatory phosphorylation events in different subcellular domains of hippocampal neurons after a variety of physiological and pharmacological treatments. Our studies will focus on phosphorylation events at the postsynaptic membrane of glutamatergic synapses following tetanic stimulation that can induce long-term potentiation, and following perfusion with modulatory agents that can alter induction of long-term potentiation. By comparing the time course of phosphorylation of different sites, we will attempt to """"""""chart"""""""" the sequence of regulatory phosphorylation events that is triggered by various stimuli. To make the proposed measurements, we will use a laser-scanning confocal immuno- fluorescence microscopy technique that we developed and have nearly perfected in the first three years of this grant. We will use phospho-site specific antibodies raised against proteins associated with the postsynaptic membrane of glutamatergic synapses, including CaM kinase II, the 2B and 2A subunits of NR2B, AMPA receptors, and densin-180. The antibodies will be engineered to recognize the proteins either when they are phosphorylated or when they are not phosphorylated at specific sites identified as targets for phosphorylation in vivo. Double-immunofluorescence labeling with these antibodies allows us to visualize the ratio of phosphorylated and nonphosphorylated protein at various cellular and subcellular sites within tissue slices. We will extend the immunofluorescence labeling method to the level of single neurons, by marking neurons that have been induced to undergo LTP by pairing of depolarization and tetanic stimulation under whole cell clamp, then examining the relative levels of phosphorylation along the dendrites and at synapses of the marked neuron. These data will help us to develop and test theories about the complex regulatory events governing control of synaptic strength in the hippocampus.
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