This proposal for an ADAMHA Research Scientist Development Award (RSDA) outlines a series of experiments which address fundamentally important questions about the mechanisms underlying long-term potentiation (LTP). LTP is along-lasting increase in the strength of synaptic transmission produced by brief, repetitive activation of excitatory pathways. It is the most compelling model in the mammalian brain for a neural mechanism related to learning and memory. Using cellular electrophysiological and biochemical techniques, the proposed experiments will examine the intracellular factors which are required for the induction and maintenance of LTP in the CA1 region of the hippocampal slice preparation. Collaboration with several neurochemists and molecular biologists will permit the utilization of biochemical methodology to test the effectiveness of experimental manipulations. This will not only make the experimental results more meaningful but also will foster my scientific growth and facilitate the development of techniques and approaches not presently used in my laboratory. The induction of LTP in the CA1 region of the hippocampus requires activation of postsynaptic NMDA (N-methyl-D-aspartate) receptors and concomitant postsynaptic depolarization leading to the influx of calcium, the presumptive critical trigger for LTP. It has been proposed that this rise in calcium may activate specific protein kinases. An initial set of experiments will examine the effects on basal synaptic transmission of non- specific kinase inhibitors, specific peptide inhibitors of calcium/calmodulin-dependent protein kinase II or protein C, and okadaic acid, a phosphatase inhibitor. Whether these compounds act primarily pre- or postsynaptically also will be determined. Biochemical assays of in situ substrate phosphorylation will be performed to determine directly the efficacy of the various kinase or phosphatase inhibitors. Intracellular biochemical pathways potentially involved in LTP will be examined by: (1) determining whether kinase inhibitors depress previously established LTP to a greater degree than basal synaptic transmission, (2) determining the effects on LTP of GTP-binding protein inhibitors or lithium, and (3) monitoring ionic conductances known to be modulated by second messenger systems. The involvement of de novo protein synthesis or RNA synthesis in LTP will be examined using specific protein synthesis and RNA synthesis inhibitors. Biochemical assays will determine the effectiveness of these compounds. Preliminary results indicate that it is possible to generate two distinct forms of NMDA receptor-dependent synaptic enhancement; LTP and a decremental short-term synaptic potentiation (STP). Experiments will be performed to determine the factors which control the duration of synaptic potentiation and the events responsible for converting STP to LTP. A final section will describe novel experimental approaches which eventually may permit more specific and potent manipulation of neuronal biochemical pathways. A detailed understanding of the mechanisms underlying synaptic transmission and LTP will yield important insights into the cellular and molecular properties underlying synaptic plasticity and thus human memory. This in turn may eventually lead to pharmacological interventions which either promote the ability to learn and remember or retard the deterioration of this ability.
