| Hippocampal long term potentiation (LTP) is a form of synaptic plasticity elicited by high frequency stimulation that is thought to underlie certain types of learning and memory in mammals. The location (pre- vs. post-synaptic) of LTP mechanisms remains highly controversial. In this thesis, a biochemical approach was used to address this issue and to better understand the molecular changes occurring during LTP. Unfortunately, conventional single electrode stimulation only potentiates a small fraction of hippocampal slice synapses, making detection of subtle biochemical changes difficult to impossible. Thus, a stimulation strategy was designed utilizing multiple placements of a multiple-electrode array (the rake) to optimize the fraction of CA1 mini-slice synapses receiving LTP stimulation.;Using this method, we investigated the effects of LTP stimulation on specific candidate molecules, carefully chosen to reveal information about the nature, location, and time course of mechanisms underlying LTP. Synapsin I, a presynaptic vesicle-associated protein, is believed to regulate the availability of synaptic vesicles for release in a phosphorylation state-dependent manner. It has been proposed that its phosphorylation by Ca$sp{2+}$/calmodulin dependent protein kinase II (CaMKII) leads to increased transmitter release, and may underlie a presynaptic component of LTP. We found that LTP stimulation produces a 6-fold increase in synapsin I phosphorylation at its CaMKII sites (from 2% to 12%), an effect which persists for 30 minutes. This effect is fully blocked by APV, an NMDA receptor antagonist which blocks LTP, and KN-62, an inhibitor of CaMKII.;We also examined the role of postsynaptic glutamate receptor expression in LTP. Late-phase (3 hour) LTP was associated specifically with an increase in synthesis and expression of postsynaptic AMPA (GluR1 and GluR2/3), and not NMDA (NR2A and NR2B), receptor subunits. Late-phase LTP and increased AMPA receptor synthesis are both blocked when inhibitors of cAMP-dependent protein kinase or gene transcription are present during and after stimulation, strengthening the correlation between LTP and the observed increases. These data also support a model of LTP in which CREB activation directs late-phase LTP. Collectively, our data show changes in both pre- and post-synaptic compartments occur with different time courses, supporting models of LTP in which temporally distinct mechanisms occur in each compartment. |
