Regulation of Molecular Composition at Glutamatergic Synapses

157 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2001.Proper targeting and clustering of synaptic molecules is necessary for the formation of functional glutamatergic synapses. Here I examined the clustering of presynaptic (Brain-Specific L-Proline Transporter, PROT) and postsynaptic (N-Methyl-D-Aspartate Glutamate Receptor, NMDAR) molecular markers in rat hippocampal cultured neurons. PROT localized specifically to presynaptic excitatory synapses. Immunostaining of PROT was detected several days following synapse formation, suggesting a regulatory function for the transporter. Approximately 60% of total pyramidal neurons expressed PROT, and PROT immunolabeling was seen at all terminals in expressing cells. In contrast, postsynaptic NMDAR was regulated by activity. Neurons treated with NMDAR antagonists showed increased synaptic NMDAR localization and 50% greater susceptibility to NMDAR-mediated excitoxicity after anagonist washout. Protein phosphorylation was necessary to induce synaptic NMDAR localization, but new protein expression was not necessary. Pharmacological blockade of phosphorylation reversed previously induced synaptic NMDAR localization. Blockade of cAMP-dependent protein kinase (PKA) was shown to prevent synaptic NMDAR localization following antagonist treatment, whereas stimulation of PKA induced synaptic localization of NMDARs in 24 hours. Thus activity blockade induces synaptic targeting of NMDAR through activation of PKA. Furthermore, activity regulation of synaptic NMDAR targeting appears to occur under physiological conditions, since the ration of inhibitory to excitatory input correlated with synaptic NMDAR localization. Low density cultures, corresponding with a low level of glutamic acid decarboxylase (GAD, the synthetic enzyme for GABA), exhibited far fewer synaptic NMDAR clusters compared to high density cultures, corresponding with a high level of GAD expression. An correlation between GABAergic innervation and synaptic NMDAR localization was also observed on individual neurons. Chronic blockade of GABAergic transmission resulted in a decrease in synaptic NMDAR localization. Interestingly, coverslips switched from high density cultured media to low density cultured media underwent a dramatic decrease in the number of synaptic NMDAR clusters, indicating the involvement of a soluble factor, perhaps BDNF, which has been shown to stimulate GAD expression in cultured neurons. Taken together, these results demonstrate that the ratio of inhibitory to excitatory activity regulates NMDAR localization in rat hippocampal neurons via phosphorylation.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD