Regulation of N-Methyl-D-Asparate receptor activity in cultured rat hippocampal neurons

NMDA receptors (NMDARs) play a crucial role in neuronal development, synaptic plasticity, and excitotoxicity, therefore regulation of NMDAR function is important in both physiological and pathological conditions. In this study, the regulation of the properties of NMDARs, including rundown and desensitization, in cultured rat hippocampal pyramidal neurons was investigated by electrophysiological, immunocytochemical, and biochemical approaches. Novel mechanisms for regulating rundown and desensitization of NMDARs were identified. NR2B-subtype NMDARs, which are mainly extrasynaptic, showed faster and more extensive peak current rundown in response to repeated agonist applications compared with NR2A-containing NMDARs, which are mainly synaptic. Moreover, rundown of the extrasynaptic, 2B-subtype receptors was largely independent of Ca²⁺ and dependent on tyrosine dephosphorylation, whereas rundown of the synaptic, 2Acontaining receptors was Ca -dependent and regulated by F-actin. The differences in rundown of the two subpopulations of NMDARs were determined by subcellular localization rather than the subunit composition, since synaptic 2B-subtype and 2Acontaining receptors were resistant to Ca2+-independent rundown, and extrasynaptic 2Bsubtype and 2A-containing receptors were vulnerable to Ca -independent rundown. Furthermore, an increase in receptor internalization and resulting decrease in numbers of NMDARs available on the cell surface were closely correlated with the Ca -independent rundown of extrasynaptic NMDARs. Subcellular localization also regulates NMDAR desensitization. The glycineindependent desensitization of NMDARs in rat cultured hippocampal neurons decreases during development. This decrease was not dependent on a switch in subunit composition, nor was it due to a change in the sensitivity of NMDARs to agonist or zinc during development. Instead, the developmental decrease in glycine-independent desensitization correlated with the synaptic localization of the receptor. Furthermore, overexpression of PSD-95 in immature neurons reduced NMDAR desensitization, and dispersion of PSD-95 away from synapses or manipulations that induced movement of NMDARs away from synapses increased NMDAR desensitization in mature neurons. We conclude that synaptic localization increases stability of hippocampal neuronal NMDAR responses to sustained agonist exposure. Our results elucidate mechanisms for regulating NMDAR function that tune receptor activity in neurons of different developmental stages, or the response of subpopulations of NMDARs in a single neuron to different stimuli.Medicine, Faculty ofGraduat