Modulation of AMPA glutamate receptor functions in primary neuronal cultures

Abstract: Primary neuronal cultures were used to investigate the properties of AMPA receptors and functional consequences of their activation, as well as AMPA receptor desensitization and possibilities for its modulation. We established the presence and spontaneous functional activity of AMPA receptors in cerebellar granule and cortical cell cultures. Stimulation of AMPA receptors under normal conditions had a limited effect on neuronal calcium metabolism. When the desensitization of AMPA receptors was blocked by cyclothiazide we monitored a dramatic increase in both [Ca2+]i and 45Ca2+ uptake after application of AMPA in both types of neurons with much higher responses in cortical cells. Application of cyclothiazide induced [Ca2+]i oscillations in single cortical neurons, probably indicating cyclothiazide amplification of AMPA receptor activation by endogenously released glutamate. We found that AMPA receptor blockade by novel uncompetitive antagonist GYKI52466 could be overcome by increasing concentrations of cyclothiazide indicating that the two compounds may act via allosterically connected binding sites on the AMPA receptor. Kainate-elevated [Ca2+]i was also antagonised by GYKI52466, although not completely, with the remaining GYKI52466-insensitive fraction of kainate-induced [Ca2+]i probably due to activation of native kainate receptors. Further we also characterised AM PA receptor-induced activator protein (AP- 1 ) transcri ption factor DNA binding. Stimulation of AMPA receptors induced AP-1 transcription factor activation both in the presence and absence of cyclothiazide. However, in the presence of cyclothiazide the maximal AP-1 binding activity was higher and was reached faster. Application of AMPA alone activated an AP-1 complex consisting of c-Fos and Jun D protein dimers. Cyclothiazide co-application induced an AP-1 complex in which the c-Fos and Jun D proteins were accompanied by c-Jun. Fos B and Jun B. These data suggest that blockade of AMPA receptor desensitization may produce both quantitative and qualitative changes in AMPA receptor-mediated glutamate effects on gene transcription. AMPA receptors may mediate glutamate neurotoxicity. To study this phenomenon, we analysed effects of AMPA receptor overactivation by using various measurements for apoptotic or necrotic cell death. Our results clearly demonstrate that AMPA receptors may be involved in the neurotoxic effects of glutamate in situations where the desensitization of AMPA receptors is impaired. AMPA receptor mediated toxicity display morphologically distinct features of both necrosis and apoptosis in the same cell population. Similar to other neuronal systems, the rate of AMPA receptor desensitization may be regulated intracellularly. Based on this assumption we examined effects of impaired energy production on AMPA-induced neurotoxicity and Ca2+ fluxes in cerebellar granule cells. No toxic effects were observed when either AMPA or low concentrations of inhibitor of oxydative phosphorylation, NaCN, were applied alone. In contrast, co-application of these compounds produced both necrotic and apoptotic cell death preceded by a pronounced elevation of [Ca2+]i suggesting that metabolic inhibition had increased the responsiveness of AMPA receptors, possibly by interferring with the rate of desensitization. Finally, we tested the possibilities for functional modulation of glutamate signaling by other transmitter systems down-stream of the ionotropic glutamate receptors, by monitoring the functional responses to NMDA and AMPA receptor stimulation in granule cells pre-exposed to M cholinoreceptor agonist carbachol. Carbachol pre-treatment potentiated both AMPA- and NMDA-induced AP-1 DNA binding but not 45Ca2+ uptake or neurotoxicity, suggesting that some signaling pathways are sensitized by chronic muscarinic receptor stimulation. These pathways appear to be located down-stream of NMDA and AMPA receptor-controlled cation channels and may be specifically involved in glutamate-mediated regulation of transcription factor activity. In conclusion, activation of AMPA receptors is short-lasting because of the rapid receptor desensitization. Blockade of desensitization dramaticaly enhances AMPA receptor-mediated responses. At least partially, the rate of desensitization may be modulated from within the cell, probably via an energy-dependent mechanism. In energetically compromised neurons normal rate of AMPA receptor desensitization can not be maintained resulting in Ca2+ overload and neuronal death. Finally, even when the rate of AMPA receptor desensitization is constant, the strength of glutamate effects via AMPA receptors is dependent on the responsiveness of signaling pathways down-stream of AMPA receptors.