AMPA receptor mediated excitotoxicity in neocortical neurons is developmentally regulated and dependent upon receptor desensitization

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) excitotoxicity was examined in cultured neocortical neurons using the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to measure cell viability. Neurons were exposed to AMPA at different culture periods during development of the neurons. In order to describe the pharmacology of AMPA-mediated toxicity, several glutamate receptor antagonists were used: MK-801, NS 394, NBQX, GYKI 52466, GYKI 53405 and GYKI 53655. Increased excitotoxicity was observed when cortical neurons cultured for 5, 8 and 12 days in vitro (DIV) were exposed to a high concentration of AMPA (500 mu M) for 6 h. However, only at DIV 12 was part of the toxicity mediated directly through AMPA receptors since 10 mu M MK-801 blocked all AMPA toxicity at DIV 5 and 8, but only some of the AMPA response at DIV 12. This indicated that NMDA receptors were being activated, causing some of the observed toxicity. The high dose of AMPA was not sufficient to damage all neurons since 59% remained viable after exposure to AMPA even for neurons that were cultured for 12 DIV. Since it is known that both glutamate and AMPA activate AMPA receptors with a fast and rapidly desensitizing response, this could explain the relatively low toxicity produced by 500 mu M AMPA. This was investigated by blocking AMPA receptor desensitization with cyclothiazide. Using a lower concentration (25 mu M) of AMPA, addition of 50 mu M cyclothiazide increased the AMPA induced excitotoxicity in cultured cortical neurons at all DIV except for DIV 2. This combination of AMPA + cyclothiazide yielded 77% cell death for DIV 12 cultures. In contrast to the results observed with 500 mu M AMPA, the neurotoxicity mediated directly by AMPA receptors when desensitization was blocked was seen as early as 5 DIV since 10 mu M MK-801 did not completely block the response whereas 10 mu M NBQX did. The 2,3-benzodiazepine GYKI compounds, which have been reported to be selective non-competitive AMPA receptor antagonists, were here observed to block the AMPA toxicity with the following rank order: GYKI 53655 > GYKI 52466 greater than or equal to GYKI 53405, which is in agreement with their published potencies. (C) 1998 Elsevier Science Ltd. All rights reserved.