AMPA RECEPTOR-MEDIATED EXCITOTOXICITY IN NEOCORTICAL NEURONS IS DEVELOPMENTALLY-REGULATED AND DEPENDENT UPON RECEPTOR DESENSITIZATION

AMPA lpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) excitoto xicity was examined in cultured neocortical neurons using the reductio n of (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to measure cell viability. Neurons were exposed to AMPA at different c ulture periods during development of the neurons. In order to describe the pharmacology of AMPA-mediated toxicity, several glutamate recepto r antagonists were used: MK-801, NS 394, NBQX, GYKI 52466, GYKI 53405 and GYKI 53655. Increased excitotoxicity was observed when cortical ne urons cultured for 5, 8 and 12 days in vitro (DIV) were exposed to a h igh concentration of AMPA (500 mu M) for 6 h. However, only at DIV 12 was part of the toxicity mediated directly through AMPA receptors sinc e 10 mu M MK-801 blocked all AMPA toxicity at DIV 5 and 8, but only so me 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% remain ed viable after exposure to AMPA even for neurons that were cultured f or 12 DIV. Since it is known that both glutamate and AMPA activate AMP A receptors with a fast and rapidly desensitizing response, this could explain the relatively low toxicity produced by 500 mu M AMPA. This w as investigated by blocking AMPA receptor desensitization with cycloth iazide. Using a lower concentration (25 mu M) of AMPA, addition of 50 mu M cyclothiazide increased the AMPA induced excitotoxicity in cultur ed cortical neurons at all DIV except for DIV 2. This combination of A MPA + cyclothiazide yielded 77% cell death for DIV 12 cultures. In con trast to the results observed with 500 mu M AMPA, the neurotoxicity me diated 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 com pounds, which have been reported to be selective non-competitive AMPA receptor antagonists, were here observed to block the AMPA toxicity wi th the following rank order: GYKI 53655 > GYKI 52466 greater than or e qual to GYKI 53405, which is in agreement with their published potenci es. (C) 1998 Elsevier Science Ltd. All rights reserved.