Development of binding sites for excitatory amino acids in cultured cerebral cortex neurons

Binding of [3H]glutamate, [3H]AMPA (RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolo-propionate) and [3H]kainate was investigated in membranes prepared from cerebral cortex of 4-day-old and adult mice and from cerebral cortex neurons cultured for different periods of time (2, 4, 8 and 14 days). For all ligands, the number of binding sites increased as a function of development both in vivo and in culture. A significant number of binding sites for the ligands could be demonstrated on the cultured neurons already after 2 days in culture. Scatchard analysis of the binding data showed a single population of binding sites for glutamate (KD approximately 200 nM) and kainate (approximately 6 nM) regardless of the developmental stage in vivo or in culture. In case of [3H] AMPA binding two binding sites with KD values of approximately 6 nM and 100-200 nM could be demonstrated both in vivo and in culture. Binding of [3H]glutamate to cultured neurons could be displaced by N-methyl-D-aspartate (100 microM) and quisqualate (3 microM) in an additive manner but D,L-4-aminophosphonobutyrate (100 microM) had no effect. AMPA binding to cultured neurons was much more (40-fold) sensitive than kainate binding to the newly developed AMPA selective antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline) indicating that kainate and AMPA bind to independent binding sites. Monitoring membrane potentials in the cultured neurons using the lipophilic cation TPP+ (tetraphenylphosphonium) it was demonstrated that potassium (55 mM) as well as glutamate, AMPA and kainate (100 microM) could depolarize the neurons both at early (2 days) and late (9 days) developmental stages in culture. The demonstration of functionally active receptors for the 3 excitatory amino acids in both immature (2 days in culture) and mature (8-9 days in culture) neurons is discussed in the light of previous studies of the development as a function of the culture period of effects of excitatory amino acids in neurons. It is concluded that no simple correlation exists between expression of binding sites for the excitatory amino acids and their ability to induce cytotoxicity and neurotransmitter release.