Excitotoxic effects of non-NMDA receptor agonists in organotypic corticostriatal slice cultures

The excitotoxic effects of the glutamate receptor agonists kainic acid (KA) and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and the corre sponding neuroprotective effects of the AMPA/KA receptor antagonist 2,3-dih ydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX) were examined in corti costriatal slice cultures. The purpose was to examine the feasibility of th ese cultures for excitotoxic studies, and to demonstrate possible different ial excitotoxic effects of KA and AMPA on striatal and cortical neurons. Sl ices of dorsolateral striatum with overlying neocortex were obtained from n eonatal rats and grown on semiporous membranes in serum-free medium for 3-4 weeks before exposure to KA or AMPA for 48 h. The uptake by injured cells of the fluorescent dye propidium iodide (PI) added to the culture medium wa s used as a quantifiable measure for neuronal degeneration and compared wit h efflux of the cytosolic enzyme lactate dehydrogenase (LDH) into the cultu re medium and loss of glutamic acid decarboxylase (GAD) activity in the tis sue. Histological sections were also stained by the fluorescent dye Fluoro- Jade (FJ), for degenerating neurons and by immunocytochemical staining for gamma-aminobutyric acid (GABA). Digitized images showed a dose (0-24 mu M K A, 0-6 mu M AMPA) and time (0-48 h) dependent increase in PI uptake in both striatum and cortex. In other cultures exposed to KA (24 mu M) or AMPA (6 mu M) together with NBQX (0.1-9 mu M), NBQX was found to exert a differenti al neuroprotective effect on striatum and cortex at low doses. NBQX was thu s more protective against KA in the cortex than in the striatum, while the opposite was seen in relation to AMPA. Regarding neurodegenerative markers, PI uptake was significantly correlated with (1) LDH release into the cultu re medium, (2) optical density of Fluoro-Jade staining, (3) loss of GAD-act ivity in tissue homogenates, and (4) loss of GABA-immunostained neurons. We conclude that both differences between compounds (AMPA vs. KA) and brain a reas (striatum vs. cortex) can be demonstrated in corticostriatal slice cul tures, which in conjunction with an established set of markers for neuronal cell damage appears to be a feasible model for studies of the neurotoxic a nd neuroprotective effects of glutamate receptor agonists and antagonists. (C) 1999 Elsevier Science B.V. All rights reserved.