Excitatory amino acid-induced neuronal cell death in rat cerebellar granule cell cultures

Abstract

Glutamate is the principle excitatory neurotransmitter in the mammalian CNS [l]. It's actions are mediated by two classes of receptor, namely the ionotropic excitatory amino add (EAA) receptors and the metabotropic EAA receptors. The ionotropic EAA receptors comprise an integral ion channel and are further subdivided into N-methyl-D-aspartate (NMDA), a-amino-3methyl-4-isoxasole propionate (AMPA) and kainate receptors on the basis of their pharmacology. In contrast, the metabotropic receptors are coupled to G-proteins, and their stimulation results in changes in the levels of cellular second messengers [2]. Excessive EAA receptor activation has been implicated in a wide variety of both acute and chronic neurological disorders including stroke, epilepsy and Huntington's disease [3,4]. As yet, the mechanisms which lead to neuronal injury are not fully understood.In order to investigate this, a model system was set up using cultured rat cerebellar granule cells. These neurons express a wide variety of EAA receptor subunits and subtypes [5-71 and have been used previously to study excitotoxicity [8,9]. The majority of these studies have been carried out using cells cultured in the presence of serum which has itself been implicated in the induction of excitotoxicity [lo] and in the regulation of EAA receptor subunit expression [ll]. For this reason, in this study, the neurons were cultured in the presence of a serum-free growth supplement (N,) to prevent any interference by serum. In addition, our technique produces relatively pure cultures of these neurons (>99%) and thus minimizes the effects of other cell types.Cerebellar granule cell cultures were prepared f m Sprague-Dawley rats on postnatal day 8 and cultured in Eaties Basal Medium supplemented with 10% (v/v) ,heat inactivated foetal calf serum, 25 mM KCI, 50 pgml gentamicin, 100 IU/ml penicillin and 100 pgml streptomycin. Cells were seeded into poly-D-lysine coated 96 well tissue culture plates at a density of 0.

Role of Group III Metabotropic Glutamate Receptors in Excitotoxin‐Induced Cerebellar Granule Cell Death

“…The excitotoxic actions of glutamate (Choi, 1987;Rothman et a!., 1987;Lysko et al, 1989;Boje et al, 1993;Platt and Bristow, 1995;Wood and Bristow, 1998) and BMAA Ross et al, 1987;Weiss et a!., 1989;Staton and Bristow, 1997) have been characterised in several neuronal preparations. However, whereas glutamate and BMAA have unequivocal neurotoxic actions, ACPD produces effects ranging from neurotoxicity (Aleppo et al, 1992;McDonald and Schoepp, 1992;Schoepp et al, 1994) to neuroprotection (Koh et al, 1991;Chiamulera et al, 1992;Siliprandi et al, 1992;Pizzi et al, 1993Pizzi et al, , 1996aColwell et al, 1996).…”