Serum and depolarizing agents cause acute neurotoxicity in cultured cerebellar granule cells: role of the glutamate receptor responsive to N-methyl-D-aspartate.

Abstract

The life span of neonatal rat cerebellar granule cells, grown in basal minimal Eagle's medium containing 10% (vol/vol) fetal calf serum, was extended to 21-30 days by weekly supplementation with glucose. Addition of 1% fetal calf serum to the culture at 14 days killed 85% of the cells within 1 hr. This lethal effect could be prevented by the Nmethyl-D-aspartate (NMDA) receptor antagonists dibenzocyclohepteneimine (MK-801) and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP). These findings suggested that the glutamate in the serum caused the dramatic neuronal death through action on the NMDA receptor. Indeed, a 5-min incubation in a Locke physiological salt solution containing 20 pM glutamate and 5 pM glycine killed 55-90% of the cells.This acute toxicity could be prevented by a lyso-GM1 ganglioside with N-acetylated sphingosine. The relatively low glutamate content of the sera analyzed suggests that factors in addition to glycine potentiate serum neurotoxicity. The above noted antagonists of the NMDA receptor also greatly reduced the lethal effect of depolarization by 90 mM KCl or 10 FaM veratridine. Therefore, it is likely that the toxicity of the depolarizing agents is mediated by glutamate released from the cells. It is concluded that survival of cerebellar neurons in primary culture may be strongly affected by unsuspected neurotoxic phenomena elicited by brief action of a rather low glutamate concentration.The possibility that primary cultures of brain neurons might be damaged by toxic actions of glutamate has apparently received little attention other than by those specifically studying excitotoxicity (1-5). However, as will be shown, rapid death of neurones may occur under conventional experimental manipulations of neuronal cultures. Similar events may affect neurons in brain slices (6, 7) and could have important implications for pathological processes in the intact brain (1-5).Excitotoxicity mediated by glutamate and its analogs has been ascribed in many instances to excessive activation of the N-methyl-D-aspartate (NMDA) receptor. Two types of processes have been distinguished: (i) an acute reaction apparently requiring action of glutamate at high concentration, which leads to swelling by influx of Na+, Cl-, and water, terminating in lysis within 1-2 hr (8-10); and (ii) a delayed toxicity extending over approximately 20 hr. Though the excitotoxicity is initiated by glutamate, it soon becomes insensitive to glutamate receptor blockers and proceeds even after glutamate removal (11). It requires extracellular Ca2l and is dependent on prolonged elevation of free cytosolic Ca2+ (10)(11)(12)(13)(14)(15)(16) (19,20). The cell concentration was 1.6 x 106 cells per ml, and the medium was not changed throughout the cultivation period. Experiments were conducted on cells grown in a 24-well multiwell dish (Nunc) with 0.5 ml of medium per well and in 35-mm dishes (Nunc) with 2 ml per dish. The life span of the cells was extended up to 31 days in vitro by addition of 1 mg ofglucose (in 40 ,ul) per ml of...

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