Analysis of glutamate uptake and monosodium glutamate toxicity in neural retina monolayer cultures

Abstract

Three different types of monolayer cultures from 8-day chick embryo neural retina can be generated by differential manipulation of culture media and substrata. They are: (i) a purified monolayer of neurons; (ii) a purified monolayer of nonneuronal, flat cells; and (iii) complex cultures containing both neurons and nonneurons. Autoradiographic studies showed that Na +-dependent, high affinity glutamate uptake occurs in about 30% of the neurons present in purified neuronal cultures, and in practically 100% of the flat cells in the purified nonneuronal monolayers or the complex cultures. These different culture preparations were used to investigate the well-known toxicity of monosodium glutamate (MSG) for retinal cells. Two different routes of administration were compared, namely: (i) in vivo preexposure of the embryos to MSG for 24 h, followed by dissociation and culture of the retina in MSG-free medium; and (ii) incorporation of MSG into the culture medium. MSG effects on flat cells were similar irrespective of the route used: flat cells showed a very dramatic reduction in number and a more spindle-like shape in MSG-treated cultures as compared with control cultures. The route of administration, however, had significant influence on the response of retinal neurons to MSG. After 3 days in vitro there were 50% fewer neurons in purified neuronal cultures when the cells were obtained from embryos pretreated in vivo with MSG than in controls, but there were no differences between controls and cultures exposed to MSG in vitro. However, MSG-containing medium preexposed for 30 min to a monolayer of nonneuronal flat cells became toxic for retinal neurons: purified retinal neurons exposed in vitro to this flat cell/MSG ‘conditioned medium’ showed a neuronal loss equivalent to that caused by an in vivo pretreatment with MSG. This observation is consistent with the hypothesis that MSG toxic effects on retinal neurons may be mediated by the nonneuronal cells. Autoradiographic studies showed that the total number of glutamate-labeled retinal neurons was similar in control cultures, in cultures from embryos pretreated in vivo with MSG and in cultures exposed in vitro to flat cell/MSG conditioned medium. This suggests that neurons with the capacity to take up glutamate via a high-affinity mechanism are not sensitive to the toxic effects of MSG.