Glial uptake of excitatory amino acids influences neuronal survival in cultures of mouse hippocampus

Abstract

Several reports have described apparently normal survival and development of hippocampal and spinal cord culture preparations grown in Ham's F-12 medium, which contains 100 μM each of l-glutamate and l-aspartate. As at this concentration these amino acids are neurotoxic, some adaptive mechanism must occur to allow neuronal survival. We have investigated the mechanism underlying such adaptation. Dissociated cultures of mouse hippocampal neurons were grown in either Eagle's minimum essential medium or Ham's F-12 medium, supplemented with 5% horse serum. Analysis of neuronal density in cultures stained for neuron-specific enolase showed that although large numbers of neurons were present in mature cultures grown in either medium, neuronal survival in cultures grown continuously in F-12 was reduced to 41%, compared to controls grown in Eagle's minimum essential medium. Physiological studies showed that those neurons which survived in F-12 did not lose their sensitivity to excitatory amino acids. In addition, the acute application of fresh, serum-free F-12 to 10–14-day-old cultures grown in either minimum essential medium or F-12 was highly neurotoxic. Three lines of evidence suggest that glial uptake of amino acids, and reduction of the extracellular concentration of glutamate and aspartate below neurotoxic levels, rather than receptor desensitization underlies the adaptive mechanism allowing neuronal survival. First, application of fresh F-12 produced large depolarizations, and profound neuronal swelling in cultures grown in F-12; however, after several hours swelling reversed suggesting a slow onset of the adaptive process. Second, pressure application of conditioned F-12 obtained from sister cultures also elicited depolarizations in neurons grown in F-12, but the amplitude of the underlying inward current was only 25–30% of that produced by fresh F-12, suggesting a loss of potency of F-12 exposed to prolonged contact with hippocampal cultures. Third, measurement by high performance liquid chromatography showed reduction of aspartate concentrations to around 10% of those present in fresh F-12, within 24h after exposing glial cell cultures to fresh F-12. It is concluded that cellular uptake mechanisms for amino acids have a strong impact on excitotoxicity in vitro, and most likely play an important role in protecting neurons from the potentially damaging action of high concentrations of excitatory transmitters in vivo. In addition our experiments help to explain the mechanisms permitting neuronal survival in cultures grown in Ham's F-12 medium, which when applied acutely to mature cultures is strikingly neurotoxic.