Differences in transmitter release, morphology, and ischemia-induced cell injury between cerebellar granule cell cultures developing in the presence and in the absence of a depolarizing potassium concentration.

Release of glutamate and aspartate was measured in mouse cerebellar granule cells in primary cultures grown for 4-16 days in serum-containing tissue culture medium with either a partially depolarizing (25 mM) or a physiological concentration of potassium (5.4 mM). The cells migrated to form aggregates connected by a network of processes during the first week in culture and both groups of cultures survived for at least 2 weeks. In cultures grown in the presence of 25 mM potassium for at least 8 days there was a large (approximately 10 nmol/min/mg protein), calcium-dependent glutamate release and a smaller aspartate release during superfusion with 50 mM potassium. This response was not present in cultures grown in the physiological medium. Nevertheless, exposure to an elevated potassium concentration caused a normal, or even enhanced calcium entry into the cells. Phase contrast microscopy showed a similar appearance of the cellular aggregates under each of the two conditions. Electron microscopy revealed that the aggregates consisted of a centrally located neuropil and peripherally located granule cell bodies. The morphology of the cell bodies and the neuropil in the cells grown at the high potassium concentration closely resembled that of cerebellar granule cells in vivo. In the cells grown at the low potassium concentration, cell bodies, axons and synaptic vesicles looked normal, but the remainder of the neuropil, especially dendrites, showed massive degeneration. Immunochemical measurements demonstrated similar amounts of synaptophysin under each of the two culturing conditions, thus confirming our impression that there were similar numbers of synaptic vesicles and hence presynaptic elements in the two types of cultures. Fluorescence microscopy, using fluorescein diacetate to stain living cells and propidium iodide to stain dead cells, indicated a much greater resistance to ischemic cell injury in the cells cultured at the low potassium concentration. Possible reasons for this difference are discussed.