Effect of a Prolonged Glutamate Challenge on Plasmalemmal Calcium Permeability in Mammalian Central Neurones. Mn²⁺ as a Tool to Study Calcium Influx Pathways

The rate of Mn²⁺-induced fluorescence quenching (RFQ) was used as a relative measure of plasma membrane Ca²⁺ permeability (Pca) in fura-2-loaded cultured hippocampal neurons and cerebellar granule cells during and after protracted (15–30 min) glutamate (GLU) treatment. Some limitations of this method were evaluated using a kinetic model of a competitive binding of Mn²⁺ and Ca²⁺ to fura-2 in the cell. In parallel experiment a contribution of Ca²⁺ influx to the cytoplasinic Ca²⁺ ([Ca²⁺]i) was repeatedly examined during and following a prolonged GLU challenge by short-duration “low-Ca²⁺ trials” (50 pM EGTA) and by measurements of ⁴⁵CaL²⁺ uptake. Experiments failed to reveal a putative persistent increase in Pca that earlier was thought to underlie Ca²⁺ overload of the neuron caused by its toxic GLU treatment. By contrast. a sustained increase of [Ca²⁺], was found to be associated with a progressive decrease in Pca and Ca²⁺ influx both in the period of GLU application and after its termination. These findings give new evidence in favour of the hypothesis that the CLU-induced Ca²⁺ overload of the neuron results mainly from an impairment of its Ca²⁺ extrusion.