Ca super(2+) release from intracellular stores induced by afferent stimulation of CA3 pyramidal neurons in hippocampal slices

Ca super(2+) imaging and simultaneous intracellular recording were performed on CA3 pyramidal neurons in hippocampal slice cultures and standard acute slices. Both fura-2 and a dextran conjugate of fura-2 (MW = 10,000) were used in the Ca super(2+) measurements to control for compartmentalization artifacts. Experiments were performed under conditions giving minimal ligand- and voltage-gated Ca super(2+) influx, with the use of competitive and noncompetitive antagonists of ionotropic glutamate receptors and steady-state depolarization, respectively. Tetanic stimulation of stratum lucidum evoked dendritic Ca super(2+) transients with rapid onset that were blocked by the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (2-5 mu M), but not by the competitive alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10-50 mu M). Zn super(2+)-containing mossy fiber terminals (assessed by Timm's staining) and postsynaptic structures (thorny excrescences) are preserved in s. lucidum of hippocampal slice cultures. A Ca super(2+) store loading protocol, consisting of brief repolarizations followed by steady depolarization, primed most of the neurons so that a subsequent tetanus gave a Ca super(2+) increase in the presence of MK-801 that was reported by both fura-2 and the dextran conjugate. The onset of the Ca super(2+) increase was significantly delayed (by 2-3 s) with respect to the MK-801-sensitive increase, and often had a different spatial pattern within the neuron. Response characteristics were similar in slice cultures and acute slices. The delayed Ca super(2+) increase showed a steep rundown with subsequent stimuli, but was restored by further priming by the Ca super(2+) store loading paradigm. Postsynaptic currents evoked by the tetani under these conditions were not correlated with the magnitude of the delayed Ca super(2+) transients. Delayed Ca super(2+) increases were observed in 44% of the neurons dialyzed with normal intracellular solution at room temperature. The success rate of observing delayed Ca super(2+) transients was increased to 86% in neurons maintained at 30 degree C, and dialyzed with an inhibitor of the inositol-triphosphate-3-kinase. The delayed Ca super(2+) transients could not be initiated after inhibition of endosomal Ca super(2+)-ATPase-mediated uptake by thapsigargin. Both fura-2 and the dextran conjugate reported increases in resting Ca su