PubMed1992
Spatiotemporal changes of the intracellular calcium ion (Ca2+) were recorded by digital ratio imaging of fura-2 in pyramidal neurons acutely isolated from the adult guinea-pig hippocampus. Increases in calcium were evoked in tetrodotoxin (2 microM) containing saline either by stimulation with the excitatory amino acids, glutamate or N-methyl-D-aspartate, or by depolarization with high potassium (50 mM). Local stimulation with excitatory amino acids, applied from a microelectrode with 1-2-s iontophoretic pulses at the dendrite, induced a rapid increase in intracellular Ca2+ predominantly supported by a Ca2+ influx at the site of stimulation (primary response). Ca2+ levels recovered within 1-2 min in about one-half of the neurons examined. In the remaining neurons the initial exposure to excitatory amino acids induced a non-recovering gradient of Ca2+, highest at the site of stimulation, that lasted for periods of minutes (secondary response). Within the population that showed recovery from the initial agonist exposure, a second, or in some cases, a third application triggered the sustained, secondary response. Pretreatment of neurons with the protein kinase inhibitor sphingosine (10 microM) blocked development of the secondary response but had no effect on the primary response to the excitatory amino acids. There were no Ca2+ increases in Ca(2+)-free medium with either agonist, and responses to N-methyl-D-aspartate were blocked by 2-amino-4-phosphovaleric acid and significantly reduced at physiological concentrations of Mg2+ (1.8 mM). The maintained gradient of Ca2+ was supported by a continuous influx of calcium from outside the cell. In contrast, dendritic gradients of Ca2+ induced by short exposures to high potassium (50 mM, 5 s) collapsed immediately at the end of the stimulus and could be repeatedly evoked. Minute-long exposures to high K, induced large, repeatable changes in Ca2+ but there was always rapid recovery in normal saline. K depolarization applied after excitatory amino acid stimulation produced larger Ca2+ changes than the same K stimulus applied before the cell was stimulated with the excitatory amino acid. Bath application of GABA (10-100 microM) reduced the magnitude of the maintained Ca2+ gradients. The functional significance of the extended, secondary response cannot be directly established from these measurements on isolated neurons, but its properties could give rise, in part, to mechanisms involved in neural plasticity, in kindling epileptogenesis or in glutamate-induced toxicity.