Application of confocal microscopy on glutamate-induced intracellular calcium transient in neurons

Intracellular calcium, as an important second messenger, plays a significant role in cell signaling transduction and metabolism. Glutamate can induce the intracellular calcium transient through triggering diverse signaling pathways. To test the effect of glutamate to neurons, we loaded Fluo-3/Am in cultured rat hippocampal neurons, and then acquired two-dimensional fluorescent image by confocal microscopy and the analyzed fluorescent intensity. In cultured neurons, we observed two types of neurons that have different morphology: bipolar-type and pyramidal-type. Inducing [Ca2+](i) transient by glutamate, we found the amplitude and time constant of the response curves of bipolar neurons are larger than those of pyramidal neurons. Further, we induced [Ca2+](i) transient under different concentrations of glutamate. Two different types of kinetic of the [Ca2+](i) transient have been found, corresponded to the two kinds of neuron. The amplitude of [Ca2+](i) transient increased when applying higher concentration of glutamate in pyramidal neurons; while it decreased in bipolar ones. Responses of neurons bathing in calcium-free extracellular solution to glutamate were different from those bathing in normal solution. [Ca2+](i) transient of pyramidal neurons caused by any concentration were totally blocked; while [Ca2+](i) transient in bipolar neurons caused by high concentration of glutamate (500 mu M) were partly inhibited. All of the phenomena suggest that different types of cultured hippocampal neurons may have different mechanism of the response to glutamate.