Membrane capacitance measurements of depolarization-triggered exocytosis in single nerve terminals from the rat neurohypophysis
Exocytosis of hormones and neurotransmitters is believed to be stimulated by a rise in intracellular calcium concentration. Membrane depolarization and entry of calcium through voltage-gated channels initiate the response, but subsequent steps leading to fusion of secretory vesicles are largely unknown. Investigation of stimulus-secretion coupling mechanisms in neurons is limited by the inaccessibility of most nerve endings to electrophysiological studies. Progress in other secretory cells has been achieved using the technique of cell membrane capacitance measurements, which is a sensitive assay of the increase in membrane area of single cells that accompanies exocytosis. Using patch clamp methods, a sinusoidal voltage stimulation is applied to the cell, and the resulting membrane current is analyzed to determine changes in membrane capacitance. An advancement called phase tracking is described here that automates one aspect of the capacitance technique and that improves the accuracy of the measurement. To investigate the mechanism of exocytosis in excitable cells, we have applied this capacitance technique to single vertebrate nerve endings isolated from the rat neurohypophysis. The nerve endings of this preparation are among the largest found in vertebrates, and are amenable to electrophysiological recording. We find that in response to brief depolarization, the membrane area is rapidly increased by an amount corresponding to the fusion of up to 100 secretory granules. We have investigated the relationship between this exocytotic signal and calcium influx during the voltage pulse. Capacitance responses are sharply reduced by increasing the concentration of intracellular Ca chelator. Also, the dependence of capacitance response amplitude on membrane potential is a bell shaped mirror image of the Ca current-voltage relation. On average, we find a linear relation between Ca influx and exocytosis in this preparation, although there is variability in the Ca sensitivity among terminals. In experiments using higher stimulation rates ($>$1Hz) we observe patterns of facilitation and depression of capacitance responses. These processes occur by a mechanism independent of the amplitude of Ca current, and indicate that steps subsequent to Ca entry are also important in stimulus-secretion coupling.
Lim, Nancy Fidler, "Membrane capacitance measurements of depolarization-triggered exocytosis in single nerve terminals from the rat neurohypophysis" (1991). Dissertations available from ProQuest. AAI9125706.