Analysis of neurosecretory activities by real-time image processing

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In order to determine the origin of the light scattering change associated with the secretory activity in nervous tissues, transparency of these tissues was examined through a video camera attached to a high power microscope. The video signal was processed to produce contrast enhanced differential images. By virtue of this system accurate site of optical response could be visualized.In the sinus gland of the lobster, the site of the optical response was nerve terminals, and not smooth muscles, blood vessels or other contractile elements. The image could be reversibly suppressed by Cd-containing or Ca-deficient medium. The response could be greatly enhanced by tetraethylammonium and ouabain.Differential images of the rat neurohypophysis was obtained with a highest spatial resolution. They consisted of many small dots of 0.3 um in diameter inside nerve terminals, suggesting that individual secretory granules inside the nerve terminals are the site of optical response. The amplitude of scattering response increased with the frequency of stimulation. Therefore, the scattering response share the same property as that of peptide release from this tissue. Furthermore, the amplitude had a linear relationship with amount of vasopressin measured by radioimmunoassay in the same preparation simultaneously. From these findings, scattering response can be most directly ascribed to the exocytosis of individual secretory granules.This mechanism was further confirmed in an isolated single chromaffin cell which showed a clear image of exocytosis directly under video enhanced bright-field microscope. The exocytosis appeared as an increase in light intensity on very localized spot on the plasma membrane. The time course of the light intensity in active part of a single cell showed a change very similar to scattering change observed by a photodiode.The present study established that the scattering response of the secretory cell is a direct measure of exocytotic activities.