Analysis of neurosecretory activities by real-time image processing

为了确定与神经组织中的分泌活性相关的光散射变化的起源，通过连接到高倍显微镜的摄像机检查这些组织的透明度。处理视频信号以产生对比度增强的差分图像。在龙虾的窦腺中，光反应的部位是神经末梢，而不是平滑肌、血管或其他可收缩的成分。含镉或缺钙的介质可可逆地抑制该图像。四乙铵和哇巴因可显著增强该反应，获得了具有最高空间分辨率的大鼠神经垂体的差分图像。它们由神经末梢内的许多直径为0.3 μ m的小圆点组成，表明神经末梢内的单个分泌颗粒是光反应的部位。散射反应的幅度随刺激频率的增加而增加。因此，散射响应与从该组织释放的肽具有相同的性质。同时，该振幅与放免法测定的同一制剂中加压素含量呈线性关系。从这些发现中，散射反应可以最直接地归因于单个分泌颗粒的胞吐作用。这一机制进一步证实了在一个孤立的单个嗜铬细胞，显示了清晰的图像，直接在视频增强明场显微镜下的胞吐作用。胞吐作用表现为质膜上非常局部的点上的光强度增加。单个细胞活性部位的光强随时间的变化与光电二极管观察到的散射变化非常相似，本研究证实分泌细胞的散射响应是胞吐活性的直接量度。

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.