Analysis of neurosecretory activities by real-time image processing

为了确定与神经组织分泌活性相关的光散射变化的来源，通过连接在高倍显微镜上的摄像机检查了这些组织的透明度。对视频信号进行处理，生成对比度增强的差分图像。利用该系统可以精确地显示光响应的位置。在龙虾的窦腺中，光反应的部位是神经末梢，而不是平滑肌、血管或其他收缩元件。含cd或缺钙介质可可逆抑制图像。四乙基铵和乌巴因可大大增强反应。以最高的空间分辨率获得大鼠神经垂体的鉴别图像。它们由神经末梢内许多直径0.3 um的小点组成，表明神经末梢内的单个分泌颗粒是光学反应的部位。散射响应的幅值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。此外，在同一制剂中，振幅与放射免疫法测定的抗利尿激素量呈线性关系。从这些发现来看，散射反应可以最直接地归因于单个分泌颗粒的胞吐作用。这一机制在分离的单个嗜铬细胞中得到进一步证实，该细胞在视频增强明场显微镜下直接显示出清晰的胞外分泌图像。胞吐表现为在质膜上非常局部的点上光强的增加。单个电池有源部分光强的时间变化过程与光电二极管观察到的散射变化非常相似。本研究证实，分泌细胞的散射反应是胞外活动的直接测量指标。

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.