MORPHOLOGICAL AND FUNCTIONAL PROPERTIES OF IDENTIFIED NEURONS IN ABDOMINAL GANGLION OF APLYSIA CALIFORNICA
MORPHOLOGICAL AND FUNCTIONAL PROPERTIES OF IDENTIFIED NEURONS IN ABDOMINAL GANGLION OF APLYSIA CALIFORNICA
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DOI：
10.1152/jn.1967.30.6.1288
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发表时间：
1967-01-01
影响因子：
2.5
通讯作者：
COGGESHALL, RE
中科院分区：
文献类型：
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作者：
FRAZIER, WT;KANDEL, ER;COGGESHALL, RE
方法用Rimmon C.来自太平洋生物海洋公司的费伊，威尼斯，加州。将动物饲养在天然或人工（海洋魔术或即时海洋）海水水族箱中，温度保持在14 ℃。动物在两种培养基中均存活良好，无需喂食，持续数周。为了进行解剖，将动物以完全伸展的姿势牢牢固定在蜡盘上，并沿动物的全长沿着切开。从动物身上解剖腹神经节及其主要外周神经（虹吸管、生殖器心包和鳃神经）和两个结缔组织，通过结缔组织囊的边缘固定在Lucite室的石蜡基底上，并用海水覆盖。该室包含五对用于神经刺激的Ag-AgCl电极。左结缔组织、右结缔组织、麸皮神经、生殖器-心包联合神经和虹吸神经分别固定在单独的电极对上。用海水缓慢地灌注腔室。实验在室温（18-22 ℃）和14 ℃（温度通过Cambion设计的热电冷却单元控制）下进行。待描述的鉴别特征在两种温度下定性相同。增益将填充有2 M柠檬酸钾的微电极通过常规的单阴极跟随器引导到多束示波器的差分直流放大器。电极具有5-10兆欧的电阻。使用简单的惠斯通电桥（25）来同时记录并使电流通过微电极。一卷浸没的氯化银线用作接地。这些研究中使用的光学和电子显微镜解剖技术在以前的论文中有描述（15）。一个主要的技术问题是将微电极插入细胞中，即使它们被厚厚的结缔组织覆盖。在先前的实验中，这通常通过用显微手术刀（41）切片结缔组织来完成。然而，这种方法总是导致暴露的细胞弹出，导致神经节结构的扭曲和地形关系的丧失。在细胞中。为了避免这种困难，将微电极压在覆盖给定细胞的未解剖结缔组织上，然后轻轻敲击显微操作器，以便刺穿结缔组织并穿透下层细胞。这个过程运行良好，经过一些实践，可以将两个独立的电极接入同一个电池或相邻的电池。在某些情况下，局部切除关节囊（27）是额外的帮助。通过以阈值或略大于阈值的强度刺激神经和连接来确定顺向反应。非常强烈的刺激往往会产生复杂的反应，本报告只介绍其中的一些反应。逆向反应与顺向反应的区别在于出现典型的、恒定潜伏期的A或AB峰电位，这些峰电位可被细胞体的直接超极化所阻断。通过离子电渗注射，使用装有5 v，w/v乙酰胆碱在蒸馏水中的微量移液器测定对乙酰胆碱的反应。在某些情况下，通过将染料离子电渗通过记录微电极来标记细胞，以确保形态学鉴定。
METHODSThese experiments were carried out, over a 2-year period, on several hundred specimens of Aplysia californica supplied by Dr. Rimmon C. Fay of Pacific Bio-Marine Corporation, Venice, California. The animals were kept in natural or artifical(Marine Magic or Instant Ocean) sea-water aquaria with the temperature maintained at 14 C. The animals survived equally well in both media without feeding, for several weeks. For dissection, the animal was firmly pinned to a wax dish in a fully extended position, and an incision was made along the full length of the animal. The abdominal ganglion, with its main peripheral nerves (the siphon, genital pericardial, and branchial nerves) and two connectives, was dissected from the animal, pinned by the edges of the connective tissue capsule to the paraffin base of a Lucite chamber, and covered with sea water. The chamber contained five pairs of Ag-AgCl electrodes for nerve stimulation. The left connective, theright connective, the bran&al nerve, the combined genital-pericardial nerve, and the siphon nerve were each pinned over separate pairs of electrodes. The chamber was slowly perfused with sea water. The experiments were carried out both at room temperature (18-22 C) and at 14 C (the temperature was controlled by means of a thermoelectric cooling unit designed by Cambion). The identifying characteristics to be described were qualitatively the same at both temperatures. gain Microelectrodes filled with 2 M potassium citrate were led through conventional unitycathode followers to the differential dc amplifiers of a multibeam oscilloscope. The electrodes had resistances of 5-10 megohms. A simple Wheatstone bridge (25) was used for simultaneously recording and passing current through the microelectrodes. A submerged coil of chlorided silver wire served as ground. The anatomical techniques for light and electron microscopy used in these studies were described in the previous paper (15). A major technical problem was to insert microelectrodes into the cells even though they were overlain by thick connective tissue. In previous experiments this was generally accomplished by slicing the connective tissue with a microscalpel(41). However, this procedure invariably caused the exposed cells to pop out, resulting in distortion of the ganglionic architecture and loss of topographical relationships a. mong the cells. In order to avoid this difficulty, the microelectrode was pressed against the undissected connective tissue overlying a given cell, and then the micromanipulator was gently tapped so that the connective tissue was pierced and the underlying cell penetrated. This proced ure worked well, and with some practice it was possible to tap two independent electrodes into the same cell or into neighboring cells. In some cases, localized dissection of the capsule(27) was of additional help.Orthodromic responses were determined by stimulating the nerves and connectives with threshold or slightly greater than threshold intensity. Very strong stimulation often produced complicated responses, only some of which will be described in-the present report. Antidromic responses were distinguished from orthodromic ones by the appearence of typical, constant-latency, A or AB spikes which could be blocked by direct hyperpolarization of the cell body. Responses to ACh were determined by means of iontophoretic injection using micropipettes filled with 5 v;, w/v ACh in distilled water. In some cases cells were marked by passing a dye iontophoretically through the recording microelectrode in order to secure a morphologic identification.