Mechanisms of neural and endocrinological signal transduction systems during adaptation toenvironmental changes.

促性腺激素释放激素（GnRH）最初被纯化和鉴定为促垂体十肽激素，促进促性腺激素从垂体释放。我们已经证明了存在三个解剖以及功能不同的GnRH神经元系统在几个硬骨鱼类。然后，我们使用热带鱼（Colisa lalia）的大脑，因为这种鱼的终末神经（TN）GnRH神经元很大，并且形成紧密的细胞簇，因此可以使用尖锐的微电极或贴片移液管在体外制备的整个大脑中容易地记录单个GnRH神经元的活动。由于大多数脊椎动物的GnRH神经元胞体很小，且分布广泛，因此很难从单个GnRH神经元中记录。在本研究项目中，我们取得了以下成果. GnRH的神经调节功能终末神经（TN）-GnRH细胞具有自发振荡活动， ...更多信息 来自于它们自身膜的固有离子通道特性。我们发现TTX抗性的持续性Na^+通道I_<Na（slow）>和TEA敏感的K^+通道在起搏活动的产生中起重要作用。接下来，我们使用全细胞膜片钳技术研究TN-GnRH细胞起搏活性的调制。鲑鱼GnRH（sGnRH），这是由TN-GnRH细胞本身产生的GnRH肽的相同分子种类的浴应用，短暂减少，然后持续增加起搏器活动的频率。因此，TN-GnRH细胞的活动可能受自分泌sGnRH的调节，结果表明与磷脂酶C偶联的Gq型G蛋白参与其中。GnRH的胞吐释放我们建立了一种用碳纤维电极安培法测定GnRH神经元分泌活动的方法。首先，我们用循环伏安法或安培法对GnRH溶液的氧化还原电流进行了电化学记录。将CFE轻轻置于硬骨鱼全脑终末神经-GnRH细胞表面，在1000 mV的保持电位下，用安培法记录电流信号，当CFE尖端接触GnRH细胞时，记录到自发峰电流。由于这些电流峰的电压依赖性与真实GnRH溶液的电流类似，我们认为电流峰代表了GnRH从终末神经GnRH细胞的胞吐释放.海鞘的GnRH神经元系统我们首次用免疫细胞化学方法对海鞘的GnRH神经元系统进行了形态学观察，发现在脑神经节的特定表面区域、背侧血窦的沿着内壁和卵巢的前表面都有许多GnRH免疫反应阳性的神经元细胞和纤维。然后，我们进行了细胞内记录在大脑神经节（GnRH神经元丰富的地区）的海鞘，玻璃海鞘的后腹侧部分的浅层。我们发现，23%（27/115）的神经元在光刺激下表现出各种类型的电压反应，包括六种类型的“开”反应和一种类型的“关”反应。结果提示，玻璃海鞘脑神经节为研究GnRH神经元的神经调节功能与光感受之间的可能关系提供了一个良好而简单的实验模型。少

The gonadotropin-releasing hormone (GnRH) was originally purified and identified as a hypophysiotrophic decapeptide hormone that facilitates gonadotropin release from the pituitary. We have demonstrated the existence of three anatomically as well as functionally different GnRH neuronal systems in several teleost species. We then used the brain of a tropical fish (Colisa lalia) because the terminal nerve (TN) GnRH neurons of this fish are large and make a tight cell cluster so that one can readily record the activities of a single GnRH neuron in a whole brain in vitro preparation using sharp microelectrodes or patch pipettes. Because the GnRH neuronal cell bodies in most vertebrate species are small and diffusely distributed, it has been extremely difficult to record from single GnRH neurons. In the present research project, we have obtained the following results.1. Neuromodulatory function of GnRHThe terminal nerve (TN)-GnRH cells have spontaneous oscillatory activities that originate … More from the intrinsic ionic channel properties of their own membranes. We found that a TTX-resistant persistent Na^+ channel, I_<Na(slow)>, and TEA-sensitive K^+ channel play important roles in the generation of pacemaker activities. Next, we investigated the modulation of pacemaker activities of TN-GnRH cells using the whole-cell patch-clamp technique. Bath application of salmon GnRH (sGnRH), which is the same molecular species of GnRH peptide produced by TN-GnRH cells themselves, transiently decreased and then persistently increased the frequency of pacemaker activities. Thus, it is possible that the activities of TN-GnRH cells are regulated by autocrined sGnRH.The results suggested the involvement of a G protein of the Gq type, which is coupled to phospholipase C.2. Exocytotic release of GnRHWe developed a method to measure secretory activities of GnRH neurons by amperometry using carbon fiber electrode (CFE). First, we performed the electrochemical recording of the redox current of GnRH by using cyclic voltammetry or amperometry of authentic GnRH solution. Oxidation current of GnRH was recorded when the holding potential of CFE was higher than 800mV.Next, CFE was gently placed on the surface of terminal nerve-GnRH cells of the teleost whole-brain in vitro preparation, and the current signals were recorded by amperometry at a holding potential of 1000mV.When the tip of CFE touched the GnRH cell, spontaneous current spikes were recorded. Because the voltage-dependence of these current spikes was similar to that in the amperometry of authentic GnRH solution, we concluded that the current spikes represent exocytotic release of GnRH from terminal nerve GnRH cells.3. GnRH neuronal system of ascidiansWe first examined the morphology of the GnRH neuronal system of ascidian, Ciona intestinalis, by immunocytochemistry and found many GnRH-immunoreactive neuronal cells and fibers in a specific surface area of the cerebral ganglion, along the inner wall of the dorsal blood sinus, as well as on the anterior surface of the ovary. We then performed intracellular recordings in the superficial layer of the posterior ventral part of the cerebral ganglion (GnRH neuron-rich area) of an ascidian, Ciona savignyi. We found that 23% (27/115) of the neurons in this area showed various types of voltage responses to light stimuli, including six types of 'on' responses and one type of 'off' response. The results suggest that the cerebral ganglion of Ciona may provide an excellent and simple experimental model to study the possible relationship between the neuromodulatory functions of GnRH neurons and photoreception. Less