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

促性腺激素释放激素(GnRH)最初被纯化并鉴定为一种促垂体促性腺激素释放的十肽激素。我们已经证明了在几个硬骨鱼物种中存在三个解剖上和功能上不同的GnRH神经元系统。然后我们使用了热带鱼(Colisa Lalia)的大脑，因为这种鱼的终末神经(TN)GnRH神经元很大，形成一个紧密的细胞团，因此在使用尖锐的微电极或贴片吸管的体外制备中，人们可以很容易地记录整个大脑中单个GnRH神经元的活动。由于大多数脊椎动物的GnRH神经元胞体较小且分布广泛，因此很难从单个GnRH神经元上记录到GnRH神经元。在本研究项目中，我们取得了以下成果。促性腺激素释放激素的神经调节功能末梢神经(TN)-促性腺激素释放激素细胞具有产生…的自发振荡活动更多的来自于自身膜的固有离子通道属性。我们发现抗TTX的持续性Na~+通道I_lt；Na(Slow)&Gt；和TEA敏感的K~+通道在起搏器活动的产生中起着重要的作用。接下来，我们利用全细胞膜片钳技术研究了TN-GnRH细胞起搏活动的调节。鲑鱼GnRH(SGnRH)是TN-GnRH细胞自身产生的GnRH多肽的同一分子物种，沐浴后，起搏器活动的频率一过性减少，然后持续增加。因此，Tn-GnRH细胞的活性可能受自分泌的sGnRH的调节。结果表明，Gq型G蛋白参与了与磷脂酶C.2偶联的G蛋白的参与。促性腺激素释放激素的胞吐释放我们建立了一种用碳纤维电极(CFE)电流法测定促性腺激素释放激素神经元分泌活动的方法。首先，我们用循环伏安法或电流法记录了GnRH的氧化还原电流。当CFE的保持电位高于800 mV时，记录GnRH的氧化电流。然后，将CFE轻轻放置在硬骨全脑终末神经-GnRH细胞的表面，在保持电位为1000 mV时用安培法记录电流信号。当CFE尖端接触GnRH细胞时，记录到自发的电流尖峰。由于这些电流尖峰电流的电压依赖性与GnRH正品溶液的电流测量结果相似，因此我们认为这些电流尖峰电流代表末梢神经GnRH细胞胞吐释放GnRH。腹水促性腺激素释放激素神经元系统我们首先用免疫细胞化学方法研究了海鞘促性腺激素释放激素神经元系统的形态，发现在脑神经节的特定表面区、沿背血窦内壁以及卵巢前表面有大量促性腺激素释放激素免疫反应神经元细胞和纤维。然后，我们在海鞘动物Ciona savignyi的大脑神经节后腹侧部分(GnRH神经元富集区)的浅层进行了细胞内记录。我们发现该区域23%(27/115)的神经元对光刺激表现出不同类型的电压反应，包括六种类型的“开”反应和一种类型的“关”反应。结果提示，脑脊神经节可能为研究促性腺激素释放激素神经元的神经调节功能与光接收之间的关系提供了一个很好的、简单的实验模型。较少

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