| Research Abstract |
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 GnRH
The terminal nerve (TN)-GnRH cells have spontaneous oscillatory activities that originate
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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 GnRH
We 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 ascidians
We 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
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