Multidisciplinary studies on the biological functions of peptidergic neuromodulatory systems : from molecules to behavior

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In the present research project, we obtained the following results concerning the biological functions of peptidergic neuromodulatory systems.1. Real-time simultaneous measurement of exocytotic release of GnRH peptide, electrical activities, and intracellular Ca^<2+> concentrationsAccording to our working hypothesis, changes in the sensory inputs from the environments are processed and fed on to the GnRH neuromodulatory system and modulate the release of GnRH peptides from the neurites of the terminal nerve GnRH neurons, which are distributed widely in the brain. Here, we developed a method for real-time simultaneous measurement of exocytotic release of GnRH peptide, electrical activities, and intracellular Ca^<2+> concentrations, using an in vitro brain block preparation with exposed GnRH neurons. We demonstrated a strong relationship between the pacemaker frequency and the intracellular Ca^<2+> concentrations, which are though to be related to the GnRH peptide release.2. Differential expression of GnRH receptor subtypes in the TN-GnRH neurons and the brainThe neuromodulatory functions of GnRH peptides released from the TN-GnRH neurons are thought to be mediated by GnRH receptors. Here, we cloned three subtypes of GnRH receptor genes from the brain of the dwarf gourami, and examined the distribution of GnRH receptor mRNAs by using the patch-clamp single-cell RT-PCR method, in which the cytoplasm of electrically recorded neurons are aspirated and analyzed by RT-PCR. We demonstrated differential expression of different GnRH receptor gene subtypes among different TN-GnRH neurons, which may suggest anto/paracrine regulation of GnRH neurons by GnRH peptides and the presence of subpopulation of TN-GnRH neurons. We also examined the anatomical distribution of GnRH receptor mRNAs expressions by using the in situ hybridization method.