Primary culture of the isolated terminal nerve-gonadotrophin-releasing hormone neurones derived from adult teleost (dwarf gourami, Colisa lalia) brain for the study of peptide release mechanisms.
Terminal nerve (TN)-gonadotrophin-releasing hormone (GnRH) neurones are suggested to release GnRH peptides from widely-branched neural processes and the somatodendritic regions, depending on their firing activities. The released GnRH may exert its neuromodulatory actions on GnRH receptors located on various target neurones. The electrophysiological and morphological characteristics of TN-GnRH neurones, which are shared with other peptidergic neurones of vertebrate brains, are thought to represent general features of neuromodulatory and ⁄ or neurosecretory neurones. To address questions concerning the ways in which the electrical activities of peptidergic (TN-GnRH) neuronal somata affect GnRH release from different neuronal compartments, we established a primary culture system of TN-GnRH neurones, which will facilitate simultaneous recordings of various physiological signals from different compartments of a single TN-GnRH neurone cultured in a flat plane. The whole brain of an adult freshwater teleost, the dwarf gourami, was dissected out. The TN-GnRH neurones were then isolated and plated on a coverslip in culture medium. The isolated TN-GnRH neurones could be cultured for up to 2 weeks. In culture, the neurones grew both axon- and dendrite-like neurites, and these processes were phenotypically similar to those found in situ. Unlike the neurones in situ, the cultured neurones had somewhat depolarised resting membrane potentials and showed no spontaneous discharge, which, however, should not be considered to comprise unhealthy culture conditions. Instead, they showed subthreshold spontaneous membrane potential oscillations and could be induced to fire in phasic or tonic patterns. In addition, stimulus-induced exocytotic events could be demonstrated in the soma and neurites using a fluorescent dye, FM1-43. Thus, the present isolated culture of TN-GnRH neurones will open up a wide range of possibilities for studying cellular mechanism of exocytosis, generation of spontaneous firing activity, and neurite outgrowth in peptidergic neurones.
Abe
Hideki
H
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Japan. habe@biol.s.u-tokyo.ac.jp
Oka
Yoshitaka
Y
D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology*; D009411:Nerve Endings / Q000378:metabolism / Q000648:ultrastructure; D009412:Nerve Fibers / Q000378:metabolism / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010473:Perciformes / Q000033:anatomy & histology* / Q000502:physiology*; D009435:Synaptic Transmission / Q000502:physiology*
D007987:Gonadotropin-Releasing Hormone