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Whole-Cell Electrophysiology of Gonadotropin-Releasing Hormone Neurons that Express Green Fluorescent Protein in the Terminal Nerve of Transgenic Medaka (Oryzias latipes)¹

Department of Physiology,⁴ University of California at Los Angeles School of Medicine, Los Angeles, California 90095 Department of Aquatic Bioscience,⁵ Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo, Tokyo 113-8657, Japan Laboratory of Reproductive Biology,⁶ National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan

ABSTRACT

Gonadotropin-releasing hormone (GnRH) controls reproduction in vertebrates. Most studies have focused on the population of GnRH neurons in the hypothalamus that ultimately controls gonadal function. However, all vertebrates studied to date have two to three anatomically distinct populations of GnRH neurons that express different forms of this hormone. The purpose of the present study was to develop a new model for studying the population of GnRH neurons in the terminal nerve (TN) associated with the olfactory bulb and then to characterize their pattern of action potential firing to provide a foundation for understanding the role of these neurons in regulating reproduction. A stable line of transgenic medaka (Oryzias latipes) was generated in which a DNA construct containing the salmon GnRH (Gnrh3) promoter linked to green fluorescent protein (GFP) was expressed in TN-GnRH3 neurons. This population of GnRH neurons is located at or near the ventral surface of the brain, making them ideally situated for electrophysiological analysis. Whole-cell and loose-patch recordings in current-clamp mode were performed on these neurons from excised, intact brains of adult males in which afferent and efferent neural connections remained intact. All TN-GnRH3-GFP neurons that we recorded showed a beating pattern of spontaneous action potential firing. Action potentials were blocked by tetrodotoxin, indicating they are generated by a voltage-sensitive Na⁺ current; however, an oscillation in subthreshold membrane potential persisted. The present results indicate that this transgenic fish will provide an excellent model for studying the cell physiology of an extrahypothalamic population of GnRH neurons.

gonadotropin-releasing hormone, neuroendocrinology

¹ Supported by seed grants from the University of California at Los Angeles and National Institutes of Health DA05010 (N.L.W.) and a grant from the National Science Foundation IOB-0414493 (N.L.W.).

² Correspondence: Nancy L. Wayne, Department of Physiology, Room 231, Center for Health Sciences, University of California at Los Angeles School of Medicine, Los Angeles, CA 90095. FAX: 310 206 5661; nwayne{at}mednet.ucla.edu

³ For requests regarding the transgenic fish: Kataaki Okubo, Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan. FAX: 81 564 55 7556; email: okubo{at}nibb.ac.jp.