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Characterization of Voltage-Gated Calcium Currents in Gonadotropin-Releasing Hormone Neurons Tagged with Green Fluorescent Protein in Rats

Department of Physiology, Nippon Medical School (M.K., M.W., Y.S.), Tokyo 113-8602, Japan; and Department of Biophysics and Biochemistry, School of Science, University of Tokyo (K.U.-T.), Tokyo 113-0033, Japan

Address all correspondence and requests for reprints to: Dr. Masakatsu Kato, Department of Physiology, Nippon Medical School, Sendagi 1, Bunkyo Tokyo 113-8602 Japan. E-mail: mkato{at}nms.ac.jp.

Functional analysis of GnRH neurons is limited, although these neurons play an important role in neuroendocrine regulation. Therefore, we decided to conduct cell physiological analysis of GnRH neurons. To identify GnRH neurons, we tagged the neurons with green fluorescence protein by a transgenic technique. A dispersed culture of GnRH neurons was prepared from the transgenic rats. After overnight culture, a perforated patch clamp was applied to the identified GnRH neurons to analyze the Ca²⁺ currents. In neonatal GnRH neurons, high voltage-activated Ca²⁺ currents were clearly observed, but low voltage-activated Ca²⁺ current was negligible. Nimodipine (L-type channel blocker) and -conotoxin GVIA (N-type channel blocker) each attenuated the current by approximately 20%. The R-type channel blocker SNX-482 attenuated the current by approximately 55%. Inhibition by the P/Q-type channel blocker -agatoxin IVA was small. In GnRH neurons around puberty, however, both high and low voltage-activated Ca²⁺ currents were observed. Inhibitions by nifedipine, -conotoxin GVIA, and SNX-482 were similar to those in the neonatal neurons, whereas the inhibition by -agatoxin IVA was clearly seen in 40–61% of the GnRH neurons examined. These results indicate that GnRH neurons functionally express L-, N-, P/Q-, R-, and T-type channels. Expressions of P/Q- and T-type channels are developmentally regulated.

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