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

Abstract

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 omega-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 omega-agatoxin IVA was small. In GnRH neurons around puberty, however, both high and low voltage-activated Ca2+ currents were observed. Inhibitions by nifedipine, omega-conotoxin GVIA, and SNX-482 were similar to those in the neonatal neurons, whereas the inhibition by omega-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.

Membrane-initiated estrogen signaling in hypothalamic neurons

“…The latter findings combined with recent technological advances, such as the development of ER mutants and transgenic animals expressing green fluorescent protein (GFP) in GnRH neurons, have greatly facilitated studies to understand the cellular mechanisms by which GnRH neurons are regulated by estrogen (Spergel et al, 1999, Suter et al, 2000, Kato et al, 2003, Han et al, 2005, Abraham et al, 2003, Smith et al, 2006, Wintermantel et al, 2006. A series of recent publications show that in mouse hypothalamic neuronal explants and in primate nasal placode cultures, E2 augments synchronous intracellular Ca 2+ oscillations in GnRH neurons (Temple et al, 2004, Temple and Wray, 2005, Abe et al, 2007.…”

Gene structures, biochemical characterization and distribution of rat melatonin receptors

“…Wistar rats, and transgenic rats that express enhanced green fluorescent protein (EGFP) under control of the GnRH promoter [14] were used in these studies. The rats had free access to water and food, and were kept under a 14-h light, 10-h dark cycle.…”

A simple integrative electrophysiological model of bursting GnRH neurons

“…According to this, we will assume that a fraction of 0 < (r d + r pd ) < 1 of Ca 2+ -dependent currents appear in the dendritic compartments, whereas the rest (1 − (r d + r pd )) flows in the soma. Additional currents injected to the somatic or to the dendritic compartment are described by I (Kato et al, 2003;Herbison et al, 2001;Watanabe et al, 2004;Krsmanovic et al, 1992;Nunemaker et al, 2003)). Although these channels exhibit different dynamical features, to keep the model as simple as possible, we try to describe their overall contribution with single HodgkinHuxley type ion channel model.…”

“…The parameters of the Hodgkin-Huxley channel describing the voltage gated Ca 2+ currents were determined to qualitatively match the voltage clamp results published in (Kato et al, 2003). The parameters of the model were adjusted to approximate the voltage dependence of the peak current corresponding to the results of the voltage step protocol described in (Kato et al, 2003).…”

“…The parameters of the model were adjusted to approximate the voltage dependence of the peak current corresponding to the results of the voltage step protocol described in (Kato et al, 2003). The peak currents of model simulation are compared to experimental data (Kato et al, 2003) in Fig.…”