Characterization of Voltage-Gated Calcium Currents in Gonadotropin-Releasing
Hormone Neurons Tagged with Green Fluorescent Protein in Rats
Masakatsu Kato, Kumiko Ui-Tei, Miho Watanabe and Yasuo Sakuma
Endocrinology 2003 144:5118-5125 originally published online Aug 13, 2003; , doi: 10.1210/en.2003-0213

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Endocrinology 144(11):5118 –5125
Copyright © 2003 by The Endocrine Society
doi: 10.1210/en.2003-0213

Characterization of Voltage-Gated Calcium Currents in
Gonadotropin-Releasing Hormone Neurons Tagged with
Green Fluorescent Protein in Rats
MASAKATSU KATO, KUMIKO UI-TEI, MIHO WATANABE, AND YASUO SAKUMA
Department of Physiology, Nippon Medical School (M.K., M.W., Y.S.), Tokyo 113-8602, Japan; and Department ofBiophysics and Biochemistry, School of Science, University of Tokyo (K.U.-T.), Tokyo 113-0033, Japan
Functional analysis of GnRH neurons is limited, althoughthese neurons play an important role in neuroendocrine regulation. Therefore, we decided to conduct cell physiologicalanalysis of GnRH neurons. To identify GnRH neurons, wetagged the neurons with green fluorescence protein by atransgenic 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 Ca2ⴙ currents. In neonatal
GnRH neurons, high voltage-activated Ca2ⴙ currents wereclearly observed, but low voltage-activated Ca2ⴙ current was
negligible. Nimodipine (L-type channel blocker) and ␻-conotoxin GVIA (N-type channel blocker) each attenuated the cur-GnRH NEURONS PLAY an essential role in reproductive neuroendocrine regulation. Despite the importance of GnRH neurons, functional analysis of these neurons
is limited. This is mainly due to difficulty in identifying
GnRH neurons in electrophysiological experiments. Recently, however, transgenic mice were produced for specificlabeling of GnRH neurons with enhanced green fluorescence
protein (EGFP) (1–3), which has facilitated the cell physiological study of GnRH neurons. Firing patterns of GnRH
neurons were studied in whole cell patch-clamp recordings(2, 4, 5), and the basic membrane properties were studied incurrent clamp analysis (3). Responses to glutamate and␥-aminobutyric acid were also reported on EGFP-tagged
GnRH neurons (1, 5, 6). Because most of these studies werecarried out in the current clamp mode, the voltage-gatedcurrents remain to be analyzed. Kusano et al. (7) reported thatmouse GnRH neurons in olfactory pit explant cultures express both low voltage-activated and high voltage activatedCa2⫹ currents. This report is to date the only one on thevoltage-gated Ca2⫹ current in GnRH neurons analyzed by
the voltage clamp experiments despite the fact that the voltage-gated Ca2⫹ channels play important roles in Ca2⫹dependent cellular functions such as transmitter release, cell
excitability, protein phosphorylation, enzyme activity, andgene transcription. We therefore decided to study the voltage-gated Ca2⫹ currents in rat GnRH neurons. We first produced transgenic rats for the identification of GnRH neurons.
Abbreviations: Aga-IVA, ␻-Agatoxin IVA; APW, action potential
waveform; EGFP, enhanced green fluorescence protein; GVIA, ␻-conotoxin GVIA; MVIIC, ␻-conotoxin MVIIC; OVLT, organum vasculosumof the lamina terminalis.

rent by approximately 20%. The R-type channel blocker SNX482 attenuated the current by approximately 55%. Inhibitionby the P/Q-type channel blocker ␻-agatoxin IVA was small. In
GnRH neurons around puberty, however, both high and low
voltage-activated Ca2ⴙ 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. (Endocrinology 144: 5118 –5125, 2003)

Here are two reasons why we chose rats instead of mice. First,
there are already several mouse lines of EGFP-tagged GnRH
neurons. If we produced a transgenic rat, we could compare
GnRH neurons in mice and rats. Second, rats have been andare still commonly used for experiments on reproductive
neuroendocrinology, as a consequence of which there is an
accumulation of useful data on rats.
In the present study we investigated the expression profile
of voltage-gated Ca2⫹ currents in neonatal and pubertal
GnRH neurons by the method of perforated patch recording
configuration with amphotericin B.
Materials and Methods
All experiments were performed with the approval of Nippon Medical School animal care committee.

Transgenic rats
The rat GnRH promoter (⫺3026 to ⫹116; a gift from Dr. M. E. Wierman, University of Colorado Health Science Center, Denver, CO) (8) wasused to express a transgene consisting of the intron of rabbit ␤-globin(640 bp; a gift from Dr. J. Miyazaki, Osaka University, Osaka, Japan), thecoding sequence for EGFP (739 bp; CLONTECH Laboratories, Inc.,Tokyo, Japan), and the polyadenylation signal. The excised transgene
was injected into the pronucleus of fertilized oocytes obtained from
Wistar rats (YS New Technology, Tochigi, Japan). Six transgenic
founders were identified through Southern blot analysis of DNA harvested from tail snips of 112 pups with a 32P-labeled EGFP probe. The
offspring of these 6 transgenic lines were cytologically examined, andone transgenic line, which had high and specific expression of EGFP in
GnRH neurons, was selected for physiological experiments. The otherfive lines were not used because they had weak EGFP fluorescence. For
cytological observation, brains were fixed with 4% paraformaldehyde.
Forty-micrometer frozen sections of the fixed brain were cut and immunostained with antisera to GnRH (a gift from Dr. K. Inoue, Saitama

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Kato et al. • Voltage-Gated Ca2⫹ Currents in Rat GnRH Neurons

University, Saitama, Japan) and Cy3-labeled second antibody (Jackson
ImmunoResearch Laboratories, West Grove, PA).

Primary culture
The brains were excised from either 1- to 7-d-old pups or 35- to
40-d-old rats under ether anesthesia. The former were used to prepareneonatal neurons, and the latter were used for the neurons around
puberty. The latter could include prepubertal animals because we did
not check for the onset of puberty. Medial septum, diagonal band of
Broca, organum vasculosum of the lamina terminalis (OVLT), and medial preoptic area were cut out with a razor and surgical blades. The
sections were minced and treated with papain (21 U/ml; Funakoshi,
Tokyo, Japan) for 30 – 60 min at 30 C with gentle agitation. The tissues
were triturated with a 5-ml plastic pipette after several washes with
MEM (Life Technologies, Inc., Tokyo, Japan). The cell suspension wasapplied to discontinuous Percoll density gradient centrifugation to remove debris. The cells were obtained from the middle layer of thedensity gradient centrifugation composed of 1.0, 1.023, and 1.078 g/ml
layers and were plated on poly-lysine-coated coverslips and incubatedovernight in Neurobasal-A medium (Life Technologies, Inc.) supplemented with 0.5 mm l-glutamine and B-27 (Life Technologies, Inc.) at
37 C. Most of the dissociated GnRH neurons were round, but some were
spindle-shaped. These neurons did not change their shape during theovernight culture.

Electrophysiology
The List EPC-9 patch-clamp system (Physio-Tech, Tokyo, Japan) wasused for electrophysiological recordings and data analysis. Whole cell
currents were measured by the perforated patch-clamp technique (9) atroom temperature (25 C). The final concentration of amphotericin B(Seikagaku Corp., Tokyo, Japan) in the pipette solution was 0.05 mg/ml.
The pipette solution consisted of 95 mm cesium aspartate, 47.5 mm CsCl,
1.0 mm MgCl2, 0.1 mm EGTA, and 10 mm HEPES (pH 7.2), and theosmolality was adjusted to 270 mosmol. The extracellular solution consisted of 116.3 mm NaCl, 10 mm tetraethylammonium chloride, 5 mm
CsCl, 10 mm CaCl2, 0.8 mm MgCl2, 0.6 mm NaHCO3, 10 mm glucose,
20 mm HEPES (pH 7.4), 0.1% BSA (fraction V, Sigma-Aldrich Corp., St.
Louis, MO), and 0.3 ␮m TTX (Seikagaku Corp.), and the osmolality was
adjusted to 300 mosmol. Pipettes were fabricated with borosilicate glass
capillaries and had a resistance of 7–9 m⍀. The pipettes were targetedto GnRH neurons in the extracellular solution without BSA. After touching the cell, slight negative pressure was applied to the pipette, which
made a seal resistance of 5–10 G⍀. Perforation with amphotericin B was
achieved within 5–10 min after giga-seal formation. Currents were filtered at 2.3 kHz, digitized at 10 kHz, and recorded. Series resistance was
70% electronically compensated. Data were taken when the series resistance was stable and less than 30 m⍀. Capacitative and leak currents
were subtracted by the p/4 protocol, and the liquid junction potential
was not compensated. Cell capacitances were 9.2 ⫾ 2.2 pF (n ⫽ 46) inmales and 9.8 ⫾ 2.4 pF (n ⫽ 34) in females in neonates, and 12.8 ⫾ 2.6
pF (n ⫽ 13) in males and 10.8 ⫾ 2.7 pF (n ⫽ 11) in females around
puberty. The input resistance of the cells ranged from 1–5 G⍀. Cells witha peak Ca2⫹ current less than ⫺100 pA were excluded from the analysis,
because it is difficult to obtain a reliable subtracted current with suchsmall currents. To confirm the perforated patch configuration, we examined the capacitative current and its change by rupturing the patch
membrane at the end of the recording. Data are expressed as the mean⫾
sd unless otherwise stated. The Kruskal-Wallis test and paired t test wereused for statistical analysis. The significance level was set at P ⬍ 0.05.

Chemicals
Nimodipine and nifedipine were obtained from Wako Junyaku (Osaka, Japan). ␻-Conotoxin GVIA (GVIA), ␻-conotoxin MVIIC (MVIIC),
␻-agatoxin IVA (Aga-IVA), and SNX-482 were purchased from Peptide
Institute, Inc. (Osaka, Japan).

Results

In the transgenic rats, EGFP fluorescence was observed
only in GnRH-immunoreactive neurons, approximately one

Endocrinology, November 2003, 144(11):5118 –5125 5119

third of which had strong EGFP fluorescence (Fig. 1). The
fluorescence was observed not only in soma, but also inprocesses including axons in the median eminence (data notshown). GnRH neurons were also identified with EGFP in a
dissociated culture (Fig. 1, D–F).
Ca2⫹ currents in neonatal GnRH neurons

In neonatal GnRH neurons Ca2⫹ currents were activatedby 100-msec voltage steps from ⫺60 to 60 mV in 10-mV
increments from a holding potential of ⫺80 mV at 0.2 Hz (Fig.
2). The maximum amplitudes were ⫺57.8 ⫾ 20.7 pA/pF (n ⫽13) in males and ⫺46.6 ⫾ 12.2 pA/pF (n ⫽ 14) in females. The
maximum current was activated at 0 –20 mV and showed a
rapid activation and a relatively slow inactivation.
The effects of several Ca2⫹ channel blockers on the maximum currents are shown in Fig. 3. The maximum currents
were elicited by 100-msec voltage pulses to 0 or 10 mV fromthe holding potential of ⫺80 mV at 0.2 Hz. After the control
currents were recorded, 10 ␮m nimodipine, 1 ␮m GVIA, 200
nm Aga-IVA, and 100 nm SNX-482 were successively applied(Fig. 3, A and B). The initial peak currents and late sustained
currents were examined. In the initial peak currents, nimodipine and GVIA each attenuated the currents by approximately 20%, and SNX-482 reduced the currents by about 55%
in both sexes. A similar inhibition by SNX-482 (60 ⫾ 7%; n ⫽4) was observed when SNX-482 was applied without priorapplication of the other Ca2⫹ channel blockers. Inhibition by
Aga-IVA was small and negligible in both sexes. Aga-IVA
exerted 7.5 ⫾ 3.3% inhibition in 4 of 11 male cells examinedand 3% inhibition in 1 of 14 female cells examined. No inhibition was observed in other cells. After combined application of all the above drugs, 6 –7% of the control current
remained. To examine the presence of T-type Ca2⫹ current,
the membrane potential was held at ⫺100 mV, and the voltage steps to ⫺70, ⫺60, and ⫺50 mV were given at 0.2 Hz. In
this voltage protocol the current density of ⫺1 pA/pF was
activated at ⫺50 mV in 2 cells among 10 male cells examined,
and that of ⫺1.4 pA/pF was activated at ⫺50 mV in 3 cells
among 9 female cells examined (Fig. 7C). No current was
activated at ⫺50 mV in the other cells. In the late sustained
currents, the inhibition caused by GVIA was approximately
30%, and that by SNX-482 was 33% in males and 47% infemales. The proportion of SNX-482-sensitive currents was
smaller in the late sustained currents than in the initial peakcurrents. This is probably due to inactivation of the SNX482-sensitive currents. The inhibition by each blocker was
significant (P ⬍ 0.01), except for that by Aga-IVA.
The action potential waveform (APW) was used for activation of the Ca2⫹ currents (Fig. 4). The half-amplitude widthof APW was set at 2.5 msec, because that of the GnRH neuron
ranged from 2.5–3 msec at room temperature (data notshown). In this voltage protocol, the inhibitory effect of nifedipine was small (9.4%), and the inhibition caused by GVIA
and Aga-IVA was 29%, whereas that by SNX-482 was 45%,so that the contribution of the nifedipine-sensitive current
was smaller in the APW than in the current activated by thesquare pulse.
The voltage-dependent activation of R-type current wasstudied by measuring tail currents at ⫺80 mV after 10-msec

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Kato et al. • Voltage-Gated Ca2⫹ Currents in Rat GnRH Neurons

FIG. 1. EGFP fluorescence in GnRH neurons. A–C, Frontal section of the OVLT, diagonal band of Broca region. The dark area in the lower
middle of each panel is the rostral part of the third ventricle. A, EGFP fluorescence. B, Double exposure for EGFP and Cy3 (GnRH). Neurons
colored yellow are positive for both EGFP and GnRH. C, Cy3-labeled GnRH neurons. D–F, Dissociated cells, D, EGFP fluorescence. E,
Cy-3-labeled GnRH neuron. F, Cells in brightfield. The cell indicated with an arrow is the EGFP-positive GnRH neuron. Scale bars, 100 ␮m(A) and 25 ␮m (F).

prepulses of ⫺60 to 60 mV in 10-mV increments from theholding potential of ⫺80 mV at 0.2 Hz in the presence of
nimodipine, GVIA, and Aga-IVA (Fig. 5). The activation
started at a prepulse of ⫺40 mV and reached full activation
at 30 – 40 mV. The half-activation voltage was 0 mV (two maleand two female neurons). Steady state inactivation was alsostudied in the R-type Ca2⫹ current (Fig. 5). The holding
potential varied from ⫺100 to 0 mV in 10-mV increments,
and a 100-ms test pulse was applied at 0.2 Hz. The inactivation started from the holding potential of ⫺80 mV andreached almost complete inactivation at 0 mV. The half-

inactivation voltage was ⫺39 mV (seven male and threefemale neurons).
Ca2⫹ currents in GnRH neurons around puberty

Ca2⫹ currents were activated by 100-msec voltage steps
from ⫺60 to 60 mV in 10-mV increments from the holding
potential of ⫺80 mV at 0.2 Hz. The activation started at ⫺40
mV and reached maximum amplitude around 0 mV. The
maximum amplitudes were ⫺49.5 ⫾ 15.1 pA/pF (n ⫽ 13) inmales and ⫺43.9 ⫾ 26.8 pA/pF (n ⫽ 10) in females. The

Kato et al. • Voltage-Gated Ca2⫹ Currents in Rat GnRH Neurons

FIG. 2. Voltage-gated Ca2⫹ currents in neonatal GnRH neurons. After perforation with amphotericin B, cells were clamped at ⫺80 mV
and given 100-msec voltage pulses from ⫺60 to 60 mV in 10-mV steps
from a holding potential of ⫺80 mV as shown at the upper left in A.
A, Current traces elicited with voltage pulses as indicated at the upper
left in each trace. Current traces at ⫺60 mV and ⫺50 mV are notshown. B, IV relationship of the peak currents with the same data as
in A.

comparisons were made in four groups according to developmental stage and sex. There was no significant differencein the control maximum current densities among these fourgroups. Nifedipine, GVIA, and SNX-482 exerted similar inhibitory effects to that in the neonatal GnRH neurons (Fig. 6).
The inhibitory effect of Aga-IVA was stronger and clearer
than that in the neonatal GnRH neurons. The overall inhibitions were 8% in males and 5% in females. The number ofcells in which Aga-IVA attenuated the peak current morethan 5 pA was eight in 13 male cells and four in 10 female
cells. A similar inhibition was exerted by 2 ␮m MVIIC (P/Q-type Ca2⫹ channel blocker) in four male and six female

Endocrinology, November 2003, 144(11):5118 –5125 5121

FIG. 3. Pharmacological characterization of the Ca2⫹ currents in neonatal GnRH neurons. A, Voltage protocol and the representative
current traces. Voltage pulses (10 mV in this cell) were applied fromthe holding potential of ⫺80 mV at 0.2 Hz. The control trace is labeled
C. The current was attenuated by 10 ␮M nimodipine (Nim), 1 ␮M GVIA(G), and 100 nM SNX-482 (S). A concentration of 200 nM Aga-IVA (A)had no effect on the current in this cell. Therefore, traces labeled G
and A are overlapped. These traces were taken from the same data
for B at time points denoted by C, Nim, G, A, or S. B, Time course of
the effect of drugs on the peak Ca2⫹ current is shown. Drugs wereapplied as indicated with horizontal bars. C, The effects of drugs arecollectively shown as a percentage of the total current (n ⫽ 11 inmales, 14 in females) for the initial peak current (peak) and the latesustained current (late). The late sustained current was measured as
the mean current of the last 5 msec of the 100-msec pulse (hatchedsquare in A). The L-type current indicates the current blocked bynimodipine. The N-type current (N) is the current blocked by GVIA.
The P/Q-type current (P/Q) is the current blocked by Aga-IVA. The
R-type current (R) is the current blocked by SNX-482. Residual indicates the current resistant to all these drugs.

neurons examined (6.6 ⫾ 9%). The proportions of the remaining currents after treatment with all of the above drugs
were 12.2 ⫾ 7.3% (n ⫽ 13) in males and 16.5 ⫾ 18.9% (n ⫽5122

Endocrinology, November 2003, 144(11):5118 –5125

Kato et al. • Voltage-Gated Ca2⫹ Currents in Rat GnRH Neurons

FIG. 4. The Ca2⫹ currents elicited by APW in neonatal GnRH neurons. The action potential waveform is composed of the 1-msec depolarization phase from ⫺60 mV to 30 mV, the 4-msec repolarization
phase from 30 to ⫺70 mV, and the 10-msec afterhyperpolarization
phase from ⫺70 mV to ⫺60 mV as shown in A. After control recording,drugs were successively applied as indicated. B, Collective presentation of the effects of drugs. The data for both sexes are combined (n ⫽8). Aga, Aga-IVA; ⫺, without drug application; ⫹, with drug application. The inhibitory effect of each drug was evaluated by comparing
the responses with and without the drug in a paired t test. *, P ⬍ 0.05;**, P ⬍ 0.01.

10) in females. These remaining currents were further attenuated by 50 ␮m Ni2⫹ to 6.5 ⫾ 4.5% and 3.8 ⫾ 4.7%, respectively, which are comparable to the remaining currents inneonatal GnRH neurons without application of Ni2⫹ (Fig. 3).
These Ni2⫹-sensitive currents were clearly seen at ⫺30 mV
in all cells examined (Fig. 7). The remaining current densitiesat ⫺30 mV were ⫺3.2 ⫾ 2.2 pA/pF (n ⫽ 8) in males and⫺6.4 ⫾ 4.3 pA/pF (n ⫽ 9) in females. These currents were
inhibited by about 90% with 50 ␮m Ni2⫹. Similar currents
were activated by a voltage step to ⫺50 mV from a holding
potential of ⫺100 mV at 0.2 Hz in all cells examined (Fig. 7C).
The values were ⫺2.2 ⫾ 1.8 pA/pF (n ⫽ 6) in males and⫺4.4 ⫾ 2.5 pA/pF (n ⫽ 6) in females. The inhibition by eachblocker was statistically significant, except for that by AgaIVA in females.
Discussion

In the present study we used isolated cells instead of cells
in acute slice preparations because we could obtain muchbetter and more reliable recordings of the Ca2⫹ currents in
isolated cells. Moreover, the isolated cells may retain their
original cellular characteristics to a certain extent even after
overnight culture. It should be noted, however, that the cells

FIG. 5. Activation and steady-state inactivation of the R-type Ca2⫹
current in neonatal GnRH neurons. A, The upper panel shows thevoltage protocol for the activation. The holding potential was ⫺80 mV.
Ten-millisecond prepulses of ⫺60 to 60 mV were applied, and the tailcurrents at ⫺80 mV were measured as indicated (F). B, Holding
potentials varied from ⫺100 to 0 mV, and the currents elicited by thetest pulse (10 mV) were measured as indicated (f). C, Activation (n ⫽4) and steady state inactivation (n ⫽ 10) are shown. The data for both
sexes were combined. The half-activation voltage was 0 mV, and thehalf-inactivation voltage was ⫺39 mV.

used in the present experiments lacked both dendrites andaxons, so that the currents originated in the cell soma.
We revealed an expression profile of the voltage-gatedCa2⫹ currents in GnRH neurons by using specific blockers forthe voltage-gated Ca2⫹ currents. In neonatal GnRH neurons,
L-, N-, and R-type Ca2⫹ currents were clearly observed in allcells examined, but P/Q- and T-type Ca2⫹ currents were
small and were seen in less than 50% of the cells examined.
In the GnRH neurons around puberty, besides L-, N-, andR-type Ca2⫹ currents, a P/Q-type Ca2⫹ current was observedin 62% of male cells examined and 40% of female cells,
whereas a T-type Ca2⫹ current was clearly observed in all

Kato et al. • Voltage-Gated Ca2⫹ Currents in Rat GnRH Neurons

Endocrinology, November 2003, 144(11):5118 –5125 5123

FIG. 6. Pharmacological characterization of the Ca2⫹ currents in
GnRH neurons around puberty. Effects of drugs are collectively
shown as a percentage of the total current for the peak current. The
L-type current (L) was the current blocked by 10 ␮M nifedipine; theT-type current (T) was the current resistant to nifedipine, GVIA,
Aga-IVA, and SNX-482 and blocked by 50 ␮M Ni2⫹. Other currents are
the same as in Fig. 3. The experimental procedure is the same as forthe neonatal neurons.

cells examined, so that the expression of P/Q- and T-type
Ca2⫹ currents was developmentally regulated. There was no
substantial sex difference in the profile of expression of thevoltage-gated Ca2⫹ currents in GnRH neurons either in neonates or around puberty. To date, the presence of L- andT-type Ca2⫹ currents has been reported in mouse GnRH
neurons in explant culture of olfactory pit (7) and GT1 cells(10, 11). No other types of Ca2⫹ current were examined inthese reports.
We identified an R-type current by two criteria. One wasa current resistant to specific blockers for L-, N-, and P/Qtype Ca2⫹ channels in high voltage-activated Ca2⫹ currents(12–14). The other was a current that was blocked by 100 nm
SNX-482 (15, 16). This concentration is specific to the R-typecurrent, but does not block the SNX-482-resistant, R-typecurrent (16). In the present results almost all of the remaining
currents were blocked by 100 nm SNX-482, suggesting thatrat GnRH neurons express no or a very small proportion of
SNX-482-resistant, R-type current. Half-activation and halfinactivation voltages of R-type current were reported to be⫺14 mV and approximately ⫺70 mV, respectively, in mouse
hippocampal and neocortical neurons by Sochivko et al. (17).
These values differ from ours mainly because they used 5 mm
Ba2⫹ without Ca2⫹ as a charge carrier instead of the 10 mm
Ca2⫹ in our experiments.
It should be noted that the proportion of R-type current
was surprisingly big both in neonates (55%) and around
puberty (⬃40%) compared with approximately 20% in magnocellular and unidentified hypothalamic neurons (18 –21)
and neocortical and neostriatal neurons (22, 23). This meansthat the R-type Ca2⫹ current greatly contributes to intracellular Ca2⫹ regulations in GnRH neurons in these developmental stages, but in adult GnRH neurons the proportion of
R-type current was approximately 30% (our preliminary results). The half-inactivation voltage was ⫺40 mV in 10 mm
Ca2⫹ in the extracellular solution (Fig. 5). This value wouldbe ⫺50 mV in a normal Ca2⫹ concentration (2.5 mm). If wetake ⫺60 mV as the resting potential value, the contributionof R-type Ca2⫹ current would be more than 30% of the total

FIG. 7. T-Type current in GnRH neurons. A, The currents were elicited by a ⫺30 mV pulse from the holding potential of ⫺80 mV inneurons around puberty. Combined application of nifedipine (10 ␮M),
GVIA (1 ␮M), Aga-IVA (200 nM), and SNX-482 (100 nM) slightly attenuated the peak amplitude of the current. This remaining current
was almost completely blocked by 50 ␮M Ni2⫹ (lower trace). B, IV
relationship of the peak current in the control (f); with nifedipine,
GVIA, Aga-IVA, and SNX-482 (E); and with the addition of Ni2⫹ (F).
C, The voltage pulses to ⫺70, ⫺60, and ⫺50 mV were given from theholding potential of ⫺100 mV at 0.2 Hz, as shown at the top. The upper
two current traces are from the neonatal neuron (male, 3 d old). The
current at ⫺70 mV is not shown. The lower three traces are from theneuron around puberty (female, 35 d old). The numbers at the upper
left in each trace indicate the amplitude of voltage pulses. For clarity,
the currents in C were filtered at 500 Hz.

Ca2⫹ current activated by the action potential. In fact, thecontribution of the R-type current was 45% in our APW
experiment (Fig. 4). Cytochemistry revealed a wide distribution of the prime candidate of R-type channel ␣1E (17) inthe brain in both mice (13) and rats (24), including the OVLT
and medial preoptic area. These findings suggest that R-type
Ca2⫹ channels must be expressed at least in the somadendritic region of GnRH neurons and contribute to Ca2⫹dependent regulation in GnRH neurons. The R-type Ca2⫹
channels might be involved in GnRH release at nerve endings, because the R-type channels are reported to contributetransmitter release at a rat calyx synapse (25), oxytocin
release from the nerve endings (26, 27), and exocytosis inmouse adrenal chromaffin cells (28).
We used the dihydropiridine antagonists nifedipine andnimodipine to block L-type current (29, 30). An L-type current was observed both in neonates and around puberty as
approximately 20% of total Ca2⫹ currents. Kusano et al. (7)reported a high voltage-activated Ca2⫹ current sensitive to
100 ␮m Cd2⫹ and 1 ␮m nifedipine expressed in mouse GnRH
neurons in explant culture of the olfactory pit, suggesting thepresence of an L-type current in these neurons. A similar typeof current has been reported in GT1 cells (10, 11). The L-type5124 Endocrinology, November 2003, 144(11):5118 –5125

current is well known to contribute hormone release in avariety of neuroendocrine cells, including pancreatic ␤ cells(31) and pituitary somatotrophs (32). In physiological conditions, an L-type current may be activated by slow depolarization, such as by an excitatory postsynaptic potential,
rather than by an action potential (33). Moreover, L-typecurrents become prominent in slow depolarization because
the inactivation process eliminates some other Ca2⫹ currents,
such as the R-type to a certain extent. Taken together withpreferential expression of L-type Ca2⫹ channels in the somadendritic region of central neurons (34), L-type currents may
regulate Ca2⫹-dependent functions, such as protein phosphorylation (33), enzyme activity, and gene expression, in
GnRH neurons in a different manner from that of the R-typecurrent.
The peptide antagonist GVIA is widely used to identify the
N-type Ca2⫹ current in physiological studies (35, 36). We
used 1 ␮m GVIA and found that the proportion of N-type
Ca2⫹ current was 15–20% of the total Ca2⫹ currents. N-Type
Ca2⫹ channels could be involved in GnRH release at nerve
endings, because the N-type channel is known to be involvedin vasopressin release (20), oxytocin release (26), and synaptic transmission in cultured hypothalamic neurons (37)and several central synapses (38). Immunostaining of N-type
Ca2⫹ channel subunit ␣1B revealed the presence of the Ntype channel not only at nerve terminals, but also in thesoma-dendritic region of central neurons (39), so that N-typechannels in the GnRH neuron may play some roles in thesoma-dendritic region besides at nerve terminals.
In the present study we did not separately identify P-type
and Q-type Ca2⫹ currents, but treated them as P/Q-type
Ca2⫹ currents by using a high concentration (200 nm) of
Aga-IVA that does not distinguish between P- and Q-typechannels (14). This was further confirmed with another P/Qtype channel blocker, MVIIC (2 ␮m). The P/Q-type Ca2⫹
current was small, but clearly observed around puberty in
40 – 62% of GnRH neurons examined. This developmental
change in the expression of P/Q-type Ca2⫹ current may havefunctional significance. For example, a P/Q-type channel
might be involved in GnRH release from nerve terminals at
the median eminence, which changes dramatically throughpuberty, thereby controlling gonadotropin release from theanterior pituitary. The P/Q-type Ca2⫹ current is shown invarious central neurons with different degrees of expression(40, 41). Q-type channels are present on a subset of the neurohypophysial terminals that release vasopressin (20). Developmental change in the contribution of P/Q-type Ca2⫹
current is also demonstrated at several central synapses (38).
Its contribution is greater on postnatal d 13–19 than on postnatal d 7–9.
Expression of T-type Ca2⫹ current also showed a clear
change in development. The T-type current is classified as alow voltage-activated current. Some R-type currents are also
activated in a similar voltage range (13, 16, 17). Therefore, inthe present study the T-type current was identified by its
sensitivity to Ni2⫹ and its insensitivity to SNX-482 (16) inaddition to the low voltage activation. This type of current
is demonstrated in mouse GnRH neurons in explant cultureof the olfactory pit (7) and GT1 cells (10, 11). T-type Ca2⫹
current in GnRH neurons possibly activates small conduc-

Kato et al. • Voltage-Gated Ca2⫹ Currents in Rat GnRH Neurons

tance, Ca2⫹-activated K⫹ channels (SK channels), such as in
midbrain dopaminergic neurons (42), thereby controlling action potential firing. According to the several reports concerning the firing pattern of mouse GnRH neurons, irregularspontaneous firing of single action potentials and irregularbursting of spikes are observed in these neurons (1– 6, 43). As
the SK channel is responsible for sustained tonic firing of
single spikes (42), the T-type Ca2⫹ current may function asa regulator of SK channels in mouse and possibly rat GnRH
neurons. The present results clearly demonstrate that theT-type current becomes active around the pubertal stage.
In conclusion, the present study revealed rat GnRH neurons functionally expressed L-, N-, and R-type Ca2⫹ channels
both in neonates and around puberty and expressed the
P/Q- and T-type Ca2⫹ channels around puberty. Cellularfunctions of these voltage-gated Ca2⫹ channels remain to beanalyzed in future experiments.
Acknowledgments
We are grateful to Dr. Koichi Ishikawa for his suggestions on themethod of dispersion of neurons. We also thank Drs. Hisashi Mori,
Tsuyoshi Hamada, Keisuke Kaneishi, Tomohiro Hamada, and Masugi
Nishihara for their help and valuable suggestions on the transgenic
technique.
Received February 14, 2003. Accepted August 6, 2003.
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@nms.ac.jp.
This work was supported in part by Grants-in-Aid for Scientific
Research (C) 10670071 and 13680883 from the Japan Society for the
Promotion of Science.

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