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Selective Modulation of Voltage-Gated Calcium Channels in the Terminal Nerve Gonadotropin-Releasing Hormone Neurons of a Teleost, the Dwarf Gourami (Colisa lalia)

Laboratory of Biological Signaling, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

Address all correspondence and requests for reprints to: Dr. Yoshitaka Oka, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: okay{at}biol.s.u-tokyo.ac.jp.

	Abstract

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GnRH neurons in the terminal nerve (TN) have been suggested to function as a neuromodulatory system that regulates long-lasting changes in the animal behavior. Here we examined electrophysiological properties of TN-GnRH neurons in a teleost (dwarf gourami, Colisa lalia), focusing on the voltage-gated Ca²⁺ channels, which are thought to be coupled to several cellular events such as GnRH release. TN-GnRH neurons showed low-voltage activated (LVA) currents and three types of pharmacologically distinct high-voltage activated (HVA) currents. The L- and N-type currents constituted 30.7 ± 3.1 and 41.0 ± 3.9%, respectively, of HVA currents, which was recorded at the holding potential of –60 mV to inactivate the LVA currents. Although P/Q-type current was small and negligible, R-type current accounted for the remaining 23.6 ± 1.6% of HVA currents. Next we examined the possibility of Ca²⁺ channel modulation induced by GnRH released in a paracrine/autocrine manner. HVA currents of up to 40% was inhibited by the application of salmon GnRH, which is the same molecular species of GnRH as is synthesized by TN-GnRH neurons themselves. However, salmon GnRH had no measurable effects on LVA currents. The inhibition of HVA currents had a dose dependence (EC₅₀ was 11.5 nM) and type specificity among different HVA currents; N- and R-type currents were preferentially inhibited, but L-type currents had by far lower sensitivity. The physiological significance of different Ca²⁺ influx pathways, and their paracrine/autocrine regulation mechanisms in TN-GnRH neurons are discussed.

	Introduction

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THERE HAS RECENTLY been a growing interest in the nature and cellular control mechanisms of release activities of GnRH neurons in vertebrate brains. However, due to the technical difficulties in identifying GnRH neurons in vertebrate brains, i.e. small cell bodies scattered in some hypothalamic and extrahypothalamic regions, physiological studies have been mainly focused on GnRH-secreting immortalized cell lines (GT1 cells) and cultured embryonic GnRH neurons (1, 2). The GnRH neurons of vertebrate brains have generally been identified in three distinct locations: terminal nerve (TN), hypothalamus, and midbrain (3, 4, 5). GnRH neurons located in the terminal nerve (TN-GnRH neurons) of the dwarf gourami (Colisa lalia) are large and make a tight cell cluster in the ventral surface of the olfactory bulb-telencephalon border and are easily identified visually. Thus, they serve as an ideal material for studying intact authentic GnRH neurons (3, 4, 5, 6, 7, 8, 9, 10). The TN-GnRH neurons are functionally distinct from the hypophysiotropic hypothalamic GnRH neurons and have been suggested to function as a neuromodulatory system that is involved in the regulation of long-lasting changes in the animal behavior (3, 5, 10). They have been well studied as a model system for the study of peptidergic neuromodulators (3, 4, 5, 8, 9, 10, 11, 12, 13, 14). The electrical activity of TN-GnRH neurons, regular pacemaker activity, has been suggested to be important for their role as a neuromodulator and has been shown to be controlled by the interaction of a variety of ion channels including Na⁺, K⁺, and Ca²⁺ channels (11, 12, 13). Despite the importance of voltage-gated Ca²⁺ channels in cellular physiological events, only a limited number of studies on the Ca²⁺ channels of hypophysiotropic GnRH neurons are available (15, 16, 17, 18, 19), and we know very little about the expression profile of Ca²⁺ channels in TN-GnRH neurons. Here we focused on the voltage-gated Ca²⁺ channels of TN-GnRH neurons and investigated the electrophysiological properties of Ca²⁺ channels with the use of specific peptide toxins.

Moreover, it has been reported that salmon GnRH (sGnRH), which is the same molecular species of GnRH as is synthesized by TN-GnRH neurons, affects the frequency of pacemaker activity (14), and this mechanism has been suggested to function as a paracrine/autocrine regulation of GnRH release in TN-GnRH neurons (4, 5). Similar effects of GnRH have also been reported in GT1 cells (15, 16, 17), and this receptor-coupled modulation of electrical activity has been suggested to be a candidate mechanism to account for the pulsatile release of GnRH. The physiological analysis of ion channels expressed in GT1 cells showed that GnRH receptor activation caused an activation of Ca²⁺-activated K⁺ currents and store-operated Ca²⁺ currents, which resulted in the transient hyperpolarization and the subsequent persistent depolarization of the membrane potential (16, 17). The mechanisms of GnRH modulation of pacemaker activity of GnRH neurons have also been studied in the TN-GnRH neurons, but the ion channels that are modulated by GnRH have not been studied in detail. Here we report that only the high-voltage activated (HVA) currents, but not the low-voltage activated (LVA) currents, are inhibited by GnRH in a dose-dependent and subtype-specific manner.

	Materials and Methods

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Preparation of brain slices with exposed TN-GnRH neurons
Adult male and female dwarf gouramis (C. lalia), approximately 4 cm in standard length, were purchased from a local dealer. Each fish tank containing up to 20 fish was maintained at 27 C and 12-h light, 12-h dark cycle. The fish were fed with worm once a day until used. They were chilled by immersing them in crushed ice and were quickly killed by decapitation. The animals were maintained and used in accordance with the guidelines of The Physiological Society of Japan and the University of Tokyo for the Use and Care of Experimental Animals. After the ventral meningeal membrane of the forebrain was carefully removed, thick brain slices of about 1 mm containing TN-GnRH cells were manually cut out with razor blades in a low-Na Ringer solution consisting of (in millimoles): NaCl 40, KCl 4.0, MgCl₂ 8.5, MgSO₄ 1.0, CaCl₂ 0.17, NaHCO₃ 26, NaH₂PO₄ 1.0, glucose 10, and sucrose 210. The slice-containing GnRH cells were allowed to recover for at least 1 h before the experiments in the standard external solution (in millimoles): NaCl 124, KCl 5.0, MgSO₄ 1.3, CaCl₂ 2.4, HEPES 10, and glucose 10 (pH 7.4). All the external solutions (including the experimental solutions, see below) were continuously bubbled with 95% O₂/5% CO₂ gas. All the experiments were performed at room temperature (22–24 C)

Recording solutions and pharmacological agents
The external recording solution used to isolate Ca²⁺ currents consisted of (in millimoles) 130 tetraethylammonium (TEA)-Cl, 1.3 MgSO₄, 10 HEPES, 5 4-aminopyridine, 5 CaCl₂, 0.001 tetrodotoxin, and 10 glucose (pH 7.4 adjusted with TEA-OH; 310–320 mOsm/liter). The patch pipette solution included the following (in millimoles): 90 CsCl, 2 MgCl₂, 20 TEA-Cl, 10 HEPES, 0.3–10 EGTA, 0.5 Li₂-GTP, 2 Mg-ATP, 13 phosphocreatine di-Tris salt, 50 U/ml creatinine phosphokinase (pH 7.2 with CsOH; 280–300 mOsm/liter).

The calcium channel antagonists (peptide toxins) and sGnRH were first dissolved in water containing 0.5% BSA, lyophilized, and then stored at –30 C. Each stock was diluted before use to the appropriate concentrations in an external recording solution containing 0.1% BSA to prevent nonspecific binding of peptides to the plastic and glassware.

The peptide toxins and sGnRH were purchased from the Peptide Institute (Osaka, Japan), and the other chemicals were from Sigma-Aldrich Corp. (St. Louis, MO). The potent GnRH analog antagonist [Ac-D-Ala[3-(2-naphthyl)]-D-Phe(4-Cl)-D-Ala[3-(3-pyridyl)]Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-OH; Ac-A[3-(2-naphthyl)]F(4-Cl)A[3-(3-pyridyl)]SYCitLRPA] (Cetrorelix) was kindly provided by Dr. T. Minegishi.

Electrophysiology
Under an upright microscope with infrared differential interference contrast (IR-DIC) optics, the exposed TN-GnRH neurons were easily identified visually in the brain slice (Fig. 1). The cell bodies of TN-GnRH neurons were large (30–40 µm in diameter) and made a tight cell cluster in the ventral surface of the olfactory bulb-telencephalon border. Whole-cell patch clamp recordings (20) were made from spherical TN-GnRH neurons that were fully exposed to the surface (not buried deep in the cluster) so as to maximize the space-clamp quality. Patch pipettes were pulled from borosilicate glass capillaries of 1.5 mm outer diameter (Narishige, Tokyo, Japan) using a Flaming-Brown microelectrode puller (P-97; Sutter Instruments, Navato, CA). The tip resistance of patch pipettes in the bath solution was approximately 2 M. Recordings were performed with a patch-clamp amplifier, Axopatch 200B (Axon Instruments, Foster City, CA), and currents were filtered at 1 kHz by low-pass four-pole Bessel filter and digitized at 100 kHz using pCLAMP software (Axon Instruments). In the recordings of HVA currents, capacitive and leakage currents were canceled by subtracting the currents not blocked by Cd²⁺ (100 µM) from the control records.

View larger version (110K): [in this window] [in a new window]   FIG. 1. DIC image of the cluster of TN-GnRH neurons in the brain slice. Sagittal brain slice of the dwarf gourami brain (rostral is to the right). Large arrowheads indicate TN-GnRH neurons. T, Telencephalon; OB, olfactory bulb.

The liquid junction potential of the pipette solution to the external solution was estimated to be as small as –4.3 mV (21). This junction potential counteracted the voltage error due to the uncompensated resistance, and we therefore neglected these voltage artifacts and did not carry out any corrections.

In some experiments, the voltage dependence of activation was assessed with voltage ramps from –60 to +40 mV (0.5 mV/msec). Using the Goldman-Hodgkin-Katz (GHK) constant field current equation, an estimate of membrane permeability was calculated as follows (22, 23):

where

and I(V_m) is the measured current divided by whole-cell capacitance (assumed to be 98.8 pF), V_m is the membrane potential (in millivolts), z is 2, F is 9.648 x 10⁴ Cmol^–1, R is 8.315 VCK^–1mol^–1, T is 273.16 + °C, [Ca²⁺]_i is assumed to be 100 nM, [Ca²⁺]_o is 5 mM, and P(V_m) is the membrane permeability to Ca²⁺ as a function of membrane potential.

Statistics
Statistical analyses were performed with GraphPad Prism (version 4, GraphPad Software, San Diego, CA) and Clampfit (Axon Instruments). Concentration-response relationships were fitted to a four-parameter logistic equation using a nonlinear curve-fitting program (GraphPad Prism). In the box plots, the median line in the box represents the median value, whereas the sides of the box represent the inner quartiles of the data set. The bars extending from the box indicate the two outer quartiles of the data set. Numerical data in the present paper are represented as mean ± SEM unless otherwise noted.

	Results

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HVA Ca²⁺ currents in TN-GnRH neurons
Figure 2A shows isolated Ca²⁺ currents evoked by depolarizing voltage steps from the holding potential of –60 mV to test potentials (–60 to 20 mV). The currents showed rapid activation with slow and small inactivation. They activated with test potentials more positive than –30 mV and peaked at about 0 mV. Current response to a slow ramp depolarization (from –60 to +40 mV in 200 msec; Fig. 2B) showed a similar current-voltage (I-V) relationship to the one constructed from the peak currents in response to step depolarizations (Fig. 2C). When the voltage dependence of activation was estimated from the ramp response by a conversion based on the GHK current equation, it was very similar to the one calculated from the relative amplitude of tail currents (Fig. 2D). In 10 cells tested using the ramp protocol, the half-activation voltage was –12.3 ± 0.9 mV, with a slope factor of 4.7 ± 0.3 mV. From these observations, the Ca²⁺ currents shown in Fig. 2 were classified into the HVA currents.

View larger version (22K): [in this window] [in a new window]   FIG. 2. HVA currents in TN-GnRH neurons. A, Current traces elicited by step depolarizations from a holding potential of –60 mV to test potentials (from –60 to +20 mV with 10-mV increments). B, Current response to 200 msec ramp depolarization from –60 to 40 mV (0.5 mV/msec). C, After the leakage and capacitive currents were subtracted, an I-V curve was constructed from the current response obtained by ramp protocol. Circles () indicate peak negative currents in A. D, I-V relationship in ramp responses was converted to permeability-voltage relationship using the GHK current equation (continuous line). Squares () indicate relative amplitudes of tail currents in A. Note that the activation curves of HVA currents constructed from ramp protocol and that from tail current analysis agree well with each other.

Pharmacological characterization of HVA currents
To examine the subtypes of HVA Ca²⁺ currents in TN-GnRH neurons, the time course of peak Ca²⁺ currents elicited by step depolarization form –60 to +10 mV were plotted during the sequential application of specific HVA channel blockers. As shown in Fig. 3A, nimodipine (Nimo, 5 µM) and -conotoxin GVIA (CgTx, 1 µM) (24) blocked 30.7 ± 3.1 and 41.0 ± 3.9% of initial HVA currents, respectively (n = 9 female and 9 male). To assess the contribution of P/Q-type calcium currents to the total HVA currents, effects of -conotoxin MVIIC (MVIIC, 1 µM) (25) and -agatoxin TK (AgTx, 100 nM) (26) were then examined. In 20 cells tested, there was no measurable effect of AgTx or MVIIC. The current component resistant to the blockade by the antagonists thus far mentioned (Nimo, CgTx, AgTx, and MVIIC) but sensitive to 100 µM Cd²⁺ accounted for 23.6 ± 1.6% (n = 18) of the initial HVA currents. SNX-482, the synthetic version of a peptide toxin isolated from the venom of the West African tarantula Hysterocrates gigas, was found to be a specific blocker of R-type Ca²⁺ currents in several types of central neurons including green fluorescent protein (GFP)-tagged rat GnRH neurons (27 , see Discussion). As shown in Fig. 3A, SNX-482 blocked a considerable fraction of the residual current in a dose-dependent manner with IC₅₀ of approximately 40 nM (n = 3). Furthermore, CgTx and Nimo reduced about the same percentage of HVA currents when SNX was applied first (Fig. 3B). Thus, the residual current component above was classified into R-type Ca²⁺ currents in the present study.

View larger version (26K): [in this window] [in a new window]   FIG. 3. Pharmacological characterization of the HVA currents in TN-GnRH neurons. A, Time course of peak inward currents (absolute values) during sequential addition of specific blockers of HVA channels. Blockers were present during the time periods indicated by the bars above the plots. The current component that was insensitive to all these blockers was blocked by SNX-482 (SNX) in a dose-dependent manner. B, The effects of SNX was tested without prior addition of the other HVA channel blockers. It should be noted that SNX does not reduce the component sensitive to CgTx or Nimo. C, Estimates for voltage dependence of activation was constructed for L-, N-, and R-type current. Each estimate was well fitted by a single Boltzman function (continuous lines). In this cell, the Vhalf was –9.5 (L-type), –11.4 (N-type), and –12.7 (R-type) mV, and the slope factor was 8.4 (L-type), 4.7 (N-type), and 7.2 (R-type) mV. D, The box plots showing the Vhalf and slope factor, constructed from data of seven cells.

Figure 3D shows the summary of half-activation voltage (V_half) and slope factor of each current component in seven cells. V_half was –11.2 ± 1.7 (L-type), –9.7 ± 1.2 (N-type), and –13.8 ± 1.5 mV (R-type), and the slope factor was 6.3 ± 1.6 mV (L-type), 5.0 ± 0.9 mV (N-type), and 6.3 ± 2.1 mV (R-type).

LVA Ca²⁺ currents in TN-GnRH neurons
LVA currents were isolated in the presence of a mixture of various HVA blockers (Nimo, CgTx, and SNX-482). Figure 4A shows the LVA Ca²⁺ currents elicited by voltage steps from the holding potential of –100 mV to the test potentials (–90 to 0 mV). LVA currents could be activated by low-voltage steps such as –70 or –60 mV and were completely inactivated during the 200-msec voltage step. Furthermore, the LVA currents were completely blocked by 200 µM Ni²⁺ (not shown). From these results, we concluded that LVA currents of TN-GnRH neurons can be classified as a conventional T-type current. The voltage dependence of activation was calculated from the I-V plot (Fig. 4B) using the GHK current equation (Fig. 4D, activation). In the seven cells tested, the V_half was –56.9 ± 1.4 mV, and the slope factor was 7.4 ± 1.3 mV. The steady-state voltage dependence of inactivation was studied by double-pulse protocols. After prepulses of 2 sec duration (–120 to –30 mV), the peak inward current was measured at the test potential of –20 mV (Fig. 4C). The current amplitudes normalized to the one evoked from –120 mV prepulse were plotted as a function of prepulse potentials, and the data were well fitted by a single Boltzman function (Fig. 4D, inactivation). For the five cells tested, the V_half was –76.0 ± 1.3 mV, and the slope factor was –9.2 ± 1.2 mV.

View larger version (28K): [in this window] [in a new window]   FIG. 4. LVA currents in TN-GnRH neurons. A, The currents were elicited by 200-msec step depolarizations from a holding potential of –100 mV to test potentials (from –90 to 0 mV, 10-mV increments) in the presence of various blockers of HVA currents. B, I-V relationship of relative amplitude of the peak negative currents (n = 4). The peak negative currents were normalized to the maximum current amplitude for each cell. C, Holding potentials were varied from –120 to –30 mV, and the currents elicited by a 100-msec test pulse to –20 mV were measured. The plot of the current amplitudes normalized to the one evoked from –120 mV prepulse gives the steady-state inactivation curve shown in D (n = 5). D, The activation curve was estimated from the peak current of B with a GHK current equation (n = 4). All data were fitted by a single Boltzmann function. The Vhalf was –56 mV, and the Vhalf was –76 mV.

View larger version (22K): [in this window] [in a new window]   FIG. 5. Effects of sGnRH on the HVA current in TN-GnRH neurons. A, Changes in the peak inward currents were plotted on a time scale. Effects of GnRH on the peak HVA currents appeared within 10 sec after GnRH application and reached the steady state within 1 min. Inset, HVA currents obtained by depolarization from –60 to –10 mV with or without GnRH (100 nM). B, I-V relationship of HVA currents obtained from ramps with or without GnRH. Leakage and capacitive currents were not subtracted in these data. It should be noted that there is a depolarizing shift of peak current along the voltage axis (5.7 mV, between arrows), whereas the reversal potential was not affected. C, Voltage dependence of activation was estimated form negative peak currents elicited by step depolarizations (from –60 to +25 mV, 5-mV increments; n = 7 cells). D, Concentration dependence of the effects of GnRH (n = 5–7 cells for each concentration, ) and Cetrorelix (GnRH analog, n = 3 cells, ) on HVA currents. In the presence of 100 nM Cetrorelix, the effect of GnRH was attenuated with a shift of EC50 from 11.4 to 53.6 nM (n = 3). Data represent means of peak currents obtained by step depolarizations form –60 to –10 mV; bars represent SEM.

The dose dependence of this inhibition was examined, and it was shown that the inhibitory effect was saturated at GnRH concentrations higher than 1 µM (Fig. 5D), and the maximum inhibition was 40.1% of the total HVA currents. EC₅₀ of sGnRH was 11.5 ± 0.3 nM. To test whether this HVA current modulation was mediated by the activation of GnRH receptors, we examined the effects of GnRH analog antagonist (Cetrorelix, [Ac-D-N al (2)l, D-Phe (4Cl)2, D-P al (3)3, D-Cit6, D-Ala10] LHRH) (28). First, we confirmed that Cetrorelix alone had no effect on HVA currents at concentrations as high as 1 µM (Fig. 5D, Cetrorelix). On the other hand, we found that a high concentration of Cetrorelix attenuated the GnRH-induced inhibition (EC₅₀ was shifted to 53.6 ± 0.6 nM in 100 nM Cetrorelix). This means that the presence of GnRH analog Cetrorelix reduced the availability of functional GnRH receptors and thus attenuated the inhibition of HVA currents, which indicates that the inhibitory effect of GnRH on the HVA currents is mediated by specific GnRH receptors.

In addition to the current amplitude and voltage dependence of activation, we evaluated the effects of GnRH on the activation kinetics of the HVA currents. As shown in Fig. 6, HVA currents activated rapidly to reach a peak and thereafter showed a slow and partial inactivation when elicited by step-depolarizing pulses. In the presence of 100 nM GnRH, we observed a significant slowing of the current activation (not shown). The slow and partial inactivation was also observed during the application of GnRH (Fig. 6). Although a voltage-dependent modulation of N-type Ca²⁺ currents by G protein ß-subunits, known as kinetic slowing, has been reported (29), the current behaviors observed above in the GnRH modulation of HVA currents seem to be somewhat different (see Discussion). We also examined the occurrence of the voltage-dependent relief from inhibition by strong depolarizing prepulses such as +100 mV and 100 msec (30, 31, 32, 33). However, the currents inhibited by GnRH in the present study never showed full recovery by strong depolarizing prepulses but only showed a small (10–20%) relief of inhibition (not shown).

View larger version (10K): [in this window] [in a new window]   FIG. 6. Effects of GnRH on the kinetics of HVA currents. A, Voltage steps from –60 to –20 mV were applied for 100 msec in the absence or presence of 100 nM GnRH. B, The trace in the presence of 100 nM GnRH was scaled up by a factor of 1.55 to match the control peak current amplitude. In the presence of GnRH, the time to peak was slightly delayed, whereas the slow and partial inactivation was similar to the one observed in the control trace.

View larger version (26K): [in this window] [in a new window]   FIG. 7. The effects of GnRH on LVA currents. A, I-V relationship of peak inward LVA currents in external solutions with or without 100 nM GnRH. Currents were elicited by step depolarizations from a holding potential of –100 mV to test potentials. B, Steady-state inactivation was examined in the absence or presence of 100 nM GnRH. There was no significant difference between the control and GnRH-perfused groups (n = 7). C, Two-pulse protocol was used to measure recovery from inactivation. Interpulse holding potential of –100 or –80 mV was tested, and the interpulse interval was changed. D, Ratio of the peak current in response to the test (second) pulse to that in response to the first one was plotted as a function of the interpulse interval. The application of GnRH did not affect the recovery from inactivation of LVA currents.

View larger version (14K): [in this window] [in a new window]   FIG. 8. Pharmacological isolation of GnRH-insensitive HVA current. A, Peak inward currents elicited by step depolarizations from –60 to –10 mV were plotted as a function of time, whereas HVA antagonists were sequentially applied (see Fig. 3 legend). GnRH-insensitive current was categorized as L- (Nimo-sensitive), N- (CgTX-sensitive), or R-type (residual, Cd2+-sensitive) current. B, Percentage of each GnRH-insensitive HVA current component was compared with that obtained without GnRH treatment. In the control as well as the GnRH-treated groups, the percentage was defined as the ratio of the current amplitude of each subtype to the total HVA current amplitude before the GnRH treatment. There were statistical significances in N and R type (*, P < 0.05) but not in L type).

View larger version (26K): [in this window] [in a new window]   FIG. 9. Effects of GnRH examined in isolated HVA currents. A, Current traces of L-type component, which is insensitive to CgTx and SNX elicited by step depolarization from –60 to test potentials (–50 to +10 mV). Leakage currents were subtracted with P/4 protocols. B and C, Current traces of Nimo and SNX-insensitive (N-type) and CgTx and& Nimo-insensitive (R-type) current components, respectively. Voltage commands are the same as that of A. D, I-V relationship of L-type currents constructed with a negative peak current in A ( control, 100 nM GnRH). E and F, I-V relationship of N- and R-type currents, respectively. Open circles and open triangles indicate peak currents in the control, whereas filled circles and filled triangles indicate the values in the presence of 100 nM GnRH.

	Discussion

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Voltage-gated Ca²⁺ channels in TN-GnRH neurons
Ca²⁺ influx through voltage-gated Ca²⁺ channels is prerequisite for the GnRH release in GT1 cells, hypothalamic culture (35, 36), and TN-GnRH neurons in the dwarf gourami (37). Ishizaki et al. (37) measured GnRH release from a brain slice containing TN and midbrain tegmentum GnRH neurons by RIA and showed that depolarization-induced release was significantly reduced by the application of nifedipine and -conotoxin GVIA, but not by AgTx. Our results showing that TN-GnRH neurons expressed L-, N-, R-, and T-type currents, but not P/Q-type current, thus agree quite well with their data.

L- and N-type currents have been observed in GT1 cells and embryonic GnRH neurons in explant culture (2, 15, 38). Recently Kato et al. (18) and Nunemaker et al. (19) reported on the expression profiles of voltage-gated Ca²⁺ channels in GFP-tagged hypothalamic GnRH neurons in rats and mice. GFP-tagged hypothalamic GnRH neurons had R- and P/Q-type currents besides L and N type. In the present study, we found that TN-GnRH neurons express L-, N-, and R-type Ca²⁺ currents. These differences in the expression patterns of different Ca²⁺ currents may be partly because of the differences among different reports in the type of GnRH neurons and several experimental conditions, such as animal species, maturities, and procedures for dissociation and culturing.

R-type currents with pharmacological properties similar to the GFP-tagged hypothalamic GnRH neurons of rats (18) were observed in TN-GnRH neurons as well. Remarkably R-type currents in both hypothalamic and TN-GnRH neurons were highly sensitive to 100 nM SNX-482, and SNX-insensitive component was very small. However, except for the pharmacological sensitivity to SNX-482, the electrophysiological properties of these currents were rather different; V_half was –14 mV in TN-GnRH neurons and 0 mV in GnRH neurons of the rat. It is unclear to what extent methodological difference of Ca²⁺ current recording could contribute to this discrepancy. Another possibility is the channel heterogeneity (39) or differential expression of Ca²⁺ channel ß-subunits (40, 41).

HVA Ca²⁺ channels are thought to be tightly coupled to active zones and involved in triggering neurotransmitter release in a very fast and localized manner (42). P/Q-type currents are reported to be most efficiently coupled to secretory machinery in neuromuscular junctions (43), and R-type current is more tightly coupled to the control of rapid secretory responses than N- or P/Q-type currents in chromaffin cells (44, 45). Thus, the study of differential distribution of different Ca²⁺ channel types and their coupling to GnRH release machinery may be an important future problem.

Among the HVA currents in TN-GnRH neurons, N-type current comprised approximately 40% of the total current. However, in other GnRH-secreting neurons, the proportion of N-type current was usually between 10 and 30% (2, 15, 18, 19). Because there is a growing body of evidence to show that N-type Ca²⁺ current is one of the major target of neuromodulation induced by a number of G protein-mediated pathways (34), the predominance of N-type currents in the TN-GnRH neurons may make them more susceptible to modulation of Ca²⁺ influx.

T-type Ca²⁺ current has been reported to be important in the generation of oscillatory activity in some neurons (46). Although TN-GnRH neurons show regular pacemaker activities, it has been shown not to be the T-type Ca²⁺ current but a tetrodotoxin-resistant persistent Na⁺ current that is mainly responsible for its generation (11, 12). On the other hand, T-type currents may contribute to the rebound action potentials after hyperpolarizing current injections due to their increased availability.

Inhibitory effects of GnRH on Ca²⁺ channels
It has been reported in the TN-GnRH neurons of the dwarf gourami that sGnRH, which is the same molecular species of GnRH as is synthesized by TN-GnRH neurons, affects the frequency of pacemaker activity (14), and this mechanism was suggested to function as a paracrine/autocrine regulation of GnRH release in TN-GnRH neurons (4, 5, 14). Therefore, we examined the effects of GnRH on the voltage-gated Ca²⁺ currents in TN-GnRH neurons. We found that HVA currents of up to 40% were inhibited by GnRH, whereas LVA currents were not. This inhibition is suggested to be mediated by the activation of GnRH receptors, considering the results of Cetrorelix experiments (Fig. 5D). Furthermore, we also confirmed the expression of GnRH receptor mRNAs in TN-GnRH neurons by in situ hybridization (our unpublished data).

We confirmed, among HVA subtypes, the differences in sensitivity to the GnRH-induced inhibition in two strategies (see Results). It should be noted that L-type current, for example, was defined as a nimodipine-sensitive current component in the first strategy, whereas it was defined as an N- and R-type blocker-insensitive current components in the second strategy. Although the sensitivities of each subtype to the GnRH-induced inhibition obtained from the second strategy may be overestimated due to a contamination by unblocked currents of other subtypes, the present results clearly show that N- and R-type currents have much higher sensitivity to GnRH-induced inhibition than L-type current.

Whereas the present report shows the first evidence to demonstrate the GnRH-induced modulation of ion channels in GnRH neurons, there have been not a few reports mainly in the peripheral ganglia that voltage-gated Ca²⁺ channels are modulated by GnRH. The major type of GnRH-induced modulation of HVA currents is via the voltage-dependent mechanism, which can be relieved by a strong depolarizing prepulse (47, 48). In the voltage-dependent inhibition, the activation of currents becomes slower, which has been referred to as kinetic slowing (29). This voltage-dependent inhibition was generally caused by activation of G_i/G_o-, G_z-, or G_s-coupled receptors. On the other hand, the activation of G_q/11-coupled receptor causes a voltage-independent inhibition, which is not accompanied by kinetic slowing (33, 49, 50, 51). Because our results indicated a lack of prominent kinetic slowing or voltage-dependent relief of inactivation, the channel inhibition by GnRH observed in the present paper may belong to the voltage-independent inhibition mediated through activation of G_q/11-coupled receptor. It has been established that GnRH receptor is equally coupled to G_q and G₁₁ subunits (52). In the TN-GnRH neurons, it has been suggested that GnRH receptor is coupled to G_q/11 (14) because Abe and Oka (53) obtained evidence to show that Ca²⁺ released from intracellular stores activates apamin-sensitive Ca²⁺-activated K⁺ current [I_K(Ca)] so that GnRH receptor activation transiently decreases the frequency of pacemaker activity in the initial phase. On the other hand, it has been reported that GnRH receptor can also be coupled to G_i/o subunits (54). The mechanisms of GnRH-induced inhibition of HVA currents in the TN-GnRH neurons remain to be clarified, and the use of inhibitors of these signal molecules will help us to identify the type of active G protein subunits and understand the intracellular signaling pathways from the activation of GnRH receptor to the modulation of HVA currents.

Physiological significance of HVA Ca²⁺ channel modulation
We suggest that the modulation of HVA currents is a part of orchestrated changes of electrical activities by GnRH that lead to accelerated pacemaking activity and altered action potential wave forms. In the physiological conditions, the application of GnRH not only increased the frequency of pacemaker activity (14) but also broadened the action potential and reduced its spike afterhyperpolarization (our unpublished observations). In TN-GnRH neurons, Ca²⁺ influx activates SK-type I_K(Ca) (53), which should underlie the fast repolarization of the action potential. It might be possible that I_K(Ca) participates in the regulation of afterhyperpolarization and/or action potential duration and that the modulation of Ca²⁺ currents plays an important role in the activation of I_K(Ca). The increase in the frequency of pacemaker activity seems not to be dependent on the Ca²⁺ current modulation but on the other ionic mechanisms, which is under investigation at present.

In GT1 cells, Van Goor et al. (15, 17, 55) reported that GnRH application broadens their spontaneous action potentials and increases their firing rate, thus enabling long-term boosting of Ca²⁺ influx, although the activity of Ca²⁺ channels per se is not affected. In TN-GnRH neurons, GnRH application also affected the shape and firing frequency of spontaneous action potentials, and the activity of Ca²⁺ channels was affected as well. Therefore, to understand the physiological significance of GnRH-induced channel modulation, it should be helpful to examine the changes in the intracellular Ca²⁺ levels and GnRH release during GnRH application in the physiological conditions. Along this line, Ca²⁺ imaging and amperometric measurement of GnRH release (56) in TN-GnRH neurons as well as computational simulation studies are necessary and are already in progress.

	Acknowledgments

 
We thank Professor T. Minegishi (Gunma University, Maebashi, Gunma, Japan) for his kind gift of the GnRH analog (Cetrorelix). We also thank Drs. H. Abe (University of Wisconsin-Madison), M. K. Park (The University of Tokyo, Tokyo, Japan), and K. Shimazaki (Jichi Medical School, Minamikawachi-machi, Tochigi, Japan) for kind technical advice and helpful discussion.

	Footnotes

 
This work was supported by Grants-in-Aid (15029206 and 15370032) from The Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to Y.O.).

Abbreviations: AgTx, -Agatoxin TK; CgTx, -conotoxin GVIA; GFP, green fluorescent protein; GHK, Goldman-Hodgkin-Katz; HVA, high-voltage activated; I_K(Ca), Ca²⁺-activated K⁺ current; I-V, current-voltage; LVA, low-voltage activated; MVIIC, -conotoxin MVIIC; Nimo, nimodipine; sGnRH, salmon GnRH; TEA, tetraethylammonium; TN, terminal nerve; V_half, half-activation voltage.

Received March 18, 2004.

Accepted for publication June 22, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Mellon PL, Windle JJ, Goldsmith PC, Padula CA, Roberts JL, Weiner RI 1990 Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5:1–10[CrossRef][Medline]
2. Kusano K, Fueshko S, Gainer H, Wray S 1995 Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures. Proc Natl Acad Sci USA 92:3918–3922[Abstract/Free Full Text]
3. Oka Y 1997 The gonadotropin-releasing hormone (GnRH) neuronal system of fish brain as a model system for the study of peptidergic neuromodulation. In: Parhar IS, Sakuma Y, eds. GnRH neurons: genes to behavior. Tokyo: Brain Shuppan; 245–276
4. Oka Y, Abe H 2002 Physiology of GnRH neurons and modulation of their activities by GnRH. In: Handa RJ, Hayashi S, Terasawa E, Kawata M, eds. Neuroplasticity, development, and steroid hormone action. Boca Raton, FL: CRC Press; 191–203
5. Oka Y 2002 Physiology and release activity of GnRH neurons. Prog Brain Res 141:259–281[Medline]
6. Amano M, Oka Y, Aida K, Okumoto N, Kawashima S, Hasegawa Y 1991 Immunocytochemical demonstration of salmon GnRH and chicken GnRH-II in the brain of masu salmon, Oncorhynchus masou. J Comp Neurol 314:587–597[CrossRef][Medline]
7. Kim MH, Oka Y, Amano M, Kobayashi M, Okuzawa K, Hasegawa Y, Kawashima S, Suzuki Y, Aida K 1995 Immunohistochemical localization of sGnRH and cGnRH-II in the brain of goldfish, Carassius auratus. J Comp Neurol 356:72–82[CrossRef][Medline]
8. Yamamoto N, Oka Y, Amano M, Aida K, Hasegawa Y, Kawashima S 1995 Multiple gonadotropin-releasing hormone (GnRH)-immunoreactive systems in the brain of the dwarf gourami, Colisa lalia: immunohistochemistry and radioimmunoassay. J Comp Neurol 355:354–368[CrossRef][Medline]
9. Oka Y, Ichikawa M 1990 Gonadotropin-releasing hormone (GnRH) immunoreactive system in the brain of the dwarf gourami (Colisa lalia) as revealed by light microscopic immunocytochemistry using a monoclonal antibody to common amino acid sequence of GnRH. J Comp Neurol 300:511–522[CrossRef][Medline]
10. Oka Y, Matsushima T 1993 Gonadotropin-releasing hormone (GnRH)-immunoreactive terminal nerve cells have intrinsic rhythmicity and project widely in the brain. J Neurosci 13:2161–2176[Abstract]
11. Oka Y 1995 Tetrodotoxin-resistant Na⁺ current underlying pacemaker potentials of fish gonadotropin-releasing-hormone neurons. J Physiol 482:1–6
12. Oka Y 1996 Characterization of TTX-resistant persistent Na⁺ current underlying pacemaker potentials of fish gonadotropin-releasing hormone (GnRH) neurons. J Neurophysiol 75:2397–2404[Abstract/Free Full Text]
13. Abe H, Oka Y 1999 Characterization of K⁺ currents underlying pacemaker potentials of fish gonadotropin-releasing hormone cells. J Neurophysiol 81:643–653[Abstract/Free Full Text]
14. Abe H, Oka Y 2000 Modulation of pacemaker activity by salmon gonadotropin-releasing hormone (sGnRH) in terminal nerve (TN)-GnRH neurons. J Neurophysiol 83:3196–3200[Abstract/Free Full Text]
15. Van Goor F, Krsmanovic LZ, Catt KJ, Stojilkovic SS 1999 Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels. Mol Endocrinol 13:587–603[Abstract/Free Full Text]
16. Van Goor F, Krsmanovic LZ, Catt KJ, Stojilkovic SS 1999 Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion. Proc Natl Acad Sci USA 96:4101–4106[Abstract/Free Full Text]
17. Van Goor F, Krsmanovic LZ, Catt KJ, Stojilkovic SS 2000 Autocrine regulation of calcium influx and gonadotropin-releasing hormone secretion in hypothalamic neurons. Biochem Cell Biol 78:359–370[CrossRef][Medline]
18. Kato M, Ui-Tei K, Watanabe M, Sakuma Y 2003 Characterization of voltage-gated calcium currents in gonadotropin-releasing hormone neurons tagged with green fluorescent protein in rats. Endocrinology 144:5118–5125[Abstract/Free Full Text]
19. Nunemaker CS, Defazio RA, Moenter SM 2003 Calcium current subtypes in GnRH neurons. Biol Reprod 69:1914–1922[Abstract/Free Full Text]
20. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ 1981 Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100[CrossRef][Medline]
21. Neher E 1992 Correction for liquid junction potentials in patch clamp experiments. Method Enzymol 207:123–131[Medline]
22. Bargas J, Howe A, Eberwine J, Cao Y, Surmeier DJ 1994 Cellular and molecular characterization of Ca²⁺ currents in acutely isolated, adult-rat neostriatal neurons. J Neurosci 14:6667–6686[Abstract]
23. Lorenzon NM, Foehring RC 1995 Characterization of pharmacologically identified voltage-gated calcium-channel currents in acutely isolated rat neocortical neurons. I. Adult neurons. J Neurophysiol 73:1430–1442[Abstract/Free Full Text]
24. McCleskey EW, Fow AP, Feldman DH, Cruz LJ, Olivera BM, Tsien RW, Yoshikami D 1987 -Conotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not in muscle. Proc Natl Acad Sci USA 84:4327–4331[Abstract/Free Full Text]
25. McDonough SI, Swartz KJ, Mintz IM, Boland LN, Bean BP 1996 Inhibition of calcium channels in rat central and peripheral neurons by -conotoxin MVIIC. J Neurosci 16:2612–2623[Abstract/Free Full Text]
26. Kuwada M, Teramoto T, Kumagaye KY, Nakajima K, Watanabe T, Kawai T, Kawakami Y, Niidome T, Sawada K, Nishizawa Y, Katayama K 1994 -Agatoxin-TK containing D-serin at position 46, but not synthetic -[L-Der46]agatoxin-TK, exerts blockade of P-type calcium channels in cerebellar Purkinje neurons. Mol Pharmacol 46:587–593[Abstract]
27. Newcomb R, Szoke B, Palma A, Wang G, Chen X-H, Hopkins W, Cong R, Miller J, Urge L, Tarczy-Hornoch K, Loo JA, Dooley DJ, Nadasdi L, Tsien RW, Lemos J, Miljanich G 1998 Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas. Biochemistry 37:15353–15362[CrossRef][Medline]
28. Schally AV 1999 Luteinizing hormone-releasing hormone analogs: their impact on the control of tumorigenesis. Peptides 20:1247–1262[CrossRef][Medline]
29. Ikeda SR 1996 Voltage-dependent modulation of N-type calcium channels by G-protein ß subunits. Nature 380:255–258[CrossRef][Medline]
30. Dolphin AC, Scott RH 1987 Calcium-channel currents and their inhibition by (–)-baclofen in rat sensory neurons—modulation by guranine-nucleotides. J Physiol 386:1–17[Abstract/Free Full Text]
31. Ikeda SR, Schofield GG 1989 Somatostatin blocks a calcium current in rat sympathetic-ganglion neurons. J Physiol 409:221–240[Abstract/Free Full Text]
32. Jeong SW, Ikeda SR 1998 G protein subunit G (z) couples neurotransmitter receptors to ion channels in sympathetic neurons. Neuron 21:1201–1212[CrossRef][Medline]
33. Hille B 1994 Modulation of ion-channel function by G-protein-coupled receptors. Trends Neurosci 17:531–536[CrossRef][Medline]
34. Elmslie KS 2003 Neurotransmitter modulation of neuronal calcium channels. J Bioenerg Biomembr 35:477–489[CrossRef][Medline]
35. Krsmanovic LZ, Stojilkovic SS, Mertz LM, Tomic M, Catt KJ 1993 Expression of gonadotropin-releasing-hormone receptors and autocrine regulation of neuropeptide release in immortalized hypothalamic neurons. Proc Natl Acad Sci USA 90:3908–3912[Abstract/Free Full Text]
36. Krsmanovic LZ, Martinez-Fuentes AJ, Arora KK, Mores N, Navarro CE, Chen HC, Stojilkovic SS, Catt KJ 1999 Autocrine regulation of gonadotropin-releasing hormone secretion in cultured hypothalamic neurons. Endocrinology 140:1423–1431[Abstract/Free Full Text]
37. Ishizaki M, Iigo M, Yamamoto N, Oka Y 2004 Different modes of gonadotropin-releasing hormone (GnRH) release from multiple GnRH systems as revealed by radioimmunoassay using brain slices of a teleost, the dwarf gourami (Colisa lalia). Endocrinology 145:2092–2103[Abstract/Free Full Text]
38. Constantin JL, Charles AC 1999 Spontaneous action potentials initiate rhythmic intercellular calcium waves in immortalized hypothalamic (GT1–1) neurons. J Neurophysiol 82:429–435[Abstract/Free Full Text]
39. Tottene A, Moretti A, Pietrobon D 1996 Functional diversity of P-type and R-type calcium channels in rat cerebellar neurons. J Neurosci 16:6353–6363[Abstract/Free Full Text]
40. Castellano A, Perezreyes E 1994 Molecular diversity of Ca²⁺ channel ß-subunits. Biochem Soc Trans 22:483–488[Medline]
41. Dewaard M, Campbell KP 1995 Subunit regulation of the neuronal (1A) Ca²⁺ in Xenopus oocytes. J Physiol 485:619–634[Medline]
42. Neher E 1998 Vesicle pools and Ca²⁺ microdomains: new tools for understanding their roles in neurotransmitter release. Neuron 20:389–399[CrossRef][Medline]
43. Urbano FJ, Piedras-Renteria ES, Jun KS, Shin HS, Uchitel OD, Tsien RW 2003 Altered properties of quantal neurotransmitter release at endplates of mice lacking P/Q-type Ca²⁺ channels. Proc Natl Acad Sci USA 100:3491–3496[Abstract/Free Full Text]
44. Albillos A, Neher E, Mose T 2000 R-type Ca²⁺ channels are coupled to the rapid component of secretion in mouse adrenal slice chromaffin cells. J Neurosci 20:8323–8330[Abstract/Free Full Text]
45. Aldea M, Jun K, Shin HS, Andres-Mateos E, Solis-Garrido LM, Montiel C, Garcia AG, Albillos A 2002 A perforated patch-clamp study of calcium currents and exocytosis in chromaffin cells of wild-type and (1A) knockout mice. J Neurochem 81:911–921[CrossRef][Medline]
46. Bal T, McCormick DA 1996 What stops synchronized thalamocortical oscillations? Neuron 17:297–308[CrossRef][Medline]
47. Bean BP 1989 Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence. Nature 340:153–156[CrossRef][Medline]
48. Elmslie KS, Zhou W, Jones SW 1990 LHRH and GTP--S modify calcium current activation in bullfrog sympathetic neurons. Neuron 5:75–80[CrossRef][Medline]
49. Shapiro MS, Wollmuth LP, Hille B 1994 Modulation of Ca²⁺ channels by PTX-sensitive G-proteins is blocked by N-ethylmaleimide in rat sympathetic neurons. J Neurosci 14:7109–7116[Abstract]
50. Shapiro MS, Hille B 1993 Substance-P and somatostatin inhibit calcium channels in rat sympathetic neurons via different G-protein pathways. Neuron 10:11–20[CrossRef][Medline]
51. Haley JE, Delmas P, Offermanns S, Abogadie FC, Simon MI, Buckley NJ, Brown DA 2000 Muscarinic inhibition of calcium current and M current in G (q)-deficient mice. J Neurosci 20:3973–3979[Abstract/Free Full Text]
52. Grosse R, Schmid A, Schoneberg T, Herrlich A, Muhn P, Schultz G, Gudermann T 2000 Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins. J Biol Chem 275:9193–9200[Abstract/Free Full Text]
53. Abe H, Oka Y 2002 Mechanisms of the modulation of pacemaker activity by GnRH peptides in the terminal nerve-GnRH neurons. Zool Sci 19:111–128[CrossRef][Medline]
54. Krsmanovic LZ, Mores N, Navarro CE, Tomic M, Catt KJ 2001 Regulation of Ca²⁺-sensitive adenylyl cyclase in gonadotropin-releasing hormone neurons. Mol Endocrinol 15:429–440[Abstract/Free Full Text]
55. Van Goor F, LeBeau AP, Krsmanovic LZ, Sherman A, Catt KJ, Stojilkovic SS 2000 Amplitude-dependent spike-broadening and enhanced Ca²⁺ signaling in GnRH-secreting neurons. Biophys J 79:1310–1323[Abstract/Free Full Text]
56. Ishizaki M, Oka Y 2001 Amperometric recording of gonadotropin-releasing hormone release activity in the pituitary of the dwarf gourami (teleost) brain-pituitary slices. Neurosci Lett 299:121–124[CrossRef][Medline]

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