According to our working hypothesis, the terminal nerve (TN)-gonadotropin releasing hormone (GnRH) system functions as a neuromodulatory system that regulates many long-lasting changes in animal behaviors. We have already shown by using in vitro whole brain preparations of a small fish (dwarf gourami) that the pacemaker activities of TN-GnRH neurons are modulated biphasically by salmon GnRH, which is the same molecular species of GnRH produced by TN-GnRH neurons themselves; the modulation consists of initial transient decrease and late increase of firing frequency. In the preset study, we investigated the possible involvement of Ca2 release from intracellular store and voltage dependent Ca2 currents in the modulation of pacemaker activities. Pharmacological blockade of Ca2 release from intracellular stores or apamin-sensitive Ca2 -activated K current inhibited the initial transient decrease of firing frequency by sGnRH. On the other hand, bath application of Ca2 channel blockers Ni2 or La3 slowed down the pacemaker frequency and attenuated the rate of the late increase of pacemaker frequency by GnRH. Furthermore, voltage-clamp experiments suggested that low-voltage-activated (LVA) Ca2 current and high-voltage-activated (HVA) Ca2 current were present in the TN-GnRH neurons, and bath application of GnRH shifted the activation threshold of HVA Ca2 current to more negative potentials. These results suggest that (1) sGnRH induces Ca2 release from intracellular stores and activates apamin-sensitive Ca2 -activated K current so that it decreases the frequency of pacemaker activity in the initial phase, (2) some kinds of Ca2 currents contribute to the generation and modulation of pacemaker activities, and (3) HVA Ca2 current is facilitated by sGnRH so that it increases the frequency of pacemaker activity in the late phase.
The gonadotropin-releasing hormone (GnRH) was originally identified as a hypophysiotropic decapeptide hormone that is produced in the preoptic area, transported to the median eminence, and facilitates the release of gonadotro-pins from the pituitary. However, GnRH neurons and their fibers have been found not only in the preoptic area but also in several brain areas outside the hypothalamic area. Such ‘extrahypothalamic’ GnRH systems have been found mainly in the terminal nerve (TN) and midbrain (Schwanzel-Fukuda and Silverman, 1990; Parhar and Iwata, 1994; Yamamoto et al., 1995), and they project widely in the brain instead of the median eminence or the pituitary.
By taking advantage of anatomical feature of the in vitro whole brain preparation of the dwarf gourami, we have been studying the electrophysiological characteristics of the TNGnRH system (reviewed by Oka, 1997; Oka and Abe, 2002). We have previously shown that (1) individual cells of TN-GnRH system project widely in the entire brain from the olfactory bulb to the spinal cord (Oka and Matsushima, 1993), and (2) single TN-GnRH neurons show spontaneous pacemaker activity (Oka and Matsushima, 1993) which consists of the depolarizing phase produced by INa(slow) (Oka, 1995, 1996) and the repolarizing phase mainly produced by TEA-sensitive persistent potassium current, IK(v) (Abe and Oka, 1999). Moreover, the frequency of pacemaker activity seems to vary according to the physiological conditions of the animal, and the firing frequency and/or firing mode may affect the efficacy of exocytosis of GnRH peptides from the GnRH neurons (Peng and Horn, 1991). On the other hand, a growing body of evidence suggests that GnRH peptide modulates the function of ion channels such as Na+ channels (Eisthen et al., 2000), K+ channels (Adams and Brown, 1980) and Ca2+ channels (Elmslie et al., 1990) and thus may regulate the excitability or neurotransmitter release of target neurons. Thus, it has been suspected that the TNGnRH system may function as a neuromodulatory system that is involved in the regulation of long-lasting changes in the animal's behavior, e.g., motivational or arousal states (Oka and Matsushima, 1993; Oka, 1997). Therefore, the study of nature and mechanisms of modulation of the pacemaker activity of TN-GnRH neurons by hormones or transmitters will give us invaluable information about the control mechanism of neuromodulatory GnRH system.
We have previously indicated that the frequency of pacemaker activity of TN-GnRH neurons was biphasically modulated by sGnRH in a dose dependent and paracrine/autocrine manner (Abe and Oka, 2000, 2002). This biphasic modulation consisted of the transient decrease (early phase) and subsequent increase (late phase) of firing frequency. Furthermore, it was suggested that the G-protein coupled GnRH receptor in the cell membrane triggers signal transduction pathway that ended up modulating the frequency of pacemaker activity of TN-GnRH neurons. This biphasic modulation of pacemaker activity may contribute to a synchronized facilitation of pacemaker activities of neighboring TN-GnRH neurons.
In other types of neurons, it has been proposed that G-protein coupled receptors can modulate the gating properties of the ion channels. Both direct effects of G-proteins and indirect effects via diffusible second messengers have been implicated in these modulations (Hille, 1994). Generally, it has been suggested that GnRH receptors are coupled to the Gq/11 type G-proteins, and the Gq/11 type G-proteins enhance inositol phosphate formation and subsequent increase in [Ca2+]i (Naor et al., 1998; Stojilkovic et al., 1994a, b; Stojilkovic and Catt, 1995a, b). In the pituitary gonadotrophs, it has been accepted that GnRH-induced activation of phospholipase C is the major signal transduction pathway of GnRH receptor-coupled processes, and the subsequent mobilization of [Ca2+]i and the activation of protein kinase C (PKC) are the key elements in the control of gonadotropin secretion by pituitary gonadotrophs (Stojilkovic and Catt, 1995a, b).
On the other hand, the dependence on the extracellular Ca2+ of episodic release of GnRH from perifused hypothalamic neurons and GT1-7 cells suggests that GnRH secretion is controlled by Ca2+ entry through the plasma membrane Ca2+ channels (Krsmanovic et al., 1992). Electrophysiological studies have demonstrated the expression of several types of plasma membrane Ca2+ channels in the embryonic GnRH neurons (Kusano et al., 1995) and GT1 cells (Bosma, 1993; Costantin and Charles, 1999; Javors et al., 1995; Van Goor et al., 1999b), including transient and sustained voltage-dependent Ca2+ channels.
Therefore, in the present paper, we examined the possible involvement of [Ca2+]i mobilization from the intracellular stores and the plasma membrane Ca2+ channels in the biphasic modulations of pacemaker activity in the TN-GnRH neurons.
MATERIAL AND METHODS
Adult male and female dwarf gourami (Colisa lalia), ∼4 cm in standard length, were purchased from a local dealer and kept at 22 ∼ 27 C° until used. The whole brain in vitro preparation was made according to the procedures described in the previous papers (Oka, 1995, 1996; Abe and Oka, 1999, 2000).
The in vitro whole brain preparation was continuously super-fused with an oxygenated Ringer solution containing (in mM) 124 NaCl, 5 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 26 NaHCO3, and 10 glucose (pH 7.4 adjusted with NaOH) in the silicone elastomer (Shin-Etsu Silicone No.KE-106, Shin-Etsu Chemical Co., Ltd., Japan) base of a small recording chamber. Whole cell voltage- and current-clamp recordings were carried out with the use of CEZ-2300 amplifier (Nihon Kohden, Japan) and pCLAMP software (Axon instruments). Pipette resistance was ∼8 MΩ, and seal resistance was >10GΩ. Series resistance was compensated as much as possible. The patch pipette was visually guided to the cluster of TNGnRH neurons exposed on the ventral surface of the brain under a dissecting microscope (Oka and Matsushima, 1993). After gigaohm seal formation and “break in” for the whole cell recording mode, characteristic spontaneous pacemaker activity was confirmed in the current-clamp mode (see Oka and Matsushima, 1993; Abe and Oka, 2000).
For current-clamp experiments, the patch pipettes contained (in mM) 110 K-gluconate, 3 MgCl2, 40 N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 0.3 ethylene glycol-bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 2 Na2ATP, and 0.2 Na2GTP (pH 7.4 adjusted with NaOH). NiCl2 (1 mM), LaCl3 (5 μM), Apamin (100 nM; Alomone Labs.) and salmon GnRH (sGnRH, 200 ∼ 300 nM; RBI) were dissolved directly in the Ringer solution. Ruthenium red (50 μM; Sigma) and heparin (300 μg / ml; Sigma) were dissolved in the pipette solution. Pacemaker activities were digitized (2 kHz), displayed on-line with Axotape software or pClamp8 (Axon Instruments), and stored on a computer.
For voltage-clamp recording of Ca2+ currents, the extracellular solution contained (in mM) 95 choline Cl, 40 tetraethylammonium chloride (TEACl), 2.4 CaCl2, 1.3 MgCl2, 5 Glucose, 5 4-aminopyri-dine (4AP), and 10 HEPES (pH 7.4 adjusted with NaOH). In addition, 0.75 μM tetrodotoxin (TTX) was added to the extracellular solution. Patch pipettes were filled with a solution consisting of (mM) 90 CsCl2, 2 MgCl2, 20 TEACl, 10 EGTA, 10 HEPES, 2 Na2ATP, and 0.2 Na2GTP (pH 7.4 adjusted with CsOH). For voltage-clamp recording of tentative Ca2+-activated K+ currents, the extracellular solution contained (in mM) 115 choline Cl, 20 TEACl, 5 KCl, 2.4 CaCl2, 1.3 MgCl2, 5 glucose, and 10 HEPES (pH 7.4 adjusted with NaOH). 0.75 μM TTX was also added to the extracellular solution. Patch pipettes were filled with a solution consisting of (mM) 110 KCl, 3 MgCl2, 40 HEPES, 0.3 EGTA, and 2Na2ATP (pH 7.4 adjusted with NaOH). The linear leakage currents were digitally subtracted, either automatically with the use of the P/4 protocol, or manually, after measuring ohmic resistance in response to hyper-polarizing command pulses. The data were not corrected for the liquid junction potentials.
Most of the TN-GnRH neurons showed regular spontaneous pacemaker activity, and it was biphasically modulated by bath application of sGnRH, as we have reported previously (Abe and Oka, 2000; also see Fig. 3Ba∼d).
Involvement in the control of pacemaker activity of Apamin-sensitive Ca2+-activated K+ currents induced by Ca2+ released from the intracellular store
First, in order to examine the possible involvement of Ca2+ released from the intracellular store in the control of pacemaker activity of TN-GnRH neuron, the cells were dialyzed with ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, and heparin, that of IP3-induced Ca2+ release, respectively, by including them in the patch pipette solution. After that, the effects of bath application of sGnRH on the pacemaker activity were examined. To ensure the diffusion of ruthenium red and heparin into the cytoplasm of the TNGnRH neuron, the data collection was started 10 min after the whole-cell recording was established; this time period was determined on the basis of the results of intracellular application of QX-314 (see Abe and Oka, 2000). Fig. 1 shows the pacemaker activity of a cell recorded with patch pipette containing ruthenium red (50 μM) and heparin (300μg / ml) in the pipette solution. In Ringer solution, the cells showed slightly irregular beating discharge pattern (Fig. 1Ba). Many cells tended to show such a firing pattern by intracellular application of the blockers of Ca2+ release from the intracellular store. In these experiments, we further applied La3+ (5 μM) to the bath solution in order to block some Ca2+ channels (see below). Bath application of La3+ slowed down the frequency of pacemaker activity (Fig. 1Bb). Further bath application of sGnRH (1 μM) failed to evoke transient decrease of firing frequency (Fig. 1Bc) but evoked subsequent increase of firing frequency (Fig. 1Bd). Figure 1Bc and Bd (20 and 120 s after the onset of sGnRH perfusion, respectively) were recorded during time periods similar to that of transient decrease and subsequent increase of firing frequency by sGnRH. Fig. 1Bf shows the time course of these changes before and after the application of sGnRH (during the period corresponding to Fig. 1Bb∼d).
We compared the degree of transient decrease of firing frequency in the presence and absence of the intracellular Ca2+ mobilization blockers. Each effect of the blockers was measured during a 20 s period that was 30 ∼ 50 s after the onset of sGnRH perfusion, which corresponded to the early transient phase of pacemaker frequency decrease, and was normalized to the frequency in Ringer solution. The transient decrease that was induced by sGnRH (0.88±0.06; n=16) was nullified by heparin (1.01±0.05; n=5). Similarly, intracellular application of ruthenium red alone or both ruthenium red and heparin nullified the transient decrease or rather increased slightly the firing frequency (1.11±0.14; n=7 and 1.16±0.11; n=4, respectively). However, we could not find statistically significant nullifying effects of blockers of Ca2+ release from the intracellular store due to the large SE. Anyway, these results suggest that the Ca2+ release from the intracellular store may be involved in the transient decrease of firing frequency that was induced by sGnRH.
In our previous studies of voltage-dependent K+ cur-rents (Abe and Oka, 1999), we found that a transient and 4AP-sensitive, but not TEA- or charybdotoxin-sensitive Ca2+-depenent outward current component was present in TN-GnRH neurons. Because this current could be observed only when Ca2+ was present in the external solution, we defined this 4AP-sensitive transient outward current that was dependent on the presence of extracellular Ca2+ ions as tentative Ca2+-activated K+ currents. When the current responses were measured in Ca2+-containing extracellular solutions containing 0.75 μM TTX and 20 mM TEA, a mixture of large transient currents and smaller persistent outward currents could be recorded in response to depolarizing pulses from a holding potential of −100 mV. The activation and steady-state inactivation curves of the mixture current containing tentative Ca2+-activated K+ current are shifted to more depolarized potentials compared with those of “4AP-sensitive transient current” (data not shown). These tentative Ca2+-activated K+ currents are only partially inactivated at membrane potentials of −60 to −40 mV, which correspond to the base membrane potentials of the pacemaker activity of TN-GnRH neurons. Thus, it is reasonable to think that tentative Ca2+-activated K+ current can be activated in the pacemaker range of TN-GnRH neurons.
Next, we examined the effect of sGnRH on the tentative Ca2+-activated K+ current. Fig. 2A shows the superimposed traces of the tentative Ca2+-activated K+ currents before, during, and after sGnRH applications elicited during a series of +50 mV test pulses from a holding potential of −100 mV. In this case, bath application of sGnRH (1 μM) facilitated current amplitude about 18% (Fig. 2A). The current amplitude recovered to the control level after washout by Ringer solution. Fig. 2B shows the time course of the tentative Ca2+-activated K+ current before, during, and after sGnRH applications. Test pulses were applied at 15 s intervals. The current amplitude showed a tendency to gradually decrease due to the rundown. However, it was rapidly increased by bath application of sGnRH (within 15 to 90 s; Fig. 2B, solid bars). This time course corresponded to the onset of the transient decrease of firing frequency of pacemaker activity induced by sGnRH. Averaged increase of the amplitude of tentative Ca2+-activated K+ current was about 9% (2 to 19%, n=4).
Finally, we examined the effects of bath application of apamin, a specific SK-type Ca2+-activated K+ channel blocker. Fig. 3 shows the effect of bath application of apamin. In Ringer, TN-GnRH neurons showed regular beating discharge (3.02±0.86 Hz; n=4). Bath application of sGnRH (20 nM) transiently decreased (2.74±0.83 Hz; Fig. 3Bb) and subsequently increased (4.98±0.60 Hz; Fig. 3Bc) firing frequency of pacemaker activity. After washout of sGnRH, bath application of apamin (100 nM) facilitated the frequency of pacemaker activity (3.60±0.68 Hz; Fig. 3Be). However, further bath application of sGnRH (20 nM) failed to evoke transient decrease of firing frequency (3.70±0.74 Hz; Fig. 3Bf) but evoked subsequent increase of firing frequency (6.59±1.15; Fig. 3Bg). Fig. 3Bd and Bh show the time course of these changes before and after the application of sGnRH. We also compared quantitatively the normalized transient decrease of firing frequency in the presence and absence of apamin. The transient decrease that was induced by sGnRH (0.92±0.07; n=4) was nullified by apamin (1.02±0.03; n=4).
From these data, it is suggested that sGnRH triggers the Ca2+ release from the intracellular store and activates apamin-sensitive Ca2+-activated K+ currents, which leads to the transient decrease of the frequency of pacemaker activity.
Involvement of Ca2+ currents in the pacemaker activity
Because the preliminary experiments that examined the effect of sGnRH upon INa(slow) and/or IK(V) did not show any noticeable modulation, we investigated the possible involvement of Ca2+ currents in the modulation of pacemaker activity. Fig. 4 shows the effects of Ca2+ channel blockers on the pacemaker frequency of TN-GnRH neurons. Bath application of various Ca2+ channel blockers slowed down the pacemaker frequency of TN-GnRH neurons. Fig. 4Aa shows the effects of the blocker of high-voltage-activated Ca2+ cur-rents, La3+ (5 μM, n=4), and Fig. 4Ab shows those of the blockers of low-voltage-activated Ca2+ current and store-operated Ca2+ current, Ni2+ (1 mM, n=4). The frequencies of pacemaker activities before and after these treatments were 3.7±0.8 and 2.6±0.5 Hz for La3+ (P<0.05, Student's two-tailed paired t test) and 4.1±0.7 and 1.7±0.2 Hz for Ni2+ (P<0.05, Student's two-tailed paired t test). Furthermore, when the bath application of the combination of La3+ (5 μM) and Ni2+ (1 mM) was examined, it completely blocked the generation of pacemaker activity (in 7/8 cells, Fig. 6Ba,c and d). The results are presented in Fig. 4B as the relative pacemaker frequencies after each treatment normalized to those before the treatment, which were defined as the ratio (firing frequency in Ringer solution containing Ca2+ channel blockers) / (firing frequency in Ringer solution). These results indicate that some kinds of Ca2+ currents are present in TNGnRH neurons and contribute to the generation of pacemaker activity.
Next, we examined the effects of Ca2+ channel blockers on the modulation of pacemaker activity by sGnRH. Fig. 5 shows the effects of Ni2+ on the modulation of pacemaker activity by sGnRH. In Ringer solution, TN-GnRH neuron showed regular beating discharge (3.5±0.5 Hz; Fig. 5Ba). Bath application of Ni2+ (1 mM) decreased the firing frequency of pacemaker activity (1.8±0.2 Hz; Fig. 5Bb). Subsequent bath application of sGnRH (300 nM) transiently decreased (1.5±0.2 Hz; Fig. 5Bc) and then increased (2.4±0.8 Hz; Fig. 5Bd) the firing frequency of pacemaker activity in a qualitatively similar manner with sGnRH application in Ringer. However, the late-phase increase of pacemaker frequency was less pronounced compared with sGnRH application in Ringer (Fig. 7; compare the left and right columns). Similarly, the late-phase increase of pacemaker frequency was less pronounced under La3+ treatment compared with sGnRH application in Ringer (Fig. 7; compare the left and middle columns). During the simultaneous bath application of La3+ (5 μM) and Ni2+ (1 mM)(Fig. 6), the pacemaker activity was stopped (Fig. 6Bb, Bc), and further bath application of sGnRH (300 nM, in 3/4 cells, Fig. 6Bd) or depolarizing DC current injection (in 2/2 cells, data not shown) did not reinstate the pacemaker activities.
Taken together, these data indicate that some kind(s) of Ca2+ channels present in the TN-GnRH neurons may be the target for the modulation by sGnRH, which somehow contributes to the late phase increase of pacemaker frequency.
Voltage-clamp recordings of voltage-dependent Ca2+ currents
To reveal the quantitative contribution of Ca2+ currents to the generation and modulation of pacemaker activity, it is important to determine the Ca2+ channel subtypes and kinetics. Depolarizing steps from a holding potential of −100 mV elicited a mixture of transient and sustained inward current components (Fig. 8Aa). They showed the characteristics of combined low voltage-activated (LVA) and high voltage-activated (HVA) Ca2+ currents. The peak inward currents (measured at peak current amplitudes during depolarizing pulses) began to appear more positive than −60 mV and reached a maximum near −20 mV. The sustained currents (measured at 200 ms after the onset of the test pulse) began to appear more positive than −20 mV and reached a maximum near 0 mV. The currents that were elicited by depolarizing steps from a holding potential of −60 mV are shown in Fig. 8Ab. The transient inward current almost disappeared, and sustained current(s) mainly remained. At this holding potential, the activation of both peak and sustained currents required depolarization more positive than −20 mV, and the maximum inward current was obtained near −10 mV. In the currents recorded from a different cell, however, the sustained current began to appear more positive than −40 mV and rapidly reached its maximum at −30 mV, when a holding potential was −60 mV (see below).
Fig. 8B shows the averaged current-voltage relationship of transient and sustained Ca2+ currents (n=40). We defined the current component that was obtained by subtraction of sustained current from peak current, as the ‘transient’ Ca2+ current. On the average, the transient and sustained currents measured at −10 mV from a holding potential of −100 mV was −1.41±0.26 and −0.87±0.12 nA, respectively (Fig. 8B ▪ and •). However, the averaged current-voltage relationship of sustained current elicited from a holding potential of -60 mV showed an activation threshold of −40 mV but also had a shoulder of current at −20 mV (Fig. 8B, arrows). This shoulder of current may reflect the difference of activation threshold and voltage dependence of sustained currents among different cells. The sustained current measured at −10 mV from a holding potential of −60 mV was −0.80±0.13 nA (n=18; Fig. 8B ○).
These results suggest that a transient inward current component that begins to activate near −60 mV and is largely inactivated when the holding potential is −60 mV represents the LVA Ca2+ current. On the other hand, HVA Ca2+ current component that are not inactivated at holding potentials of −60 mV consist of two types of sustained currents. The one current begins to activate from −20 mV and reaches its maximum amplitude near −10 mV. The other current begins to activate near −40 mV and rapidly reaches maximum at −30 mV.
Actions of GnRH on Ca2+ currents
Previous preliminary studies suggested that neither INa(slow) nor IK(v) is significantly modulated by sGnRH applications (our unpublished observations). Therefore, we suspected that the Ca2+ currents may be modulated by sGnRH. Fig. 9 shows an example of the effects of sGnRH on the Ca2+ currents. The modulation was examined in 10 cells held at −100 mV. In this case, when 1 μM sGnRH was added to the bath solution, the current amplitude and activation threshold of transient current were not changed (Fig. 9Ba □ and ▪). However, the amplitude of sustained current increased (−0.3 to −1.3 nA) in a wide voltage range. Furthermore, the activation threshold and maximum amplitude of sustained current were shifted to more negative potentials (Fig. 9Bb ○ and •). Responses of Ca2+ currents to GnRH were variable among the recorded cells. In another case, the amplitudes of both transient and sustained Ca2+ currents did not increase. However, the activation threshold and the peak amplitude of sustained Ca2+ current were shifted to more negative potentials (data not shown). On the average, the amplitude of the transient Ca2+ current measured at its maximum was decreased from −1.4 nA±0.3 to −1.1±0.4 nA (P>0.05, Student's two-tailed paired t-test; Fig. 10A □ and ▪), and those of the sustained Ca2+ current was increased from −1.6±0.4 to −1.9±0.4 (P<0.05, Student's two-tailed paired t test; Fig. 10B ○ and •), before and after bath application of 1 μM sGnRH, respectively (n=10). Moreover, the activation threshold and the voltage that evokes the maximum amplitude of the sustained Ca2+ currents were shifted to more negative potentials (Fig. 10B ○ and •), while those of transient Ca2+ current were not changed. These results indicate that the sustained HVA Ca2+ current is facilitated by sGnRH, i.e., the relative contribution of HVA Ca2+ current to the pacemaker activity may be increased by sGnRH. It is then suggested that this increased availability of HVA Ca2+-current increases the frequency of pacemaker activity.
Involvement of Ca2+ release from intracellular store and apamin-sensitive K+ current in the transient decrease of firing frequency of TN-GnRH neurons
Intracellular application of ruthenium red and heparin inhibited the transient decrease but not late-phase increase of the pacemaker frequency, both of which were induced by bath application of sGnRH. This result suggests that [Ca2+]imobilization is involved in the transient decrease but not the late-phase increase of firing frequency of pacemaker activity. Then, the presence of tentative Ca2+-activated transient K+ current was suggested from the result of present voltage-clamp experiments. This current was TEA-insensitive but 4AP-sensitive, and could be only evoked when the Ca2+ is present in the extracellular bath solutions. However, the activation and steady-state inactivation curves of this current were shifted to more depolarized potentials compared with the 4AP-sensitive A-like currents of TN-GnRH neurons (Abe and Oka, 1999), and the two currents are considered to be different. The amplitude of the tentative Ca2+-activated transient K+ current was rapidly increased by bath application of sGnRH. Furthermore, bath application of apamin inhibited the transient decrease but not the late-phase increase of the pacemaker frequency by sGnRH. These results suggest that the apamin-sensitive Ca2+-activated transient K+ current was activated by Ca2+ released from the intracellular store, which had been induced by sGnRH. This may explain the early-phase transient decrease of pacemaker activity by sGnRH.
It has been reported that the Ca2+-activated K+ current exist in the embryonic GnRH neurons (Kusano et al., 1995) and GT1-7 cells (Spergel et al., 1996; Van Goor et al., 1999). The transient decrease of firing frequency by GnRH, which was induced by the activation of SK type Ca2+-activated K+ current, has been reported in the GT1-7 cells (Van Goor et al., 1999). It has been generally accepted that the activation of Gq/11-coupled GnRH receptor increases the intracellular Ca2+ concentration via phosphoinositide signaling pathway (Naor, 1990; Naor et al., 1998; Stojilkovic et al., 1994b), and the increased [Ca2+]i may open Ca2+-activated K+ current (Sah, 1996). All of these reports are in favor of the above-mentioned mechanisms for the transient decrease of pacemaker activity of TN-GnRH neurons by sGnRH.
Ca2+ current component is involved in the pacemaker activity
We have shown that some kind(s) of Ca2+ current contribute(s) to the modulation of the pacemaker activity of TNGnRH neurons. Furthermore, the result that simultaneous bath application of 5 μM La3+ and 1 mM Ni2+ completely inhibited the generation of pacemaker activity and its modulation by sGnRH, suggests that Ca2+ current components that were blocked by simultaneous application of La3+ and Ni2+ may be somehow involved in the generation of pacemaker potentials and may be the target of modulation by sGnRH.
The present results may appear to be partly inconsistent with those of the previous study that the Ca2+ currents are not essential for the generation of pacemaker potentials (Oka, 1995). One possible explanation may be the difference in the recording methods, intracellular recording (Oka, 1995) vs. whole-cell patch-clamp recording (present study). It may be possible that the intracellular dialysis of EGTA, which was introduced by the patch pipette solution, may prevent Ca2+ channels from Ca2+-dependent inactivation (Hille, 2001). Thus, a relatively large proportion of Ca2+ channel may be available for the generation of the pacemaker activity in the whole-cell patch-clamp recording. Second possibility is the presence of diffusion barrier. While the ventral meningeal membrane of the brain was not removed in the previous study, it is always completely removed in the patch-clamp experiments. The meningeal membrane may have served as a diffusion barrier for the drug delivery. Thirdly, the bath application of Ca2+ channel blockers may have simultaneously blocked other currents (for example, INa(slow)). Preliminary experiments suggest that Ni2+ may inhibit INa(slow) (data not shown). It has also been reported that Ni2+ and La3+ also blocks Na+/Ca2+ exchanger, store-operated Ca2+ current, and other voltage-gated ion channels (Nowycky, 1991; Taylor and Brond 1998). In the present study, we used supramaximum concentrations of these Ca2+ channel blockers to block the Ca2+ influx completely. Especially, the concentrations of Ni2+(1 mM) and La3+(5 μM) used here are sufficient to block store-operated Ca2+ currents (Skryma et al., 2000). Thus, it may be possible that these Ca2+ channel blockers blocked not only voltage-gated Ca2+ channels, but also store-operated Ca2+ channels, other channels, and/or exchangers. In fact, it has been reported in GT 1-7 cell-lines that store-operated Ca2+ current contributes to the modulation of firing activity by GnRH (Van Goor et al., 1999a). Further studies using more specific Ca2+ channel blockers are necessary to clarify the quantitative contributions of voltage-dependent Ca2+ current and store-operated Ca2+ current.
Both LVA and HVA Ca2+ currents are present in the TNGnRH neurons
Voltage-clamp experiments of whole-cell patch-clamp recording suggested that TN-GnRH neuron has at least one LVA Ca2+ current component and two HVA Ca2+ current components, and the relative prevalence of each current component seems to be different among different TN-GnRH neurons. LVA Ca2+ current was a transient current evoked from membrane potentials more positive than −60 mV and reached its maximum at about −20 mV (Figs. 8; Transient current). This current was almost inactivated at a holing potential of −60 mV. It most probably corresponds to the T-type Ca2+ current (Nowycky et al., 1985). In contrast, both of the two HVA Ca2+ currents inactivated slowly and could be observed at holding potentials of −100 mV and −60 mV. One of the HVA Ca2+ current activated at potentials more positive than −40 mV. The other HVA Ca2+ current activated at potentials more positive than −20 mV. Thus, the averaged I/V curve of the sustained current had a shoulder and reached its maximum around 0 mV (Fig. 8B). Preliminary experiments showed that the amplitude of HVA Ca2+ current was reduced (= was not completely blocked) by bath application of nifedipine (data not shown). Therefore, at least one of the HVA Ca2+ current may correspond to the L-type Ca2+ current.
It has been demonstrated that embryonic GnRH neurons (Kusano et al., 1995) and GT1 cells (Bosma, 1993; Hales et al., 1994; Javors et al., 1995; Costantin and Charles, 1999) express several types of plasma membrane Ca2+ channels, including transient and sustained voltage-dependent Ca2+ channels. Kusano et al. (1995) reported that embryonic GnRH neurons possess T- and L-type Ca2+ channels, and GT1 cells possess T-, N-, and L-type Ca2+ channels. On the other hand, Costantin and Charles (1999) and Van Goor et al. (1999b) reported that GT1 cells possess T- and L-type Ca2+ channels. The present study used for the first time the adult authentic GnRH neurons, and the results basically agreed well with these reports. However, pharmacological isolation and detailed analysis of kinetic properties of each Ca2+ current component have not yet been done in the present study mainly due to the poor space-clamp of the in vitro whole-brain preparations of TN-GnRH neurons. Therefore, the use of dissociated TN-GnRH neurons may be necessary to evaluate quantitatively the contribution of each Ca2+ current component to the control of pacemaker and secretory activities of TN-GnRH neurons.
Modulation of HVA type Ca2+ channels by GnRH
Bath application of sGnRH shifted the activation threshold of sustained Ca2+ current component to more hyperpolarized potentials. Moreover, the increase of sustained current amplitude was observed in some recordings, although complete recovery of Ca2+ current amplitude by washout could not be obtained due to the rundown of Ca2+ currents. It is generally accepted that GnRH receptors are coupled to Gq/11 type G-proteins, and Gq/11 type G-proteins are coupled to phosphoinositide-mediated signaling pathways (Stojilkovic et al., 1994a,b). It has also been reported that Ca2+ channels (especially, N- and L-type Ca2+ channel) are positively modulated by PKC (Bourinet et al., 1992; Dolphin, 1998; Meir et al., 1999; Stea et al., 1995; Swartz, 1993; Yang and Tsien, 1993; Zamponi et al., 1997; Zhu and Ikeda, 1994). Bosma and Hille (1992) reported that immortalized gonadotrope (aT3-1 cell), which also has GnRH receptors, express Ca2+ channels, and these Ca2+ channel were augmented by application of GnRH or phorbol ester.
From these observations and present results, it is highly possible that certain type(s) of Ca2+ currents that contribute to the generation of pacemaker activity are modulated by sGnRH and are involved in the late-phase increase of the pacemaker frequency.
Mechanisms of the generation and modulation of pacemaker activity and possible physiological functions
From the results of the present study, we present a model about the generation and modulation mechanisms of pacemaker activity in the TN-GnRH neuron (Fig. 11). In the intact brain, TN-GnRH neurons show regular beating pacemaker activity. The INa(slow), INa(fast), and IK(V) interact in the following manner to generate the pacemaker potentials. The INa(slow), which is persistently active in the subthreshold membrane potential range, always supplies the persistent depolarizing drive and gradually depolarizes the membrane potentials. When the membrane potential reaches the activation threshold for IK(V), outward current develops, and the net flux of current reverses to outward. Then, the membrane potential becomes hyperpolarized and deactivates the K+ current, and the next cycle begins. This is the subthreshold pacemaker activity, and when the membrane potential reaches the activation threshold for the INa(fast), the spiking pacemaker activities ensues. In addition, some kind(s) of Ca2+ currents and apamin-sensitive Ca2+-activated K+ cur-rent may be also involved in the generation of pacemaker potentials. Although INa(slow) and IK(v) are essential for the generation of the basic rhythm of pacemaker activities, they do not seem to be modulated by the process described below. When the GnRH peptide binds to the GnRH receptor located on the cell surface of TN-GnRH neurons, Gq/11 protein-coupled process starts and activates phospholipase C, which leads to the production of IP3 and diacylglycerol. IP3 stimulates the release of Ca2+ from the IP3-sensitive intracellular Ca2+ store, and the increased [Ca2+]i then opens apamin-sensitive Ca2+-activated K+ channel. Thus, the pacemaker frequency of TN-GnRH neuron is transiently decreased. On the other hand, diacylglycerol activates protein kinase C, and protein kinase C then phosphorylates and modulates the Ca2+ current. Thus, the pacemaker frequency of TN-GnRH neurons is increased in the late phase.
What is the physiological significance of the pacemaker activity of TN-GnRH neurons and its modulation? Unfortunately, the functional link between the electrical activities of peptidergic neurons and the peptide release has not been firmly established thus far. A recent amperometric and RIA studies showed that the membrane depolarization triggers the secretion of GnRH from the TN-GnRH neuron in the teleost brain-pituitary slice preparation (Ishizaki and Oka, 2001 and in preparation). In the intact brain, the pacemaker activity of TN-GnRH neurons is characterized by low frequency (<10 Hz) regular beating discharge. The depolarization that is produced by single action potential of pacemaker activity may not be strong enough to induce GnRH release from the dense cored vesicles. However, it has been reported that such low frequency firing activity enables vasopressin release from rat neurohypophysis (Bondy et al., 1987), substance P or thyrotropin releasing hormone release from rat ventral spinal cord (Iverfeldt et al., 1989), and GnRH release from preganglionic C-neurons of the bullfrog (Peng and Horn, 1991). Furthermore, increase of firing frequency potentiated peptide release from these cells. Thus, it may be possible to think that the frequency of beating pacemaker activity of TN-GnRH neurons may affect the release of GnRH.
What then is the physiological significance of the modulation by sGnRH of pacemaker activity of TN-GnRH neurons? TN-GnRH neurons of the dwarf gourami make tight cell clusters with no intervening glial cells (Oka and Ichikawa, 1991; Oka and Matsushima, 1993; Oka, 1997), and the possibility of active exocytotic release from the cell body and its vicinity has also been suggested (Oka and Ichikawa, 1991). Other studies have shown that GnRH receptors are widely distributed throughout the brain (Jennes et al., 1997; Stojilkovic et al., 1994b). In addition, considerable overlap of the brain areas that contain GnRH-producing cells and GnRH receptor mRNA-expressing cells has been reported (Jennes et al., 1996). Also, cultured hypothalamic GnRH neurons have GnRH receptors (Krsmanovic et al., 1999). Furthermore, it has been reported using an in vivo analysis of multiunit activities in ovariectomized rats that GnRH injected in and around the median eminence is able to cause a population of GnRH neurons to fire synchronously (Hiruma and Kimura, 1995). From these observations and the present results, it is suggested that GnRH released from GnRH neurons facilitates the activities of their own (autocrine) and/or neighboring GnRH neurons (paracrine) and may cause synchronized positive feedback facilitation of multiple GnRH neurons. It is well known that in oxytocin neurons the release of oxytocin from single neuron into the brain environment stimulates its own activity and thus further release (Freund-Mercier and Richard, 1984; Moos et al., 1984). A similar effect has been reported for insulin-stimulated insulin release in pancreatic β cells (Aspinwall et al., 1999). Therefore, this mechanism is probably common to all neurosecretory neurons or secretory cells, whose synchronized facilitation of firing leads to facilitated release.
Comparison with the autoregulation in the other putative GnRH neurons
Possible autoregulation mechanisms of TN-GnRH cells suggested in the present study are discussed here in relation to references that described studies on putative GnRH neurons. The concept of an ultrashort feedback mechanism in the control of neurosecretion was first suggested by Hyyppa et al. (1971) in studies on the control of FSH secretion. Similarly, it was revealed from in vivo (Valenca et al., 1987) and in vitro studies (DePaolo et al., 1987) that GnRH release from hypothalamic GnRH system was negatively autoregulated. It was postulated that negative feedback could be mediated via axo-dendritic/axo-somatic synapses on adjacent GnRH or other types of neurons (DePaolo et al., 1987), and histological evidence for such connections has been reported (Leranth et al., 1985; Witkin and Silverman, 1985).
However, the results using recording of multiunit activity (MUA) in the hypothalamus, which are considered to reflect the secretory activity of GnRH, have been very complicated. Hiruma and Kimura (1995) reported that intravenous injection of GnRH or microinjection of GnRH into the median eminence immediately evoked a MUA volley of the rat. However microinjection of GnRH into the medial preoptic area did not cause these effects. Moreover, intravenous or intracerebroventricular injections of GnRH did not affect MUA volleys of the rhesus monkeys (Kesner et al., 1986; Ordog et al., 1997). Unfortunately, the neuronal elements responsible for the MUA volley, i.e., whether the MUA volley represent the activity of GnRH neurons themselves or other neuronal elements, have not been determined. Therefore, it is difficult to directly compare the results of the present paper with the results of MUA studies.
Exposure of perifused GT1-7 cells to a GnRH agonist (analog) caused a transient elevation of GnRH release and subsequent suppression of the basal pulsatile secretion. During further continuous exposure to the agonist, the cells recovered from inhibition and exhibited infrequent but increasingly prominent peaks, with a net increase in GnRH release (Krsmanovic et al., 1993). Recently, similar autoregulation of GnRH secretion has been reported in hypothalamic culture (Krsmanovic et al., 1999). However, the time courses of such autoregulation are much slower than those of TN-GnRH neurons. The autoregulation of GnRH release in GT1-7 cell and hypothalamic culture takes dozens of minutes to several hours to occur. However, the biphasic auto-regulation of pacemaker activity of TN-GnRH neurons took only a few minutes. The former has been suggested to result from the internalization of receptor molecules and downregulation of the expression of GnRH receptor gene that was induced by GnRH (Park, 1998; Stojilkovic et al., 1994a). On the other hand, the biphasic autoregulation of pacemaker activity of TN-GnRH neurons is considered to be due to the modulations of ionic channels induced by the activation of cell signaling pathways downstream of GnRH receptor activation. Thus, the two phenomena are considered to be based on quite different mechanisms.
Recently, Van Goor et al. (1999a, b) reported that the membrane excitability of GT1 cells was modulated by GnRH peptide. In this modulation of membrane excitability, they suggested that the activation of GnRH receptor induces [Ca2+]i mobilization, and this [Ca2+]i mobilization activates SK-type Ca2+-activated K+ channel and activates store-operated Ca2+-channel, which transiently hyperpolarizes and then persistently depolarizes membrane potentials, respectively. They further suggested that this sustained membrane depolarization is explained by complex interplay of Na+ channel, K+ channel, store-operated Ca2+ channel, and L-type Ca2+ channel (Van Goor et al., 2000). Furthermore, LeBeau et al. (2000) showed the contribution of cAMP-operated inward current to this modulation in GT1 cells. The mechanism of the transient decrease of firing frequency in TN-GnRH neurons (the present study) basically agrees well with these reports. However, detailed mechanism of the sustained increase of firing frequency seems to differ from each other. To further understand this mechanism, more detailed kinetic and pharmacological investigation of the control of pacemaker activities of TN-GnRH neurons are under way.
This research was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to Y. Oka (No. 12440237), and the Sasakawa Scientific Research Grant from the Japan Science Society to H. Abe.