Misaki Marine Biological Station, Graduate School of Science,
University of Tokyo, Kanagawa 238-0225, Japan
 |
INTRODUCTION |
Unlike the septo-preoptico-infundibular
gonadotropin-releasing hormone (GnRH) system that projects to the
pituitary and facilitates gonadotropin release, terminal nerve
(TN)-GnRH system projects throughout the brain instead of the pituitary
(Yamamoto et al. 1995
) and shows endogenous pacemaker
activity that is dependent on the physiological conditions of the
animal (Oka and Matsushima 1993
). On the other hand, a
growing body of evidence suggests that GnRH peptide modulates the
function of ion channels such as 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. The
morphological characteristics of TN-GnRH neurons appear to be
appropriate for simultaneous modulation of the target neurons in wide
areas of the brain, and the modulation may vary according to the
frequency of pacemaker potentials. Thus we suspect that the TN-GnRH
system may function as a neuromodulator system that is involved in the regulation of many long-lasting changes in the animal's behavior e.g.,
motivational or arousal states (Oka and Matsushima
1993
). As to the mechanisms of the generation of the pacemaker
activity, we have already shown by using whole-cell patch clamp
recordings that the tetrodotoxin-resistant persistent sodium current,
INa(slow), and the
tetraethylammonium-sensitive potassium current,
IK(v), contribute to the
depolarizing and repolarizing phase of the pacemaker potentials,
respectively (Abe and Oka 1999
; Oka 1995
,
1996
). Here, we show that the pacemaker activity of TN-GnRH
neurons is modulated by salmon GnRH (sGnRH), which is the same
molecular species of GnRH peptide produced by TN-GnRH neurons
themselves. Thus the pacemaker activity may be modulated by GnRH
peptide that is produced by TN-GnRH neurons, and the TN-GnRH system may
be a good model system to study the modulatory effect of GnRH peptides
on the target neurons as well.
 |
METHODS |
Adult male and female dwarf gourami (Colisa lalia, a
freshwater tropical fish), ~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 previously published protocols (Abe and Oka 1999
; Oka 1995
).
This whole brain preparation was continuously superfused 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). Patch pipette contained (in mM): 110 K-gluconate, 3 MgCl2, 40 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), 0.3 ethylene glycol-bis
(
-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 2 Na2ATP, and 0.2 Na2GTP (pH 7.4 adjusted with NaOH). The patch
pipette was visually guided to the cluster of TN-GnRH 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
). The voltage was not
corrected for liquid junction potentials, which were of the order of 5 mV. Pacemaker activities were digitized (2 kHz), displayed on-line with
Axotape software (Axon Instruments), and stored on a computer. Thereafter, the data were analyzed off-line by using Axograph software
(Axon Instruments). Drugs that were used are as follows: salmon GnRH
(0.2-200 nM, RBI), mammalian GnRH (mGnRH, 20 or 50 nM, Peptide
Institute. Inc.), GnRH antagonist ([Ac-pCL-DPhe1,2, DTrp3, DArg6,
DAla10] luteinizing hormone-releasing hormone (LH-RH) (American
Peptide Company, Inc.), GDP-
-S (1 mM, Sigma), guanosine 5'-0-(3-thiotri-phosphate) (GTP-
-S) (0.1 mM, Sigma). All
data in this report are presented as means ± SE.
 |
RESULTS |
Most of the TN-GnRH neurons showed regular spontaneous beating
discharge pattern (Oka and Matsushima 1993
). The
pacemaker activities of TN-GnRH neurons were modulated by bath
application of sGnRH. Figure 1 shows an
example of the changes in pacemaker activity by sGnRH (20 nM). In
Ringer, TN-GnRH neuron showed slow regular beating discharge (3.06 ± 0.20 Hz, n = 53, range 0.8-9.0 Hz; Fig.
1Ba). During the bath application of sGnRH, the firing frequency of pacemaker activity was transiently decreased (early phase;
Fig. 1Bb shows 70 s after the onset of bath
application), and subsequently increased (late phase; Fig.
1Bc shows 140 s after the onset of bath application).
Figure 1 shows a typical example of this "biphasic" modulation, and
it is clearly seen in a trace on a slower time scale (Fig.
1A). It should be noted that this modulation occurred
without detectable membrane potential changes in most cases, which
precludes the possibility that the modulation of pacemaker activity,
decrease and then increase of frequency, may be simply caused by
hyperpolarization and depolarization of the membrane, respectively. The
time course of this biphasic modulation of the frequency of pacemaker
activity is plotted in Fig. 1C. The latency of the transient
decrease in firing frequency was about 20-90 s, but this varied from
neuron to neuron even at the same concentration of sGnRH. This
relatively long latency may be partly due to the delivery time during
which the adequate quantity of perfusion solution reaches TN-GnRH
neurons and partly due to the biochemical nature of GnRH responses (see
DISCUSSION). The duration of the transient decrease
in firing frequency was 34.7 ± 3.2 s for 200 nM sGnRH
(n = 16). It increased steadily with decreasing sGnRH
concentration, reaching about 87.8 ± 11.8 s at 2 nM
(n = 9). Furthermore, when the sGnRH concentration was
0.2 nM, this decrease in firing frequency became persistent and the late-phase frequency increase was not observed (n = 7/11). This suggests that the onset of the late-phase increase of the
pacemaker potential may be dependent on the sGnRH concentration and the early-phase decrease less dependent on it, although it is difficult to
assess quantitatively. The early-phase transient decrease in firing
frequency was 11.7 ± 2.6% (n = 52) on the
average and was not significantly influenced by the sGnRH
concentrations used in the present study. On the other hand, the
late-phase increase in the firing frequency showed a clear
dose-dependence (Fig. 1D). Normalized increase of the firing
frequency of pacemaker activity was plotted against concentration of
sGnRH. Normalized increase in firing frequency was defined as
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The frequency here indicates the number of spikes during a 30-s
period, counted immediately before the application of GnRH and at the
peak of the late-phase increase (2 min after the onset of sGnRH
application). The concentration-response curve (Fig. 1D,
solid line) could be well fitted with the formula
where R is the normalized increase of the firing
frequency. The EC50 was 7.18 nM. Finally, the
frequency of the pacemaker activity recovered to the control level
within 5 min of washout in normal Ringer (Fig. 1Bd). Such
biphasic modulation of pacemaker activity was also shown by bath
application of mGnRH (20 or 50 nM; data not shown). In addition, we
also examined whether GnRH antagonist affects this biphasic modulation
of pacemaker activity. Bath application of GnRH antagonist (100 or 200 nM) alone did not evoke any modulation of pacemaker activity
(n = 11). However, pre- and coperfused GnRH antagonist
inhibited the modulation of pacemaker activity by bath application of
sGnRH (20 nM; data not shown).

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Fig. 1.
Effects of bath application of salmon gonadotropin-releasing hormone
(sGnRH). A: in a current-clamp whole cell recording from
a terminal nerve (TN)-GnRH neuron, bath application of sGnRH, the same
molecular species of GnRH peptide produced by TN-GnRH neurons
themselves, biphasically modulated their pacemaker activity.
B: bath application of sGnRH transiently decreased
(b) and subsequently increased the frequency of
pacemaker activity (c). After washout, the firing
frequency of pacemaker activity recovered (d).
C: frequency of pacemaker activity plotted against the
time course. D: dose-response relationships between the
sGnRH concentration and the normalized increase in firing frequency.
Numbers in parentheses by the filled squares represent the numbers of
cells tested for each sGnRH concentration. The smooth curve was drawn
assuming a one-to-one binding relationship with an
EC50 = 7.18 nM.
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To elucidate possible mechanisms underlying this modulation of
pacemaker activity, we next examined whether a GTP-binding protein
(G-protein) process was involved in this modulation. To examine this
possibility, we dialyzed the cell with GDP-
-S, a GDP derivative
which is a competitive inhibitor of many G-protein-mediated processes,
by including it in the patch pipette solution. After that, we examined
the effect of bath application of sGnRH under the same conditions as in
the control. To ensure the diffusion of GDP-
-S into the cytoplasm of
the TN-GnRH neuron, we first calibrated the likely diffusion time by
dialyzing the cell with a pipette solution containing QX-314, a sodium
channel blocker which is effective from inside of the cell. Under these
conditions, the action potentials were blocked within 5 min (data not
shown), although subthreshold pacemaker potentials remained because of the presence of a TTX-resistant persistent sodium current
(INa(slow)) (Oka 1996
).
On the basis of this result, we started data collection 10 min after
the whole-cell recording was established. Figure 2 shows the pacemaker activity of a cell
recorded with a large-tip patch pipette containing 1 mM GDP-
-S in
the pipette solution. In the Ringer solution, this cell showed a
slightly irregular beating discharge pattern (Fig. 2Ba).
Many cells tended to show such a firing pattern by intracellular
application of GDP-
-S, but the firing frequency did not change
significantly under these conditions. After the diffusion of GDP-
-S
into the TN-GnRH neuron, bath application of sGnRH (20 nM) failed to
evoke any modulation of the firing frequency of pacemaker activity
(Fig. 2A). Figure 2, Bb and Bc (60 and
120 s after the onset of sGnRH perfusion, respectively) was
recorded during similar time periods of transient decrease and
subsequent increase of firing frequency (corresponding to Fig. 1,
Bb and Bc, respectively). The time course of the
frequency of pacemaker activity is plotted in Fig. 2C. The
normalized increase in firing frequency was significantly blocked by
the intracellular application of GDP-
-S to the TN-GnRH neurons
[Fig. 2D; 0.77 ± 0.1 (control) versus 0.16 ± 0.1 (+GDP-
-S), n = 17, P < 0.0001, two-tailed alternate Welch t-test]. Similarly, when the
pipette solution contained GTP-
-S and dialyzed the cell well, bath
application of sGnRH did not show any modulation of the firing
frequency of pacemaker activity (data not shown). Under these
conditions, we would expect that intracellular application of GTP-
-S
alone increases the frequency of pacemaker activity after the rupture
of the membrane patch. However, we could not observe such changes
because the pacemaker activity became unstable immediately after the
rupture of cell membrane. Presumably, GTP-
-S modulated the pacemaker activity maximally soon after the rupture and occluded the effects of
sGnRH. From these results, we suggest that G-protein coupled process
mediates this biphasic modulation of the pacemaker activity by sGnRH in
TN-GnRH neurons.

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Fig. 2.
Effects of intracellular application of guanosine
5'-0-(2-thiodi-phosphate) (GDP- -S). A,
B: intracellular application of 1 mM GDP- -S blocked
the modulation of pacemaker activity by bath application of sGnRH.
Under these conditions, TN-GnRH neuron showed neither decrease
(Bb) nor increase (Bc) in firing
frequency of pacemaker activity. C: frequency of
pacemaker activity plotted against the time course. D:
normalized increase in firing frequency was significantly blocked by
the intracellular application of GDP- -S (n = 17, P < 0.0001, two-tailed alternate Welch
t-test).
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DISCUSSION |
In this study, we have demonstrated that the pacemaker activity of
TN-GnRH neurons was biphasically modulated in a dose-dependent manner
by sGnRH, which is the same molecular species of GnRH peptide that is
probably produced by TN-GnRH neurons themselves (Yamamoto et al.
1995
). This biphasic modulation of pacemaker activity was also
evoked by bath application of another kind of GnRH peptide (mGnRH) but
was not evoked by inactive GnRH analogue (GnRH antagonist alone) and
was inhibited or attenuated by GnRH antagonist. The present results
strongly suggest that modulation by GnRH peptide of pacemaker activity
of TN-GnRH neurons is caused by GnRH receptor activation, although
there does not seem to be a selectivity of different molecular species
of GnRH for the receptor activation. This is in agreement with the
report that GnRH receptors of nonmammalian species respond to any types
of GnRH peptides (King and Millar 1997
). This does not,
however, mean that GnRH released from the GnRH neurons that belong to
the other GnRH systems (other molecular species of GnRH) activates the
TN-GnRH neurons, because immunoreactive fibers of the other GnRH
systems are not distributed near the TN-GnRH neurons (Yamamoto
et al. 1995
).
The biphasic modulation consists of the transient decrease and
subsequent increase in the firing frequency of pacemaker activity. Such
biphasic modulations of the electrical activities have been reported
for the changes in membrane potentials of clonal GH3 cell lines induced
by TRH (Ozawa and Sand 1986
), and those of gonadotropes
and immortalized GnRH cell line (GT1-7 cell) induced by GnRH
(Zheng et al. 1997
). In such cases, it has been
suggested that a transient hyperpolarization arises from the activation of Ca2+-activated K+
currents induced by the elevation of
[Ca2+]i. In fact, a
similar mechanism may exist in TN-GnRH neurons because we have
preliminary data indicating that the transient decrease in firing
frequency of pacemaker activity is blocked by intracellular application
of heparin, an inhibitor of the IP3-induced calcium release (Abe and Oka, in preparation). Future studies should
analyze the changes of
[Ca2+]i induced by GnRH
application and determine the target ion channel(s) modulated by the
GnRH-induced signaling pathway.
There are, however, alternative possible mechanisms of such biphasic
modulations of the frequency of pacemaker activity of TN-GnRH neurons.
First, GnRH receptors may exist on the cell surface of TN-GnRH neurons,
and the pacemaker activity of TN-GnRH neurons may be directly modulated
by the downstream cell signaling pathways. Second, GnRH receptors may
exist on the cell surface of non-GnRH neurons, and the pacemaker
activity of TN-GnRH neurons may be indirectly modulated by these
neurons. In the present study, the biphasic modulations of the
frequency of pacemaker activity were blocked by intracellular
application of GDP-
-S contained in the patch pipette solution. It
has been already established that the GnRH receptors are members of the
G-protein-coupled receptors (Stojilkovic et al. 1994b
).
Also, it has been reported that GT1-7 cells express GnRH receptors
(Krsmanovic et al. 1993
; Stojilkovic et al.
1994a
,b
). Moreover, GnRH neurons of hypothalamic culture have
been shown by double immunostaining to coexpress GnRH and GnRH
receptors (Krsmanovic et al. 1999
). Taken together, it
is highly possible that the GnRH receptor exists on the cell surface of
TN-GnRH neurons and plays a triggering role in modulating the ion
channel(s) underlying the pacemaker activity via G-protein-mediated signaling pathways. To confirm this possibility, we are now trying to
identify the molecular nature of the GnRH receptors expressed on the
cell surface of TN-GnRH neurons by using in situ hybridization and
patch-RT-PCR methods.
What then is the physiological significance of such modulations of
pacemaker activity by sGnRH on TN-GnRH neurons? TN-GnRH neurons of the
dwarf gourami make tight cell clusters with no intervening glial cells
(Oka 1997
; Oka and Ichikawa 1991
;
Oka and Matsushima 1993
), 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 those that exhibit expression of
GnRH receptor mRNA has been reported (Jennes et al.
1996
). Also, cultured hypothalamic GnRH neurons have GnRH receptors (Krsmanovic et al. 1999
). Furthermore, it has
been reported by using an in vivo analysis of multiunit activities in
ovariectomized rats, that GnRH, especially in and around 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
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.
We thank Dr. R. Williamson for critical reading of the manuscript.
This research was supported by Grants-in-Aid from the Ministry of
Education, Science, Sports and Culture of Japan to Y. Oka, and the
Sasakawa Scientific Research Grant from the Japan Science Society to H. Abe.
Address reprint requests: Y. Oka, Misaki Marine Biological Station,
Graduate School of Science, University of Tokyo, Misaki, Miura,
Kanagawa 238-0225, Japan.
The costs of publication of this article were defrayed in part
by the payment of page charges. The article must therefore be hereby
marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.