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Endocrinology, doi:10.1210/en.2003-0960
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Endocrinology Vol. 145, No. 4 2092-2103
Copyright © 2004 by The Endocrine Society

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)

Mami Ishizaki, Masayuki Iigo, Naoyuki Yamamoto and Yoshitaka Oka

Misaki Marine Biological Station (M.Is., Y.O.), Graduate School of Science, The University of Tokyo, Miura, Kanagawa 238-0225, Japan; Department of Anatomy (M.Ii.), St. Marianna University School of Medicine, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan; and Department of Anatomy (N.Y.), Laboratory for Comparative Neuromorphology, Nippon Medical School, Bunkyo-ku, Tokyo 113-8602, 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
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has become a general notion that there are multiple GnRH systems in the vertebrate brains. To measure GnRH release activities from different GnRH systems, we conducted a static incubation of brain-pituitary slices under various conditions, and GnRH released into the incubation medium was measured by RIA. The slices were divided into two parts, one containing GnRH neurons in the preoptic area and axon terminals in the pituitary (POA-GnRH slices), and the other containing the cell bodies and fibers of terminal nerve-GnRH neurons and midbrain tegmentum-GnRH neurons (TN-TEG-GnRH slices). We demonstrated that GnRH release was evoked by high [K+]o depolarizing stimuli (in both POA-GnRH and TN-TEG-GnRH slices) via Ca2+ influx through voltage-gated Ca2+ channels. The most prominent result was the presence of conspicuous sexual difference in the amount of GnRH release in the POA-GnRH slices. The GnRH release from TN-TEG-GnRH slices also showed a small sexual difference, which was by far more inconspicuous than that of POA-GnRH slices. Immunohistochemical analysis using an antiserum specific to the seabream GnRH (sbGnRH; suggested to be specific to POA-GnRH neurons) revealed the presence of a much larger number of POA-GnRH neurons in males than in females. This clear morphological sexual difference is suggested to underlie that of GnRH release in the POA-GnRH slices.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE HAVE BEEN a number of studies on GnRH as a peptide hormone that has critical functions in reproduction in vertebrates. The GnRH neurons in the preoptic area (POA) (hypothalamo-hypophyseal GnRH neuron is a more general term) release GnRH and facilitate gonadotropin release from the pituitary. Recent studies have shown that GnRH functions not only as a hypophysiotropic hormone that controls reproductive functions but also as a neuromodulator that controls motivational or arousal states of the animal (for review, see Refs. 1, 2, 3). However, our knowledge about functions of GnRH other than the hypophysiotropic function in the brain is still limited at present.

Based mainly on the results obtained in the teleost brain, it has been generally accepted that there are at least two or three anatomically as well as functionally distinctive GnRH systems in the brain of most vertebrates (1, 3, 4, 5, 6). GnRH neurons exist in several brain areas such as POA, midbrain tegmentum (TEG), and terminal nerve (TN). The POA-GnRH system has been most intensively studied from neuroendocrinological viewpoints among the different GnRH systems. Tegmental GnRH neurons (TEG-GnRH neurons) project their axons mainly to the posterior parts of the brain. TN-GnRH neurons project their axons widely in the brain but not to the pituitary. Recently it has been suggested that the TN-GnRH system has neuromodulatory functions by releasing GnRH in wide area of brain (for reviews, see Refs. 1, 2, 3). Because the TEG-GnRH system also projects widely in the brain, it is also suggested to have neuromodulatory functions. The study of physiological control mechanisms of GnRH release is critical for our understanding of each system’s functions.

In mammals, it is extremely difficult to examine GnRH release activities of GnRH neurons because GnRH neurons are sparse and diffusely distributed in the brain. Among vertebrate species the GnRH system is most developed and best studied in teleost brains. We selected a tropical anabantid teleost fish, the dwarf gourami (Colisa lalia) to study the GnRH release in the present paper. This fish has many characteristics advantageous for the study of GnRH neurons. The brain of the dwarf gourami has a well-developed TN-GnRH system (7, 8). TN-GnRH cell bodies are large and make tight cell clusters beneath the ventral meningeal membrane. This is especially of great advantage over brains of other vertebrates, in which peptidergic neurons are small and diffusely distributed and are extremely difficult to study individually. We have already shown that TN- and TEG-GnRH neurons in the dwarf gourami are immunoreactive to salmon [Trp7Leu8] GnRH (sGnRH) and chicken II [His5Trp7Tyr8] GnRH (cGnRH-II) (6), respectively. POA-GnRH cells also make clusters in the dwarf gourami (9, 10), and their axons project directly to the pituitary (general feature of hypophysiotropic neurons in teleosts lacking the portal system). Furthermore, electrophysiological properties of TN-GnRH neurons of the dwarf gourami have been most intensively studied among vertebrate brains so far (8, 11, 12, 13, 14, 15). Thus, the brain of the dwarf gourami may be one of the best experimental systems to analyze physiological mechanisms of GnRH release and its relationship with the electrical activity of the GnRH neurons.

From electrophysiological studies, it has been shown that GnRH, which is probably released by TN-GnRH system, modulates the pacemaker activity of TN-GnRH neurons themselves in the dwarf gourami (11, 15). These studies suggest that TN-GnRH system may have some neuromodulatory functions (1, 2, 3). In accordance with this idea, there have been some studies on the modulatory functions of TN-GnRH neurons thus far. Walker and Stell (16) showed that retinal ganglion cells of the goldfish (Carassius auratus) respond to several compounds present in TN (including GnRH), and Umino and Dowling (17) showed that retinal ganglion cells of the goldfish change their responses to light when they were perfused with GnRH. Yamamoto et al. (18) reported that the lesion of the TN-GnRH cells changed the threshold for initiation of one of the reproductive behavior repertory, male nest-building behavior, in the dwarf gourami. Eisthen et al. (19) found that GnRH affects ionic channel properties of olfactory receptor neurons and suggested neuromodulatory effects of the TN-GnRH neurons on the olfactory receptor neurons in amphibians. Before concluding that the TN-GnRH system exerts modulatory functions in vertebrate brains, however, it is necessary to demonstrate GnRH release activities from the TN-GnRH neurons. GnRH release from the brain has been determined by RIA in POA-GnRH system of the goldfish brain slices (20) and the rat median eminence fragment (21). However, the GnRH release from TN-GnRH neurons and its control mechanisms have not been studied thus far. It is also important to study the difference in GnRH release activities between POA- and other (TN- and TEG-) GnRH systems because they are suggested to have quite different functions, e.g. neuroendocrine vs. neuromodulatory.

In the present study, we first measured GnRH release from the brain-pituitary slices of the dwarf gourami separated into functionally different two parts (POA- and TN-TEG-GnRH slices), by a static incubation system and RIA using a single antibody that almost equally recognizes various molecular species of GnRH (see Materials and Methods). This way, we wanted to compare the similarities and differences of GnRH release from the two functionally different systems. We took advantage of the distinctive anatomical features of the three GnRH systems of the dwarf gourami (1, 6, 18) and separated the slices into two, one containing the POA-pituitary region (POA-GnRH slices) and the other containing TN-GnRH neurons, TEG-GnRH neurons, and their projection areas in wide areas of the brain (TN-TEG-GnRH slices). The GnRH release from the former represents that related to the reproductive functions, whereas that from the latter represents that related to the neuromodulatory functions. We found a characteristic sexual difference in GnRH release from POA-GnRH neurons. The sexual difference in GnRH release from TN-TEG-GnRH slices was less prominent than that of POA-GnRH slices. Because the preoptic GnRH molecular form is seabream [Ser8] GnRH (sbGnRH) in the percomorph teleosts (22, 23, 24, 25) and the presence of sbGnRH has been suggested by an HPLC analysis combined with RIA in the dwarf gourami (6), we performed GnRH-immunohistochemistry with a sbGnRH antiserum to examine sexual dimorphism in POA-GnRH cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The dwarf gouramis were purchased from a local dealer and kept in the aquaria at 25–28 C under a photoperiod of 12-h light/12-h dark cycle until used. The animals were fed tropical fish food purchased commercially. The animals were maintained in accordance with the guidelines of The Physiological Society of Japan and the University of Tokyo for the Use and Care of Experimental Animals.

Preparation of brain and pituitary slices
The male and female fish were killed by decapitation, and the brain with pituitary was removed and immersed rapidly into a chilled Ringer solution. The Ringer solution contained (in millimoles) NaCl 124, KCl 5, CaCl2 2.4, KH2PO4 1.2, MgSO4 1.3, NaHCO3 26, glucose 10 (pH 7.4, adjusted with NaOH). The brain with pituitary was sliced into sagittal sections of 250 µM using a microslicer (DTK-1000, DOSAKA EM Co. Ltd., Osaka, Japan). Four parasagittal slices per fish were obtained so that each slice has a pituitary fragment connected to the brain. The slices were divided into two parts under a dissection microscope using a razor blade as shown in Fig. 1Go, the one containing the POA and the pituitary (POA-GnRH slice), and the remaining part of the brain (TN-TEG-GnRH slice), which contained the cell bodies of TN and TEG GnRH neurons, and most of their fibers. Because the fibers of the both TN- and TEG-GnRH systems are distributed in wide areas of the brain and are intermingled extensively, it was technically impossible to separate the two systems.



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  		FIG. 1. Preparation of brain-pituitary slices for the measurement of GnRH release with RIA. The brain-pituitary slice (250 µm) was divided into two parts. The TN-TEG-GnRH slice contains the cell bodies of TN-GnRH (denoted by star) and midbrain TEG-GnRH neurons (denoted by triangle) and most of their axons distributed in wide areas of the brain. The POA-GnRH slice contains the cell bodies of POA-GnRH neurons (denoted by closed circle) and the pituitary where the axon terminals of POA-GnRH neurons exist. Eight slices (from two fish) were collected for each type of slice and incubated in Ringer solution. The media were sampled using a static incubation system to measure the GnRH release from slices.

 
Each experimental replicate contained eight slices obtained from two fish. That is, one replicate of POA-GnRH slices corresponds to the POA regions and the entire pituitaries from two fish. Four to six replicates were examined in each experiment.

Static incubation of brain-pituitary slices in vitro
The slices in each replicate were preincubated in an oxygenized Ringer solution in a well of the 24-well plate over 50 min for recovery from injury by brain slicing. After preincubation, slices were washed with fresh Ringer solution. The medium was replaced with 200 µl of experimental solution (see below). After incubation for 10 min (20 min in thapsigargin experiment) at 27 C, the medium was transferred to sample tubes, mixed with HCl (0.1 N in final concentration), frozen on dry ice and stored at -80 C until assayed for GnRH. After the collection of experimental solutions, the slices were washed with the Ringer solution three times, and each well was filled with fresh Ringer. The next treatment was done after the slices were allowed to recover for 50 min. One experimental series consisted of two to five different treatments.

Experimental design of static incubations
Experiment 1. Depolarization-induced GnRH release.
We examined depolarization-induced GnRH release in response to high [K+]o Ringer solutions [NaCl was partly replaced with KCl to make (K+)o = 20, 40, 60, or 100 mM]. POA- and TN-TEG-GnRH slices from male and female fish were incubated first in Ringer solutions for 10 min to measure spontaneous GnRH release. Next, the slices were incubated for 10 min in high [K+]o Ringer solutions. Series of high [K+]o tests were done in the order of increasing [K+]o.

Experiment 2. Contribution of extracellular Ca2+ to the GnRH release in response to depolarization.
Extracellular Ca2+ dependence of depolarization-induced GnRH release was tested. The slices were incubated with Ringer solutions or high [K+]o solutions (100 mM) that contained nominally no [Ca2+]o (Ca2+-free) or Ca2+-free Ringer solutions and high [K+]o solutions (100 mM). Each treatment was done for 10 min.

To assess contribution of different types of Ca2+ channels to depolarization-induced GnRH release, the high [K+]o stimulation (100 mM) was done in the presence of specific Ca2+ channel blockers. L-type (10 µM nifedipine), N-type (1 µM {omega}-conotoxin GVIA), and P/Q-type (250 nM {omega}-agatoxin TK) Ca2+ channel blockers were used in this experiment. The slices were incubated for 10 min with Ringer solutions, then high [K+]o solutions (100 mM) and high [K+]o solutions (100 mM) with specific Ca2+ channel blockers. Nifedipine and {omega}-conotoxin were tested in the same series of experiment, and {omega}-agatoxin was tested in another series.

Experiment 3. Effects of Ca2+ release from intracellular Ca2+ stores on spontaneous GnRH release.
Effects on spontaneous (basal) GnRH release were examined by using caffeine to induce Ca2+ release from intracellular Ca2+ stores. First, the slices were incubated for 10 min with Ringer solutions and next with Ringer solutions that contained 10 mM of caffeine.

Experiment 4. Effects of store-operated Ca2+ influx on spontaneous GnRH release.
Store-operated Ca2+ influx (Ca2+ influx induced by depletion of intracellular Ca2+ store, see Ref. 26) was induced by thapsigargin (an inhibitor of endoplasmic reticulum Ca2+-ATPase so that it will eventually deplete the intracellular Ca2+ stores). The slices were first incubated in a Ca2+-free Ringer solutions (control) for 20 min, and the intracellular Ca2+ store was depleted by Ca2+-free Ringer with thapsigargin (20 µM) for 20 min. Thereafter, the slices were returned to Ca2+-containing [(Ca2+)o = 2.4 mM] normal Ringer for 20 min. If GnRH release occurs when [Ca2+]o is restored, it is supposed to be due to a store-operated Ca2+ influx. Then slices were incubated in Ringer that contained 2 mM Zn2+ (inhibitor of store-operated Ca2+ influx, see Refs. 27, 28, 29) for 20 min.

Chemicals used in the static incubation
Stock solutions of {omega}-conotoxin GVIA and {omega}-agatoxin TK (Alomone Labs, Jerusalem, Israel) were dissolved in double-distilled water. Stock solutions of nifedipine (Alomone Labs) and thapsigargin (Sigma, St. Louis, MO) were dissolved in dimethyl sulfoxide. The stock solutions of {omega}-conotoxin, {omega}-agatoxin, nifedipine, and thapsigargin were diluted in Ringer solution before the experiments. The vehicle (dimethyl sulfoxide) was added to control Ringer solutions in nifedipine and thapsigargin experiments. Caffeine was obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan) and dissolved in Ringer solution.

RIA
The sGnRH, cGnRH-II, and mammalian GnRH (mGnRH) peptides were obtained from Peninsula Laboratories, Inc. (Belmont, CA). The sbGnRH peptide was kindly donated by Dr. K. Okuzawa (National Research Institute of Aquaculture, Tamaki, Mie, Japan). [125I]-labeled mGnRH (2200 Ci/mmol, NEN Life Science Products, Boston, MA) was used as the tracer. The rabbit anti-GnRH serum (anti-GnRH, lot R-II, see Ref. 30) and antirabbit {gamma}-globulin goat serum (HAC-RBA2–05GTP91) were obtained from Dr. Y. Hasegawa (Kitasato University, Towada, Aomori, Japan) and Prof. K. Wakabayashi (Gunma University, Maebashi, Gunma, Japan), respectively. BSA (fraction V, RIA grade) was purchased from Sigma.

The amount of immunoreactive GnRH released into the medium from brain slices was determined by RIA according to the procedure of Okuzawa et al. (31), with slight modifications. A standard curve (4.9–2500 pg/ml) was constructed by using serial 2-fold dilutions of sGnRH dissolved in PBS [50 mM phosphate buffer containing 140 mM NaCl and 0.1% sodium azide (pH 7.5)] containing 1% BSA (BSA-PBS). For the initiation of RIA, anti-GnRH serum (1:20,000 dilution with PBS containing 50 mM EDTA and 1% normal rabbit serum, 100 µl) were added to test tubes [Eiken RIA tubes (no. 2), Eiken Kizai, Tokyo, Japan] containing BSA-PBS (300 µl) and the standard or samples (100 µl) and incubated for 5 d at 4 C. [125I]-mGnRH (approximately 10,000 cpm in BSA-PBS, 100 µl) was added to each tube and further incubated for 24 h at 4 C. Antirabbit {gamma}-globulin goat serum (1:50 dilution with PBS containing 50 mM EDTA, 200 µl) was added to each tube. After incubation for an additional 24 h at 4 C, these tubes were centrifuged (2000 x g, 30 min, 4 C) and the supernatant was aspirated. Radioactivity was then determined with a {gamma}-counter (Aloka, Tokyo, Japan).

For the validation of the RIA, parallelism and quantitative recovery studies were performed. The inhibition curve for serial 2-fold dilution of the samples was parallel to the curve for the sGnRH standard. The relationship between the quantity of sGnRH added (0–312.5 pg/ml) to the samples and the recovered amount was analyzed using linear regression analysis. A significant correlation was obtained between the two [Y = 1.094 x +0.22 (r = 0.962, n = 7, P < 0.001); X: sGnRH added (pg/ml); Y: sGnRH recovered (pg/ml)]. Intra- and interassay coefficients of variation of the RIA were 5.0–12.9% (n = 3–10) and 10.7% (n = 5) at 98.7 pg/ml, respectively. The minimum detectable level defined as 2 SD from the buffer controls was 8.5 pg/ml (corresponding to 1.7 pg/sample). The antiserum used in this study is known to cross-react with various molecular species of GnRH (6, 26). The displacement curves for GnRHs were parallel to that of the sGnRH standard. The cross-reactivities investigated in the present RIA system were as follows: sGnRH, 100%; cGnRH-II, 117.9%; sbGnRH, 156.0%; mGnRH, 187.7% (Fig. 2Go). Fifty percent inhibition doses for sGnRH were 40.8 ± 0.8 pg/ml (n = 16).



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  		FIG. 2. The competition curves for GnRH RIA. Four GnRH subtypes (mGnRH, sGnRH, sbGnRH, and cGnRH-II) were tested. This RIA system using antiserum against cGnRH-II (lot R-II) recognized all four GnRH subtypes.

 
Current clamp recording of electrical activity of terminal nerve-GnRH cells
We examined the dose dependence in frequency of electrical activity of TN-GnRH neurons on depolarizing stimuli. A whole-cell patch clamp recording was carried out to record the pacemaker activity of TN-GnRH neurons. The whole brain of male dwarf gourami was dissected and pinned ventral side up to a trough, the ventral meningeal membrane was removed, and the cluster of TN-GnRH cells were exposed. The preparation was perfused with Ringer solution or high [K+]o Ringer solutions (20, 40, 60, and 100 mM). To quantify the increase of spike frequency, the number of spikes during 10-sec periods before and after the perfusion of high [K+]o solution was counted, and the ratios between them were plotted. Whole-cell current-clamp recording was carried out with a patch clamp amplifier (PC-501A, Warner Instruments, Inc., Hamden, CT) and pCLAMP software (Axon Instrument, Inc., Union City, CA). The direct visual identification and electrophysiological characterization of TN-GnRH neurons have already been well established by our previous studies (2, 8, 11, 12, 13, 14, 15, 32), and there were no electrophysiological differences between males and females.

Immunohistochemistry
Ten mature dwarf gourami (five males and five females) ranging 3–4 cm in standard length were used for sbGnRH immunohistochemistry. The fish were deeply anesthetized in tricaine methanesulfonate (300 mg/liter) and perfused through the conus arteriosus with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed from the skull and postfixed in a fresh solution of the same fixative at 4 C for 12 h. The brains were then immersed in 20% sucrose in 0.1 M phosphate buffer (pH 7.4) for 6 h and embedded in 5% agar (Sigma, type IX) containing 20% sucrose. Tissue blocks were frozen in n-hexane at -60 C, and 30-µm-thick serial sections were cut on a cryostat either sagittally (one male and one female) or frontally (four males and four females). Thaw-mounted sections on gelatin-coated slides were dried by fans, washed with 0.05 M PBS containing 0.3% Triton X-100 (PBST), and reacted with an affinity-purified anti-sbGnRH antiserum (generous gift of Dr. I. S. Parhar, Nippon Medical School, Tokyo, Japan) for 20 h. The primary antiserum was diluted 1:50,000 with PBST containing 1% normal goat serum. Sections were then washed three times in PBST and reacted with biotin-labeled antirabbit IgG (1:200) for 2 h. After the reaction with secondary antibody, the sections were incubated for 10 min in 0.3% H2O2 in methanol to block remaining endogenous peroxidase activity. The sections were washed three times in PBST and reacted with avidin-biotin-horseradish peroxidase complex solution [1:50; Sigma (Vectastain), ABC elite kit] for 3 h. After three washes in 0.05 M PBS, the sections were incubated with diaminobenzidine containing 0.04% nickel ammonium sulfate for 30 min. All the reactions were carried out at room temperature. After dehydration through a series of graded concentrations of alcohol, the sections were cleared and coverslipped, and immunoreactive neurons were counted under the microscope. All the sections were used for cell counting. A positive cellular structure with a clearly identifiable nucleus (immunonegative, round structure in the center of cytoplasm) was regarded as one cell.

To test the specificity of the primary antiserum and immunohistochemical procedures of the present study, the following control experiments were also performed: 1) use of primary antiserum preabsorbed with 2.5 µg/ml sbGnRH peptide (generous gift of Dr. M. Kobayashi, International Christian University, Tokyo, Japan) for 20 h at 4 C; 2) omission of primary antiserum from the incubating solution; 3) omission of secondary antiserum from the incubating solution; and 4) omission of avidin-biotin-horseradish peroxidase complex from the incubating solution. All of these tests resulted in no positive structures. Alternate series of sections, which were processed normally, exhibited immunopositive neurons as in specimens used for cell counting. These observations strongly support the sbGnRH-specific immunoreaction of the present study.

Statistics
The data are expressed as the mean ± SEM. Effects of sex and drug treatment were analyzed by two-way ANOVA in RIA experiments. In Experiment 1, the dose-dependence of GnRH release on [K+]o was analyzed by one-way ANOVA followed by Dunnett’s test. In Experiments 2–4, comparison among treatment groups was performed with one-way ANOVA followed by Student-Newman-Keuls test. Effects of high-[K+]o stimuli on electrical activities of TN-GnRH neurons were analyzed by one-way ANOVA, and paired t test was used to analyze the increase in the number of spikes. The number of positive cells in immunohistochemistry was analyzed by Student’s t test. Differences were considered significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Depolarization-induced GnRH release
Figure 3Go shows the GnRH release when the POA-GnRH slices and TN-TEG-GnRH slices from male and female fish were stimulated with high [K+]o Ringer solutions [(K+)o = 20, 40, 60, and 100 mM]. The high [K+]o Ringer solutions significantly increased the GnRH release from POA- and TN-TEG-GnRH slices in a dose-dependent manner [Fig. 3Go, two-way ANOVA, F(4,29) = 5.36; P < 0.005 in POA-GnRH slices; F(4,30) = 33.22; P < 0.0001 in TN-TEG-GnRH slices]. In POA-GnRH slices, the GnRH release was by far larger in male fish than in female, and the sexual difference was highly significant [two-way ANOVA, sex: F(1,29) = 36.21; P < 0.0001]. In TN-TEG-GnRH slices, the sexual difference of the GnRH release was much smaller than in POA-GnRH slices, but still the sexual difference was significant [two-way ANOVA, sex: F(1,30) = 6.42; P < 0.05]. When we compared TN-TEG-GnRH slices with POA-GnRH slices, POA-GnRH slices released a larger amount of GnRH than TN-TEG-GnRH slices [two-way ANOVA, type of slice: F(1,59) = 15.39; P < 0.0005] despite the fact that POA-GnRH slices were much smaller in size than TN-TEG-GnRH slices. It also should be noted that there was a considerable amount of spontaneous (basal) release of GnRH (GnRH release in controls) in both POA- and TN-TEG-GnRH slices, and it was also sexually different in POA- but not in TN-TEG-GnRH slices.



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  		FIG. 3. GnRH release from POA- and TN-TEG-GnRH slices in response to high K+ depolarizing stimuli. GnRH release (pg/sample; note different scales in different graphs) was dependent on the extracellular [K+]o (P < 0.005 in POA-GnRH slices; P < 0.0001 in TN-TEG-GnRH slices, two-way ANOVA). A, In POA-GnRH slices, sexual difference in GnRH release was highly significant (P < 0.0001; not marked by symbols) at every [K+]o, and the male (blank bars) released larger amounts of GnRH than female (hatched bars). B, In TN-TEG-GnRH slices, the sexual difference of GnRH release was considerably smaller (P < 0.05) than in POA-GnRH slices. The bars and vertical lines represent means ± SEM (n = 4 in each group). Asterisks indicate means significantly different from the control (*, P < 0.05; **, P < 0.01; ANOVA followed by Dunnett’s test).

 
The electrical activity of TN-GnRH cells was also examined by whole-cell current clamp recording using a whole-brain in vitro preparation of the dwarf gourami. The perfusion of high [K+]o Ringer solution ([K+]o = 20, 40, 60, 100 mM) increased the frequency of pacemaker potentials of TN-GnRH cells in a dose-dependent manner [Fig. 4Go, one-way ANOVA; F(3,12) = 9.81, P < 0.005]. The results shown in Figs. 3Go and 4Go suggest that the GnRH release in TN-TEG-GnRH slices is dependent on the frequency of pacemaker potentials of TN-GnRH cells.



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  		FIG. 4. Electrical activities of TN-GnRH cells in response to high [K+]o Ringer solution. A whole cell current clamp recording was carried out from TN-GnRH cells using a whole brain preparation. A, Recording of the pacemaker potential of TN-GnRH cells. High [K+]o solution ([K+]o = 100 mM) was applied by perfusion during the period indicated by the bar. TN-GnRH cells were depolarized in response to the application of high [K+]o solution, and frequency of the pacemaker activities was increased. B, The ratios of number of spikes between before and after the perfusion of high [K+]o were plotted. The frequency of the action potentials was increased in a dose-dependent manner (P < 0.005, one-way ANOVA). The columns and the vertical lines represent means ± SEM of the ratios (n = 4). Asterisks indicate that the number of spikes were significantly different from controls by paired t test (*, P < 0.05; **, P < 0.01).

 
Next, the dependence on extracellular Ca2+ of GnRH release was examined (Fig. 5Go). Figure 5Go clearly shows that in both POA- and TN-TEG-GnRH slices of males and females the GnRH release evoked by the depolarizing stimulus is dependent on [Ca2+]o. Spontaneous GnRH release was observed in the controls in Fig. 5Go, A and B. The spontaneous release from POA and TN-TEG-GnRH slices was independent of the [Ca2+]o.



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  		FIG. 5. Effects of extracellular Ca2+ on GnRH release in response to high K+ depolarizing stimulus. GnRH release (pg/sample; note different scales in different graphs) evoked by high K+ (100 mM) was abolished by eliminating extracellular Ca2+ (-Ca2+) in both slices in both sexes. A, GnRH release from POA-GnRH slices from male (A1) and female (A2) fish. B, GnRH release from TN-TEG-GnRH slices of male (B1) and female (B2) fish. The bars and vertical lines represent means ± SEM (n = 4) of control, high K+ solution (100K), Ca2+-free solution (-Ca2+), Ca2+-free-high K+ solution (100K-Ca2+), respectively. Bars with different superscript letters are significantly different (P < 0.05; ANOVA followed by Student-Newman-Keuls test).

 
From these results, it was shown in both POA and TN-TEG-GnRH slices that the depolarization-induced GnRH release was dependent on Ca2+ influx. Therefore, we assessed contribution of different types of Ca2+ channels to the GnRH release in response to depolarizing stimuli (Figs. 6Go and 7Go). In POA-GnRH slices, {omega}-conotoxin almost completely abolished the GnRH release in response to high K+ (100 mM), whereas nifedipine increased the GnRH release significantly (Fig. 6AGo). {omega}-Agatoxin caused no significant changes (Fig. 7AGo). In contrast, the GnRH release from TN-TEG-GnRH slices in response to the high [K+]o stimuli was abolished by {omega}-conotoxin in males, significantly decreased in females, and was decreased by application of nifedipine in both sexes (Fig. 6BGo). GnRH release from TN-TEG-GnRH slices in response to the high [K+]o stimuli was not affected by agatoxin (Fig. 7BGo). Thus, it is suggested that the GnRH release from POA-GnRH slices induced by depolarization is mainly dependent on the Ca2+ influx through N-type Ca2+ channels and that from TN-TEG-GnRH slices induced by depolarization is mainly dependent on both L- and N-type Ca2+ channels.



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  		FIG. 6. Effects of Ca2+ channel blockers on GnRH release in response to high K+ depolarizing stimulus. A, In POA slices, GnRH release (pg/sample; note different scales in different graphs) evoked by high K+ solution (100 mM [K+]o; 100K) was diminished by {omega}-conotoxin (Ctx; N-type Ca2+ channel blocker). Nifedipine (Nif; L-type Ca2+ channel blocker) facilitated GnRH release in both males (A1) and females (A2). B, In TN-GnRH slices, GnRH release evoked by high K+ solution was significantly reduced by {omega}-conotoxin and nifedipine in both males (B1) and females (B2). The bars and vertical lines represent means ± SEM (n = 4). Bars with different superscript letters are significantly different (P < 0.05; ANOVA followed by Student-Newman-Keuls test).

 


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  		FIG. 7. Effects of P/Q-type Ca2+ channel blockers on GnRH release in response to high K+ depolarizing stimulus. In POA-GnRH slices (A) and TN-TEG-GnRH slices (B), GnRH release (pg/sample; note different scales in different graphs) evoked by high K+ solution (100 mM [K+]o; 100K) was not affected by {omega}-agatoxin (Aga; P/Q-type Ca2+ channel blocker); there was no significant difference in the amount of GnRH release between high K+-treated slices and high K+ with {omega}-agatoxin-treated slices; A1, B1, males; A2, B2, females. The bars and vertical lines represent means ± SEM (n = 4). Bars with different superscript letters are significantly different (P < 0.05; ANOVA followed by Student-Newman-Keuls test).

 
Effects of Ca2+ release from intracellular Ca2+ stores and store-operated Ca2+ influx
The contribution to GnRH release of Ca2+ released from intracellular Ca2+ stores was tested. The effects of caffeine (10 mM) and thapsigargin (20 µM) are shown in Figs. 8Go and 9Go. No significant increase of GnRH release was observed by the application of caffeine for 10 min to either POA- or TN-TEG-GnRH slices (Fig. 8Go). Thapsigargin application for 20 min decreased the GnRH release in TN-TEG-GnRH slices (Fig. 9Go, B1 and B2) but not in POA-GnRH slices (Fig. 9Go, A1 and A2). Thus, it is suggested that Ca2+ release from Ca2+ store contributes to the GnRH release only from TN-TEG-GnRH slices. Then, we examined whether store-operated Ca2+ influx affects GnRH release activities. GnRH release was increased by this treatment in TN-TEG-GnRH slices of females (Fig. 9Go, B2, SOC). This increase was abolished when 2 mM Zn2+ (inhibitor of store-operated Ca2+ influx) was added to the Ca2+-containing normal Ringer (Fig. 9Go, B2, SOC+Zn2+).



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  		FIG. 8. Effects of caffeine on GnRH release. GnRH release (pg/sample; note different scales in different graphs) was not affected significantly by caffeine treatment (10 mM) in either slice and in either sex. A, POA-GnRH slices. B, TN-TEG-GnRH slices; A1, B1, males; A2, B2, females. The bars and vertical lines represent means ± SEM (n = 6) of control and caffeine solution (Caf).

 


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  		FIG. 9. Effects of thapsigargin and store-operated currents on GnRH release (pg/sample; note different scales in different graphs). Thap, thapsigargin treatment (20 µM); SOC, extracellular Ca2+ restoration after depletion of intracellular Ca2+ stores (to assess the effect of store-operated currents); SOC+Zn2+, restoration of extracellular Ca2+ in the presence of Zn2+. Thapsigargin treatment decreased GnRH release from TN-TEG-GnRH slices of females. The effects of Ca2+ restoration and Ca2+ restoration in the presence of Zn2+ were significantly different in TN-TEG-GnRH slices of females. A, POA-GnRH slices. B, TN-TEG-GnRH slices; A1, B1, males; A2, B2, females. The bars and vertical lines represent means ± SEM (n = 4 in males, n = 5 in females). Bars with different superscript letters are significantly different (P < 0.05; ANOVA followed by Student-Newman-Keuls test).

 
Immunohistochemistry
Anti-sbGnRH serum-labeled neurons in the POA and their axon terminals in the pituitary. Minor populations of sbGnRH-immunoreactive cells were also observed in the area ventralis telencephali pars ventralis and nucleus lateralis tuberis pars posterior of Peter et al. (33). Somata of TN-GnRH cells were only faintly labeled, and faintly stained axons could be traced for very short distances. TEG-GnRH cells and their fibers were not labeled at all. This result supports sbGnRH production in this GnRH cell group as in other percomorph teleosts (22, 23, 24, 25). Because we have already shown that TN- and TEG-GnRH neurons are immunoreactive to salmon and chicken II type GnRH (6), respectively, it is concluded that there are at least three different types of GnRH neuronal systems in the dwarf gourami brain.

Two major groups of sbGnRH-immunoreactive cells were identified in the POA: rostral and caudal populations (Fig. 10AGo). The rostral population was darkly stained and present in the most rostral part of the POA surrounding the preoptic recess. No sexual differences were detected with regard to this cell group (Fig. 10Go, B and C). The numbers of rostral neurons were 26.9 ± 4.8 neurons/side in males (mean ± SEM; n = 5; see Fig. 10BGo) and 24.9 ± 2.1 neurons/side in females (n = 5; see Fig. 10CGo; P > 0.5 vs. male), and the staining intensity and distribution of these cells were much the same in both sexes. The caudal population appeared slightly caudal to the rostral population and showed a marked sexual difference (Fig. 10Go, D and E). In contrast to many moderately stained neurons in males (170.6 ± 19.6 neurons/side; n = 5; see Fig. 10DGo), labeled cells were rarely encountered in the corresponding area in females (4.4 ± 0.9 neurons/side; n = 5; see Fig. 10EGo; P < 0.005 vs. male). Also, positive cells in females were only very faintly stained.



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  		FIG. 10. Immunohistochemistry of male and female dwarf gourami brains using a specific antiserum to sbGnRH. A, Sagittal section of male POA region. In the POA, two groups of sbGnRH immunoreactive cells were stained. Rostral, Rostral cell group; caudal, caudal cell group. B–E, Frontal sections. The rostral group of neurons (B, C) was darkly stained in both sexes (B, male; C, female), and the number of cells did not differ (P > 0.5; t test). On the other hand, the caudal group of neurons (D, E) showed a marked sexual difference in number of cells (P < 0.005; t test). There were many immunoreactive cells in the caudal POA of males (D) but by far fewer immunoreactive cells in the corresponding area of females (E). Scale bar, 100 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we found that control mechanisms of GnRH release from POA- and TN-/TEG-GnRH systems have some characteristics in common but that there are also some characteristics different from each other. We found prominent sex differences of GnRH release in POA-GnRH slices but not in TN-TEG-GnRH slices. The Ca2+ mobilization pathways in GnRH release mechanisms of POA- and TN-/TEG-GnRH systems were also different.

Depolarizing stimuli caused GnRH release from both POA- and TN-TEG-GnRH slices in a dose-dependent manner. Interestingly, there was a characteristic sexual difference in spontaneous and depolarization-induced GnRH release from the POA-GnRH slices: the POA-GnRH slices from male fish released, by far, a larger amount of GnRH than those of female fish. This is consistent with our immunohistochemical data: the number and staining intensity of sbGnRH-immunoreactive cells in the caudal population of POA showed a marked sexual difference with an overwhelmingly larger number in males than in females. This morphological sexual difference most probably underlies the sexual differences in GnRH releasing activities from the POA-GnRH slices. Less pronounced but statistically significant sexual differences were also observed in the GnRH release from TN-TEG-GnRH slices. Because the male brains were slightly larger than female brains (around 3%) and we did not make corrections for this size difference, this sexual difference of TN-TEG-GnRH slices may be simply because of such sexual difference in the brain volume. In contrast, the sexual differences in the GnRH release from POA-GnRH slices and in the number of GnRH cells in caudal POA are too large to be explained by the brain volume.

Some previous studies have reported on sexual differences in several aspects of POA-GnRH neurons. The size and number of GnRH cells were larger in males than in females in the ballan wrasse (34). Sexual difference was observed in the activation of GnRH synthesis by 17{alpha}-methyltestosterone administration in yearling masu salmon (35), and GnRH immunoreactivity was different between sexes in the musk shrew brain (36). On the other hand, GnRH contents in brain slices of the rainbow trout are apparently equivalent in both sexes (31). It will be important in future experiments to determine whether there are seasonal differences in GnRH contents and/or GnRH release from the POA-GnRH system of the dwarf gourami in both sexes. One possibility is that males have larger amounts of GnRH in POA-GnRH neurons and release higher amounts of GnRH in all seasons, but females have GnRH fluctuations and release smaller amounts of GnRH except for the spawning period.

Morphological analyses in the present study revealed two populations of POA-GnRH cells: rostral and caudal populations. The rostral population did not show sex-dependent differences in immunostaining, whereas the caudal population exhibited clear sexual dimorphism. On the other hand, it has been shown that the gonadotropins of teleosts consist of two types, FSH (GTHI) and LH (GTHII) (37). It is thus an interesting possibility that each preoptic GnRH cell group is involved in the control of either FSH or LH specifically.

In POA-GnRH slices, GnRH release evoked by high [K+]o depolarizing stimuli was dependent on extracellular Ca2+ and was dependent on Ca2+ influx mediated mainly by N-type Ca2+ channels but not by L or P/Q type. In general, L-type Ca2+ channels are found in cell types such as endocrine cells, muscles, and neurons, and the Ca2+ influx via these channels can induce exocytosis in several cells such as chromaffin cells (38) and GT1 cells (39). On the other hand, N-, P-, and Q-type Ca2+ channels are generally found in neurons and are suggested to trigger transmitter release at conventional synapses in the brain (40, 41).

The involvement of N-type Ca2+ channels for POA-GnRH slices is different from previous studies that described that Ca2+ channels involved in GnRH release from GnRH-secreting cells are L-type Ca2+ channels, not N-type Ca2+ channels (39, 42). This may be due to differences among different vertebrate species or may be from differences in the degree of maturity of GnRH cells. We used POA-GnRH slices from adult mature dwarf gourami. It has been reported that the types of Ca2+ channels coupled to secretion change with degree of maturity (43, 44, 45).

In the present study, there was an unexpected but significant increase of GnRH release from POA-GnRH slices by the application of nifedipine, an L-type Ca2+ channel blocker. It is possible that the L-type Ca2+ channel is not directly involved in the depolarization-induced GnRH release from POA-GnRH neurons per se and that high [K+]o depolarization stimulated release of factors that inhibit GnRH release or some inhibitory synaptic transmitters that heavily depends on L-type Ca2+ channels and that nifedipine removed this inhibition.

It has not been reported which type of Ca2+ channels are involved in GnRH release from TEG- or TN-GnRH neurons. It has been suggested that TN-GnRH neurons release GnRH from various parts of the neurons including somatodendritic areas and axonal varicosities (3, 46). The present results suggest that GnRH release from these parts of TN-GnRH neurons is dependent on Ca2+ influx via N- and L-type Ca2+ channels.

Ca2+ release from intracellular stores has been reported to contribute to the exocytosis in several cell types (47, 48, 49). In the sympathetic neurons, intracellular Ca2+ and GnRH release are increased by agents that cause Ca2+-induced Ca2+ release such as caffeine (50, 51), whereas in GT1 cells, such agents have no effect on Ca2+ signaling (52). In the present study, caffeine application had little effect on the GnRH release from POA- and TN-TEG-GnRH slices. Thapsigargin treatment for 20 min decreased spontaneous GnRH release from TN-TEG-GnRH slices of females. Because thapsigargin is an inhibitor of endoplasmic reticulum Ca2+-ATPase, thapsigargin treatment should have reduced Ca2+ concentration in the endoplasmic reticulum, which resulted in the decrease of intracellular Ca2+ concentration in 20 min. In TN-TEG-GnRH slices of females, when extracellular Ca2+ was restored after depletion of intracellular Ca2+ stores with thapsigargin, GnRH release from TN-TEG-GnRH slices was higher than when extracellular Ca2+ was restored in the presence of Zn2+, a blocker of store-operated current. These results suggest that store-operated Ca2+ entry may contribute to the GnRH release in TEG- and/or TN-GnRH neurons. There are some studies that report on the contribution of store-operated Ca2+ influx to exocytosis (53, 54). In the present study, the amount of GnRH released possibly by store-operated Ca2+ influx after depletion of intracellular store did not exceed that of the control level in which intracellular stores was not depleted. Therefore, it is possible that Ca2+ in the intracellular Ca2+ store may be somehow related to the spontaneous or basal GnRH release from TEG- and/or TN-GnRH neurons.

It has been reported that peptidergic neurons and endocrine cells not only receive several inputs from other neurons but they are also modulated by peptide hormones released by themselves, which implies the possible role of the peptide in modulating its own secretion (autocrine or paracrine control) (11, 55, 56 ; see also Ref. 2). In the present study, effects of GnRH agonist on GnRH release were not investigated. Simultaneous recording of the GnRH release and electrical activities is necessary to test GnRH effects on GnRH release and the relationship between pacemaker activity and GnRH release activity. Recently we developed a method for real-time electrochemical recording of GnRH release using carbon fiber electrodes (57). An amperometric current was recorded in response to high [K+]o stimulation in a dose-dependent manner. This method will provide much information on the relationship between pacemaker activity and GnRH release activity.

In summary, we found similarities as well as differences in the GnRH release activities of TEG- and TN-GnRH neurons and POA-GnRH neurons of the dwarf gourami. TEG- and TN-GnRH neurons are considered to play neuromodulatory roles, and from the present study, we found multiple Ca2+ mobilization pathways related to the GnRH release in TN-TEG-GnRH slices. It is thus possible that these multiple Ca2+ mobilization mechanisms are related to the neuromodulatory function of TEG- and TN-GnRH neurons. Additional future studies are needed to determine how GnRH release from the POA-, TEG-, and TN-GnRH systems is differently regulated and how this release may be involved in reproductive functions of the POA-GnRH system and neuromodulatory functions of the TEG- and TN-GnRH systems.


   Acknowledgments
 
We thank Professor K. Aida (University of Tokyo) for allowing us to use the facilities for the RIA; Mr. T. Masuda for help in RIA; Dr. Y. Hasegawa (Kitasato University School of Veterinary Medicine and Animal Sciences), Professor K. Wakabayashi (Gunma University), and Dr. I. S. Parhar (Nippon Medical School) for their kind supply of the antisera (anti-GnRH, lot R-II, HAC-RBA2-05GTP91 and anti-sbGnRH, respectively); and Mr. K. Haneda for help in analysis of the data. We also would like to thank Drs. K. Okuzawa (National Research Institute of Aquaculture), M. K. Park (University of Tokyo), M. Kobayashi (International Christian University), and M. Amano (Kitasato University) for kind technical advice and discussion, and K.-I. Maeda, H. Tsukamura, and S. Tsukahara (Nagoya University) for their kind help in the preliminary studies.


   Footnotes
 
This work was supported by Grants-in-Aid from the MEXT of Japan (to Y.O. and M.Ii.).

Present address for M.Is.: National Agricultural Research Center, Kannondai, Tsukuba, Ibaraki 305-8666, Japan.

Present address of M.Ii.: Department of Applied Biological Chemistry, Faculty of Agriculture, Utsunomiya University, 350 Mine-machi, Utsunomiya, Tochigi 321-8505, Japan.

Present address for Y.O.: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Abbreviations: cGnRH-II, Chicken II [His5Trp7Tyr8] GnRH; mGnRH, mammalian GnRH; PBST, PBS containing 0.3% Triton X-100; POA, preoptic area; sbGnRH, seabream [Ser8] GnRH; sGnRH, salmon [Trp7Leu8] GnRH; TEG, tegmentum; TN, terminal nerve.

Received July 29, 2003.

Accepted for publication December 29, 2003.


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K. Haneda and Y. Oka
Selective Modulation of Voltage-Gated Calcium Channels in the Terminal Nerve Gonadotropin-Releasing Hormone Neurons of a Teleost, the Dwarf Gourami (Colisa lalia)
Endocrinology, October 1, 2004; 145(10): 4489 - 4499.
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