Journal of Reproduction and Development, Vol. 53, No. 2, 2007 —Full Paper— The Effects of GABA on Embryonic Gonadotropin-Releasing Hormone Neurons in Rat Hypothalamic Primary Culture Hitomi FUJIOKA1,2), Keitaro YAMANOUCHI1), Tatsuo AKEMA2) and Masugi NISHIHARA1) 1) Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Tokyo 113-8657 and 2)Department of Physiology, St. Marianna University School of Medicine, Kawasaki 216-8511, Japan Abstract. Gonadotropin-releasing hormone (GnRH) neurons arise in the olfactory placode, migrate into the preoptic area (POA), and then extend axons to the median eminence during embryogenesis. Little information is available concerning the properties of GnRH neurons during the late gestational period when GnRH neurons reach the POA and form neuronal networks, although many studies have examined such properties during earlier developmental stages or the postnatal period. The present study was performed to elucidate the involvement of γ-aminobutyric acid (GABA), one of the major neurotransmitters modifying GnRH neural activity, in regulation of GnRH gene expression on embryonic day 18.5 (E18.5) using transgenic rats expressing enhanced green fluorescence protein (EGFP) under the control of GnRH promoter. First, using RT-PCR, the mRNA of two isoforms of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), GAD65 and GAD67 was detected in E18.5 embryonic POA-containing tissues. GAD67-positive cells were also demonstrated in close vicinity to GnRH-positive cells by immunohistochemistry, and immunoreactivity for both the GABAA and GABA-B receptor subunits was detected in GnRH neurons. Next, primary cultures derived from anterior hypothalamic tissue of E18.5 embryos were prepared, and the effects of GABA and its agonists on GnRH promoter activity were evaluated using EGFP expression as a marker. GABA and the GABA-A receptor agonist muscimol, but not the GABA-B receptor agonist baclofen, significantly increased the EGFP-positive/GnRH-positive cell ratio. These results suggest that GABA plays a role in stimulating GnRH gene expression through GABA-A receptors in embryonic GnRH neurons in late gestational stages. Key words: Enhanced green fluorescence protein (EGFP), Embryo, γ-aminobutyric acid (GABA), Gonadotropin-releasing hormone (GnRH) neuron, Primary culture, Rat (J. Reprod. Dev. 53: 323–331, 2007) onadotropin-releasing hormone (GnRH) neurons in the hypothalamus play important roles in regulating reproductive function. Unlike the majority of neurons in the central nervous system (CNS), GnRH neurons arise outside the CNS. In mammals, they first become detectable in Accepted for publication: November 6, 2006 Published online: December 20, 2006 Correspondence: M. Nishihara (e-mail: amnishi@mail.ecc.utokyo.ac.jp) the olfactory placode, migrate into the preoptic area (POA), and then extend axons to the median eminence during embryogenesis [1, 2]. After maturation, they secrete GnRH into the pituitary portal blood in a pulsatile fashion in both sexes in a pattern regulated by the GnRH pulse generator [3– 5]. In addition to pulsatile secretion, GnRH is secreted in surges during the preovulatory period in females, presumably as induced by the GnRH surge generator [6]. These patterns of secretion are 324 FUJIOKA et al. influenced by numerous factors, such as amino acids, neuropeptides, cytokines and steroid hormones [7–14]. Gamma-aminobutyric acid (GABA) is one of the major neurotransmitters modifying GnRH neural activity and GnRH secretion. GABA is synthesized from glutamic acid by glutamic acid decarboxylase (GAD) [15] and acts as the principal inhibitory neurotransmitter in the adult CNS. Several studies have shown that GABA hyperpolarizes GnRH neurons [16], alters GnRH gene expression [9, 10], and suppresses LH secretion [9, 10, 17]. In contrast to its inhibitory effect on mature GnRH neurons, GABA depolarizes embryonic GnRH neurons, suppresses GnRH gene expression, and promotes GnRH secretion in olfactory explants [18–20]. Furthermore, GABA affects migrating GnRH neurons through their entire route of migration [21, 22]. Taken together, these findings indicate that GABA is one of the key modulators of GnRH neurons during both adulthood and embryogenesis. Although there are numerous reports on the effects of various regulators on the properties of GnRH neurons, many of them have focused on the properties of pre-migratory embryonic GnRH neurons, migratory embryonic GnRH neurons, or adult GnRH neurons. Little information is available on the properties of GnRH neurons forming neuronal networks in the perinatal hypothalamus. Although it has recently been reported that fibroblast growth factor (FGF)-2 may be involved in the neurite outgrowth of GnRH neurons after migration into the POA [23], the regulation of GnRH gene expression during this period remains unclear. In the rat, GnRH neurons are present in the nasal placode on embryonic day 13.5 (E13.5), migrate through the vomeronasal nerve into the developing forebrain vescicle, and reach the POA on E18.5 [2]. GnRHimmunoreactive (ir) fibers are detectable in the median eminence after E18.5 [2]. It is therefore assumed that GnRH neurons are forming a neural network with cells derived from the central nervous system at this stage. The present study was undertaken to elucidate the effects of GABA on GnRH gene expression in GnRH neurons on E18.5 using transgenic rats expressing enhanced green fluorescence protein (EGFP) in GnRH neurons. Materials and Methods Animals Wistar-Imamichi rats (Imamichi Institute for Animal Reproduction, Ibaraki, Japan) were kept in a room with controlled illumination (light on 0630– 1830 h) and given food and water ad libitum. A homozygous GnRH-EGFP rat strain, in which EGFP is expressed under control of the rat GnRH promoter, was previously generated in our laboratory [24] and was maintained by sisterbrother mating. The rats were kept in plastic cages with hardwood chip bedding and a 12-h light/dark cycle, with lights on at 0700 h. They had ad libitum access to food and water. The rats were mated on the night of proestrous, and the day on which a vaginal plug was found was designated day 0.5 of gestation. All experiments in this study were conducted according to the Guidelines for the Care and Use of Laboratory Animals, Graduate School of Agriculture and Life Sciences, The University of Tokyo. RT-PCR for GAD genes Pregnant GnRH-EGFP rats were sacrificed at 18.5 days of gestation under deep anesthesia, and their fetuses were removed in an aseptic fashion. Brains from the fetuses were immediately removed, and POA-containing regions were separated microscopically. The tissue blocks containing the POA region extended 2 mm rostral to the optic chiasm, 1 mm caudal to the optic chiasm, 1 mm lateral from the center, and was around 2 mm deep. The tissues were immediately frozen in liquid nitrogen and stored at –80 C until RNA extraction. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. RNA (3 µg) from each sample was reverse-transcribed into cDNA using SuperScript II. PCR was performed on 1 µl cDNA using α-Taq Polymerase (BioUniverse, Tokyo, Japan), according to the manufacturer’s instructions. The primers for GAD65, 5’-CGC CCC TGT ATT TGT ACT AC-3’ (sense) and 5’-GCC AAG AGA GGA TCA AAA GC-3’ (antisense) yielded a 419 bp product, while those of GAD67, 5’CAC ACC AGT TGA TGG AAG-3’ and 5’-ACA AAC ACG GGT GCA ATT-3’, yielded a 237 bp product. Amplification of the cDNA was performed under the following conditions: 95 C for 2 min for denaturation followed by 32 cycles of GABA EFFECTS ON EMBRYONIC GNRH NEURONS denaturation (at 95 C) for 15 sec, annealing at 55 C for 30 sec, and extension at 72 C for 30 sec. Reactions were completed with an additional 10min extension at 72 C. PCR products were analyzed by electrophoresis on 2% agarose gels. Immunohistochemistry for GAD and GABA receptors The brains of E18.5 Wistar-Imamichi rats were fixed with 4% formaldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.3) overnight. They were then placed in 30% sucrose in 0.1 M PBS overnight and frozen. Sections (30 µm) were subsequently cut on a cryostat, immediately mounted on glass slides, and stored at –80 C until double-label immunohistochemistry for GAD67, GABA-A receptor β -chain, or GABA-B receptor 1 subunit and GnRH. To visualize GAD67, the sections were rinsed with PBS and incubated with blocking solution (2% BSA/0.4% Triton X-100/PBS) for 30 min. They were then incubated for 24 h at room temperature with an anti-GAD67 mouse monoclonal antibody (Chemicon, Temecula, CA, USA) diluted in blocking solution, rinsed in PBST (0.4% Triton X-100/PBS), and incubated overnight at 4 C with Alexa Fluor 568-conjugated anti-mouse IgG secondary antibody (Invitrogen). For immunostaining for GABA-A receptor β chain, sections were blocked with blocking solution (5% FBS/0.2% Triton-X 100/PBS) for 30 min, incubated with an anti-GABA-A receptor mouse monoclonal antibody (Chemicon) overnight at room temperature, washed with PBS, and incubated with Alexa Fluor 568-conjugated anti-mouse IgG overnight. To visualize GABA-B receptor 1 subunit, sections were blocked with blocking solution (2% BSA and 0.4% Triton-X 100 in PBS) for 30 min, incubated with anti-GABA-B receptor g u i n e a p i g p o l y c l o n a l a n ti s e r u m ( A B 1 5 3 1 ; Chemicon) overnight at room temperature, washed with PBS, and incubated with Alexa Fluor 594labeled anti-guinea pig IgG (Invitrogen) for 4.5 h at room temperature. To detect GnRH, sections were incubated with anti-GnRH rabbit serum generated in our laboratory or an anti-GnRH mouse antibody (LRH13; kindly provided by Dr. M. K. Park, The University of Tokyo). A biotin-labeled secondary antibody (anti-rabbit IgG; Vector Laboratories, Burlingame, CA, USA, or anti-mouse IgG, Invitrogen) and Avidin-conjugated Cy2 or Alexa Fluor 488-conjugated secondary antibody 325 (Invitrogen) were used to visualized GnRHimmunoreactive (ir) signals. After immunohistochemistry, the sections were washed and coverslipped with mounting media. Fluorescence was visualized using a confocal laser scanning microscope (LSM-510, Carl Zeiss, Oberkochen, Germany). Primary cultures and detection of EGFP in GnRH neurons Brain tissues containing the POA were obtained from E18.5 fetuses as described above and placed in ice-cold HEPES containing Dulbecco’s modified Eagle’s medium (DMEM). The tissue blocks were washed and then trypsinized at 37 C for 15 min. Following dispersion, the cells were plated at a density of 5.0 × 105 cells per glass on polylysine/ laminin-coated coverslips. Th e cells were maintained in culture medium [DMEM/Ham’s F12 (7:3) containing 2% B27 supplement, 50 U/ml penicillin and 50 µg/ml streptomycin] at 37 C and 5% CO2. For study of high K+-stimulation, cells were plated for 3 h and then the cultures were treated with high K+-medium, which was prepared by adding 16 mM KCl to culture medium that contained 4.66 mM K + and 153.14 mM Na + , or osmotic control medium (which contained 16 mM NaCl added to the culture medium) for 48 h. For study of GABA, the cultures were treated with GABA (10 µ M-1 mM; Sigma-Aldrich, St. Louis, MO, USA), the GABA-A receptor agonist muscimol (10 µ M-1 mM; Sigma-Aldrich), or the GABA-B receptor agonist baclofen (10 µ M-1 mM; SigmaAldrich) for 24 h. The cultures were then fixed with 4% formaldehyde in 0.01 M phosphate buffer saline (PBS) at room temperature for 20 min, and subjected to immunohistochemistry for GnRH as described above. EGFP and Alexa Fluor 594 fluorescence was observed using a fluorescence microscope (BX50; Olympus, Tokyo, Japan), and the numbers of GnRH-ir and EGFP-positive cells were counted. Statistical analysis Student’s t-test was used for comparisons between two groups. One-way analysis of variance (ANOVA) followed by Dunnett’s test was performed for comparisons between more than two groups with comparable variances. Differences of P<0.05 were considered significant. 326 FUJIOKA et al. Results Expression of GAD and GABA receptors in E18.5 POA tissues As shown in Fig. 1A, both GAD65 and GAD67 mRNAs were detected in embryonic rat POA regions by RT-PCR. Fig. 1B shows that GAD67-ir signals were present in nearby GnRH neurons. To determine whether GABA-A receptors or GABA-B receptors were expressed in the GnRH neurons of E18.5 rat fetuses, double immunohistochemistry for GABA-A receptor β-chain or GABA-B receptor 1 and GnRH was performed. GnRH-ir cells were found to have overlapping GABA-A (Fig. 2A, C and E) and GABA-B receptor signals (Fig. 2B, D and E). Effects of high K+ and GABA on EGFP in E18.5 primary cultures As shown in Fig. 3, EGFP signals were detected in some of the GnRH-ir cells (Fig. 3A and B) but not in others (Fig. 3C and D) in embryonic rat hypothalamic cultures. The EGFP-positive/GnRHir cell ratio was significantly increased (P<0.01) when cultures were incubated in high K+ medium (Fig. 3E). Incubation of cultures with GABA (10 µM-1 mM) also significantly increased the EGFPpositive/GnRH-ir cell ratio (Fig. 4A). Muscimol at concentrations above 100 µM similarly increased this ratio (Fig. 4B), while baclofen had no effect (Fig. 4C). Discussion In the present study, we first demonstrated that GAD65 and GAD67 mRNA is present in E18.5 embryonic POA-containing regions and that GAD67-ir cells are located in nearby GnRH neurons. These findings suggest that GABA is synthesized in cells adjacent to GnRH neurons in the POA during the late gestational period. In addition, we demonstrated that the GABA-A and GABA-B receptor subunits are expressed in GnRHir cells. It is thus likely that GABA affects the properties of GnRH neurons during this period. So far, many studies have shown that GABA affects GnRH neurons at other stages. For example, electrophysiological studies have demonstrated that GABA acts on juvenile and adult GnRH neurons in hypothalamus and olfactory placode- Fig. 1. GAD expression in the E18.5 rat anterior hypothalamus. A shows RT-PCR products of RNA extracted from E18.5 rat POA-containing regions. Each lane shows the results for one individual rat (lanes 1–3). Lane 4 shows the negative control. B shows visualization of immunoreactive signals for GAD67 (red) and GnRH (green). Arrowheads indicate typical GnRH neurons. derived GnRH neurons [16, 18, 25]. GABA-A receptor subunit mRNA is expressed in olfactory placode-derived GnRH neurons [26] and postnatal [27], juveniles, and adult GnRH neurons [28], and GABA-A receptor immunoreactivity has been detected in migrating GnRH neurons [21]. In addition, it has been reported that GABA-B receptor mRNA is also expressed in olfactory placode-derived GnRH neurons [29], and GABA-B receptor immunoreactivity has been detected in adult sheep GnRH neurons [30]. Taken together, these findings suggest that GABA acts on GnRH neurons from development to adulthood via both GABA EFFECTS ON EMBRYONIC GNRH NEURONS 327 Fig. 2. Visualization of GABA receptors and GnRH-immunoreactive cells in the E18.5 rat anterior hypothalamus. A and D show GABA-A receptor- and GABA-B receptor-immunoreactive signals (red), respectively. B and E show GnRHimmunoreactive cells (green) in the same areas as A and D, respectively. C is a merged image of A and B, and F is a merged image of D and E. Arrowheads indicate typical GnRH neurons. Fig. 3. The upper 4 panels show visualization of GnRH-immunoreactive cells (A and C, red) and EGFP fluorescence (B and D, green) in E18.5 hypothalamic primary cultures derived from GnRH-EGPF transgenic rats. Solid arrowheads indicate typical EGFP-positive GnRH neurons (A and B). The open arrowhead indicates an EGFP-negative GnRH neuron (C and D). E shows the effect of high K+ on EGFP-positive/GnRH-immunoreactive cell ratios. Cells were treated with 16 mM KCl or 16 mM NaCl (osmotic control, Ct) for 48 h. Each column and vertical bar represents the mean ± SEM (n=6). *P<0.05 vs. Ct. 328 FUJIOKA et al. Fig. 4. Effects of GABA (A), muscimol (B), and baclofen (C) on EGFP expression in GnRH neurons in E18.5 hypothalamic primary cultures derived from GnRH-EGFP transgenic rats. The data are expressed as EGFPpositive/GnRH-immunoreactive cell ratios. Cells were treated with each substance or vehicle (Ct) for 24 h. Each column and vertical bar represents the mean ± SEM (n=9). *P<0.05 vs. Ct. the GABA-A and GABA-B receptors. We used GnRH-EGFP transgenic rat embryos to observe the effects of GABA on GnRH promoter activity. These transgenic rats have transgene constructs comprising approximately 3 kb of rat GnRH promoter and EGFP gene and express EGFP in a GnRH neuron-specific fashion [24]. Although around 75% of GnRH neurons in adult females have been found to be EGFP-positive [24], fewer GnRH neurons (25–35%) exhibited EGFP fluorescence in primary embryonic culture in the present study. On this point, the findings of this study are consistent with a previous study that reported less GnRH expression in the fetal anterior hypothalamus than in adults [31]. These observations suggest that the transcriptional activity of the GnRH gene in embryos is lower than that in mature rats. We also examined whether EGFP expression in GnRH neurons is altered by high K+-stimuli, since we have previously found that high K+-stimuli promote GnRH gene expression in adult rat hypothalamic slices in vitro [32]. The present finding that high K + -stimuli enhanced EGFP expression in GnRH neurons indicates that changes in EGFP expression can be used to evaluate GnRH gene expression. We then demonstrated that GABA and the GABA-A receptor agonist muscimol, but not the GABA-B receptor agonist baclofen, enhanced EGFP expression in GnRH neurons of GnRH-EGFP transgenic rat embryos in vitro. These findings suggest that GABA can activate GnRH gene expression via at least GABA-A receptors in embryonic GnRH neurons. In adulthood, GABA is the main inhibitory transmitter in the CNS, and its hyperpolarizing effect is caused by the inward flow of chloride ion through ion-specific channels opened by GABA-A receptor activation. In contrast, it has been reported that during early neural development, GABA-A receptor-mediated responses are often depolarizing due to immature chloride equilibrium [33]. It has also been reported that GABA depolarizes olfactory placode-derived GnRH neurons and postnatal day 10–17 GnRH neurons [18] and that the effect of switching of GABA on GnRH neurons from depolarization to hyperpolarization occurs during the peripubertal period. Therefore, under our conditions, GABA appears to depolarize GnRH neurons [25]. High K+ treatment, used as a means of depolarization, also induced GnRH gene expression. Increase in GABA EFFECTS ON EMBRYONIC GNRH NEURONS membrane potential is assumed to affect the spontaneous firing of GnRH neurons. Thus, the promotion of GnRH gene expression by GABA mediated via GABA-A receptors could be due to an increase in GnRH neural activity. Fueshko et al. reported the opposite findings; they found that GABA-A receptor activation decreased GnRH mRNA levels in nasal explants [19]. Although the reason for the discrepancy between their results and ours is unclear, there were many differences in the experimental conditions between the two studies, such as the tissues from which the GnRH neurons were harvested (nasal epithelium vs. POA), culture conditions (organ culture vs. cell culture), and drug reaction periods (7 days vs. 24 h). Thes e di fferences may account for the discrepancy. In the present study GABA-B receptor agonist did not change the EGFP expression in GnRH neurons despite expression of the GABA-B receptor subunit. GABA-B receptors are G-protein-coupled receptors that contribute to neuronal inhibition due to an increase in K + conductance or decrease in v ol t a g e - d e pe n de n t C a 2 + c u r r e n t [ 3 4 ] . T h e differences in the effects of the GABA-A and GABA-B agonists on GnRH gene expression may be due to differences in their effects on membrane potential. The signal transduction pathways 329 involved in GABA-induced GnRH gene expression remain unclear, and further studies are needed to determine them. During CNS development, numerous molecules affect neural proliferation, differentiation and migration; neurite genesis; axon guidance; and cell survival in time- and region-specific fashions [35– 40]. Together, they create complex neural networks. It has been reported that GABA plays an important role in neural proliferation, migration and cell survival. Our findings suggest that GABA is involved in stimulating GnRH gene expression in the perinatal hypothalamus. 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