Masakatsu Kato

Activation of A-type γ-amino butyric acid receptors excites gonadotrophin-releasing hormone neurones isolated from adult rats

Gonadotrophin‐releasing hormone (GnRH) neurones represent the final output neurones in the neuroendocrine control of reproduction, and γ‐amino butyric acid (GABA) is one of the major players in the regulation of GnRH neurones. GABA inhibits a large proportion of brain neurones in adult animals by acting on A‐type GABA receptors (GABAARs). Two contradictory reports on the action of GABA in the GnRH neurones of adult mice have been published. DeFazio et al. (Mol Endocrinol 2002; 16: 2872) demonstrated that activation of GABAARs excites the GnRH neurones of adult mice, whereas Han et al. (Endocrinology 2002; 143: 1459) showed that the response to GABA on GnRH neurones switches from depolarisation to hyperpolarisation around puberty in female mice. Therefore, we examined the reversal potential of GABAAR currents by means of perforated patch‐clamp recording with gramicidin in overnight‐cultured GnRH neurones isolated from adult GnRH‐enhanced green fluorescent protein transgenic rats. The reversal potential was −26 ± 1.4 mV (mean ± SEM, n = 42) in GnRH neurones, whereas it was −57 ± 2.7 mV (n = 34) in unidentified neurones, and GABA depolarised the GnRH neurones in current‐clamp condition. The GABAAR currents in rat GnRH neurones were augmented by neurosteroids, allopregnanolone and 3α,21‐dihydroxy‐5α‐pregnan‐20‐one, at submicromolar concentrations. In addition, the expression patterns of GABAAR subunit mRNAs were determined by multi‐cell reverse transcription‐polymerase chain reaction, which revealed that the α2, β3, γ1 and γ2 subunits were dominant and the α6 and γ3 subunits were negative in rat GnRH neurones. These results indicate that GABAARs in the soma of rat GnRH neurones are comprised mainly of α2, β3 and γ1 or γ2 subunits and that they are sensitive to neurosteroids; moreover, they suggest that activation of these receptors depolarises GnRH neurones. Thus, GABA and neurosteroids influence the electrical activity of GnRH neurones.

The SK channel blocker apamin inhibits slow afterhyperpolarization currents in rat gonadotropin-releasing hormone neurones.

Gonadotropin‐releasing hormone (GnRH) neurones play an essential role in the hypothalamo‐pituitary‐gonadal axis. As for other neurones, the discharge pattern of action potentials is important for GnRH neurones to properly function. In the case of a luteinizing hormone (LH) surge, for example, GnRH neurones are likely to continuously fire for more than an hour. For this type of firing, GnRH neurones must have a certain intrinsic property. To address this issue, we investigated the voltage‐gated Ca2+ currents and Ca2+‐activated voltage‐independent K+ currents underlying afterhyperpolarization, because they affect cell excitability. Dispersed GnRH neurones from adult GnRH‐EGFP (enhanced green fluorescent protein) transgenic rats were cultured overnight and then used for an electrophysiological experiment involving the perforated patch‐clamp configuration. The GnRH neurones showed five subtypes of voltage‐gated Ca2+ currents, i.e. the T‐, L‐, N‐, P/Q‐ and R‐types. The GnRH neurones also showed a slow afterhyperpolarization current (IsAHP), but not a medium one. It is reported that the SK channel blocker apamin (10 pm–100 nm) blocks a medium afterhyperpolarization current but not an IsAHP. In contrast to previous reports, the IsAHP observed in rat GnRH neurones was potently blocked by apamin. In addition, the GnRH neurones expressed transcripts for SK1–3 channels. The results indicate that rat GnRH neurones express all five subtypes of voltage‐gated Ca2+ channels and exhibit an apamin‐sensitive IsAHP, which may allow continuous firing in response to a relatively strong depolarizing input.

GABAA Receptors Mediate Excitation in Adult Rat GnRH Neurons

Abstract Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction. Gamma-amino butyric acid (GABA), the main inhibitory neurotransmitter in the adult brain, has long been implicated in playing key roles in the regulation of GnRH neurons. Two groups reported recently that GABA depolarizes GnRH neurons, although one group reported a hyperpolarizing action of GABA. In this study, we investigated the GABA-induced changes in [Ca2+]i of GnRH neurons from GnRH-enhanced green fluorescent protein (GnRH-EGFP) rats both to confirm the depolarizing action of GABA and to further examine the developmental and estrous cycle-dependent modulations of GABA action. GABA increased [Ca2+]i in GnRH neurons at all developmental stages of both sexes. GABA also increased [Ca2+]i in adult female GnRH neurons prepared in the afternoon at each estrous cycle stage. The percentages of neurons with increased [Ca2+]i were 90% in proestrus, 59% in estrus, 84% in diestrus I, and 89% in diestrus II. In GnRH neurons prepared from adult females in the morning, however, the percentage was significantly lower than in those prepared in the afternoon, except in estrus. The percentage was also lower in adult males than in adult females. GABA responses were mimicked by muscimol and blocked by bicuculline. In addition, removal of extracellular Ca2+ completely suppressed the GABA action, and bumetanide attenuated the response. These results indicate that GABA depolarizes GnRH neurons by activating GABAA receptors, thereby activating voltage-gated Ca2+ channels and facilitating Ca2+ influx. In addition, the response to GABA is modulated according to the estrous cycle stage, diurnal rhythm, and sex.

Neural interaction between galanin-like peptide (GALP)- and luteinizing hormone-releasing hormone (LHRH)-containing neurons

Galanin-like peptide (GALP), commonly known as an appetite-regulating peptide, has been shown to increase plasma luteinizing hormone (LH) through luteinizing hormone-releasing hormone (LHRH). This led us to investigate, using both light and electron microscopy, whether GALP-containing neurons in the rat brain make direct inputs to LHRH-containing neurons. As LHRH-containing neurons are very difficult to demonstrate immunohistochemically with LHRH antiserum without colchicine treatment, we used a transgenic rat in which LHRH tagged with enhanced green fluorescence protein facilitated the precise detection of LHRH-producing neuronal cell bodies and processes. This is the first study to report on synaptic inputs to LHRH-containing neurons at the ultrastructural level using this transgenic model. We also used immunohistochemistry to investigate the neuronal interaction between GALP- and LHRH-containing neurons. The experiments revealed that GALP-containing nerve terminals lie in close apposition with LHRH-containing cell bodies and processes in the medial preoptic area and the bed nucleus of the stria terminalis. At the ultrastructural level, the GALP-positive nerve terminals were found to make axo-somatic and axo-dendritic synaptic contacts with the EGFP-positive neurons in these areas. These results strongly suggest that GALP-containing neurons provide direct input to LHRH-containing neurons and that GALP plays a crucial role in the regulation of LH secretion via LHRH.

Rat GnRH neurons exhibit large conductance voltage- and Ca2+-Activated K+ (BK) currents and express BK channel mRNAs.

Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction. As in other neurons, the discharge pattern of action potentials is important for these neurons to function properly. Therefore it is important to elucidate the expression patterns of various types of ion channels in these neurons because they determine cell excitability. To date, voltage-gated Ca2+ channels and SK channels have been reported to be expressed in rat GnRH neurons. In this study, we focused on K+ channels and analyzed their expression in primary cultured GnRH neurons, prepared from GnRH-EGFP transgenic rats, by means of perforated patch-clamp recordings. GnRH neurons exhibited delayed-rectifier K+ currents and large conductance voltage- and Ca2+-activated K+ (BK) currents. Moreover, multicell RT-PCR (reverse transcriptase-polymerase chain reaction) experiments revealed the expression of BK channel mRNAs (alpha, beta1, beta2, and beta4). The results show the presence of delayed-rectifier K+ currents and BK currents besides previously reported slow afterhyperpolarization currents. These currents control the action potential repolarization and probably also the firing pattern, thereby regulating the cell excitability of GnRH neurons.

Purinergic regulation of intracellular Ca2+ concentration of rat pituitary folliculo-stellate cells in primary culture.

Pituitary folliculo‐stellate cells (FSCs) are glia‐like cells in the anterior pituitary and are believed to modulate the activity of the pituitary endocrine cells. However, little is known what regulates the activity of FSCs. We hypothesized that ATP could act on FSCs, because ATP is coreleased with pituitary hormones from endocrine cells. To test this possibility, we examined the effect of ATP by measuring intracellular Ca2+ concentration [Ca2+]i of FSCs in primary culture. Both ATP and UTP increased the [Ca2+]i in a concentration‐dependent manner in a range between 0.1 µM and 10 µM. The response was completely suppressed by thapsigargin, an inhibitior of endoplasmic reticulum Ca2+‐ATPase, and was significantly suppressed by U‐73122, an inhibitor of phospholipase C. The response was also suppressed by caffeine, a blocker of IP3 receptor, whereas that was not suppressed by ryanodine, an antagonist of ryanodine receptor. These results indicate that ATP increases [Ca2+]i of FSCs by activating phospholipase C via P2Y purinergic receptor and suggest that ATP would be one of paracrine factors to FSCs in the anterior pituitary.

Impaired Cytosolic Ca2+ Response to Glucose and Gastric Inhibitory Polypeptide in Pancreatic β-Cells from Triphenyltin-Induced Diabetic Hamster

Oral administration of a single dose of triphenyltin compounds induces diabetes with decreased insulin secretion in rabbits and hamsters after 2–3 days without any morphological changes in pancreatic islets. In the present study, to test the possibility that the impaired insulin secretion induced by triphenyltin compounds could result from an impaired Ca2+ response in pancreatic β-cells, we investigated the effect of triphenyltin-chloride (TPTCl) administration on the changes in the cytoplasmic Ca2+ concentration ([Ca2+]i) induced by secretagogues, such as glucose, high K+, gastric inhibitory polypeptide (GIP), and acetylcholine (ACh) in hamster pancreatic β-cells. TPTCl administration caused partial suppression in 10 mm K+-induced rise in [Ca2+]i without suppressing the increase in [Ca2+]i evoked by 20–50 mm K+. Administration of TPTCl strongly inhibited the rises in [Ca2+]i induced by 27.8 mm glucose, 100 μm ACh in the presence of 5.5 mm glucose, and by 100 nm GIP in the presence of 5.5 mm glucose. In t...

Direct Evidence of Gonadotropin-Releasing Hormone (GnRH)-Stimulated Nitric Oxide Production in the LβT-2 Clonal Gonadotropes

An immortal cell line (LβT-2) with characteristics of gonadotropes, such as LH-containing secretory granules, and LH release responsiveness to GnRH, was used to investigate the effect of GnRH stimulation on nitric oxide (NO) production. RT-PCR analysis showed that mouse nNOS mRNA was expressed in cultured LβT-2 cells. LβT-2 cells were treated with the calcium ionophore, A23187, and NO levels were measured as nitrite using the Griess assay. The data clearly showed that NO production was increased dose-dependently by A23187 treatment (0–10-5 M). Next, changes in the intracellular concentration of ionized calcium ([Ca2+]i) in LβT-2 cells induced by GnRH were analyzed by quantitative fluorescence microscopy, and [Ca2+]i was found to be increased markedly by GnRH treatment. In fact, exposure of LβT-2 cells to increasing concentrations of GnRH from 0 to 10-6 M was found to enhance NO production in a dose dependent manner, with maximal augmentation at 10-6 M. However, the stimulation of NO production by GnRH at this concentration was significantly attenuated by pretreatment with NG-nitro-L-arginine methyl ester hydrochloride (L-NAME), a NO synthase inhibitor.Taken together, the present results suggest that GnRH treatment results in increased NO production in LβT-2 clonal gonadotropes, and intracellular calcium augmentation produced by GnRH may be participate in this process. Our findings also indicate that the LβT-2 cell line is a useful tool for in vitro studies of the autocrine and paracrine roles of NO in the anterior pituitary gland.

Somatostatin inhibition of GnRH neuronal activity and the morphological relationship between GnRH and somatostatin neurons in rats

In rodents, GnRH neurons are diffusely distributed from the medial septum through to the medial preoptic area and control gonadal functions through the pituitary. The activity of GnRH neurons is regulated by a variety of bioactive substances, including the inhibitory peptide somatostatin. In the present study, we focused on somatostatin because intracerebroventricular injection of somatostatin inhibits the LH surge in rats and reduces LH secretion in ewes. Somatostatin also decreases GnRH release from rat hypothalamic slices. In mice, somatostatin is also thought to suppress GnRH neuronal activity through contact on the soma of GnRH neurons. However, similar data are missing in rats. Moreover, rat GnRH neurons receive only a few synaptic inputs. In this study, we assessed the morphological relationship between GnRH and somatostatin neurons. Confocal microscopy on the sections from the medial septum through medial preoptic area revealed about 35 close contacts per rat between the GnRH and somatostatin neuronal fibers in the organum vasculosum of the lamina terminalis region. No contact of somatostatin fibers on the GnRH neuronal somata was observed. Multicell RT-PCR for somatostatin receptor mRNA in rat GnRH neurons was also performed, which revealed moderate expression of somatostatin receptor subtypes 1-5. In addition, patch clamp experiments were carried out in acute slice preparations. Somatostatin suppressed neuronal firing in cells recorded in a cell-attached configuration and also induced whole-cell outward currents in GnRH neurons. These findings suggest that somatostatin directly inhibits the activity of rat GnRH neurons through volume transmission in the organum vasculosum of the lamina terminalis region.

Sexually Dimorphic Modulation of GABAA Receptor Currents by Melatonin in Rat Gonadotropin-Releasing Hormone Neurons

Gonadotropin-releasing hormone (GnRH) neurons represent the final output neurons in the central control of reproduction. gamma-Amino butyric acid (GABA), one of the major regulators of GnRH neurons, depolarizes GnRH neurons isolated from adult rats via GABA(A) receptors. The presence of GABA(A) receptors in GnRH neurons has also been demonstrated morphologically. Furthermore, the pineal hormone melatonin is involved in the regulation of reproductive function, including the timing of the luteinizing hormone surge. The suprachiasmatic nucleus and the GABAergic system in the medial preoptic area are considered as possible sites of the action of melatonin. Until now, however, a direct action of melatonin on GnRH neurons has not been reported. Therefore we examined the effect of melatonin on GABA(A) receptor currents in GnRH neurons isolated from GnRH-EGFP transgenic rats by means of perforated patch-clamp experiments. The GABA(A) receptor currents were modulated by melatonin in a sex-specific manner. In GnRH neurons from adult males, melatonin augmented these currents in 67% of the neurons examined, but attenuated the currents in only 19% of them. These modulations were blocked by the melatonin receptor antagonist luzindole, suggesting an involvement of melatonin receptors. The modulation by melatonin was not observed in GnRH neurons isolated from infantile rats. These findings indicate that GABA affects the excitability of GnRH neurons in adult rats through GABA(A) receptors, and that melatonin modifies this excitability via melatonin receptors in a sex-specific manner.