Kumiko Suyama

Immortalized Gonadotropin-Releasing Hormone Neurons (GT1-7 Cells) Exhibit Synchronous Bursts of Action Potentials

Although it has been assumed that synchronized firing of gonadotropin-releasing hormone (GnRH) neurons is necessary for pulsatile GnRH secretion, there is no clear evidence for this. In the present study we simultaneously recorded spontaneous action potentials from multiple cells. Immortalized GnRH neurons (GT1-7 cells) were cultured on a multi-electrode dish (MED) and action potentials recorded by an extracellular recording method. One to two weeks after the beginning of culture, spontaneous action potentials appeared, exhibiting bursts composed of 5–10 action potentials. Burst activity was intermittent and periodic with mean burst intervals of 13.3 s. Furthermore, burst activity was recorded almost simultaneously from several micro-electrodes, suggesting that electrical activities of GT1-7 cells were synchronized with each other. Periodic bursts were completely and reversibly blocked by 1–5 µM tetrodotoxin, indicating that voltage-dependent Na+ channels are involved in their generation. γ-Aminobutyric acid (GABA) given at a 10-µM concentration shortened inter-burst intervals, whereas 10 µM bicuculline lengthened them. Finally, the gap junctional blockers n-octyl alcohol (1 mM) and carbenoxolone (100 µM) reversibly blocked periodic burst activity. The present study provides direct evidence that the electrical activity of GT1-7 cells exhibits synchronous and periodic bursts composed of action potentials. In addition, endogenous GABA is involved in GT1-7 cells in determining burst frequency. Although the precise mechanism of synchronized burst activities needs to be clarified, gap junctional communications among GT1-7 cells are at least partially involved.

Phasic synaptic incorporation of GluR2-lacking AMPA receptors at gonadotropin-releasing hormone neurons is involved in the generation of the luteinizing hormone surge in female rats☆

Reproductive success depends on a robust and appropriately timed preovulatory luteinizing hormone (LH) surge, which is induced by the activation of gonadotropin-releasing hormone (GnRH) neurons in response to positive feedback from increasing estrogen levels. Here we document an increase in postsynaptic GluR2-lacking Ca2+ -permeable AMPA-type glutamate receptors (CP-AMPARs) at synapses on GnRH neurons on the day of proestrus in rats, coincident with the increase in estrogen levels. Functional blockade of CP-AMPARs depressed the synaptic responses only on the day of proestrus and concomitantly attenuated the LH surge. Thus, the phasic synaptic incorporation of postsynaptic CP-AMPARs on GnRH neurons is involved in the generation of the LH surge.

Gene expression and specific binding of platelet-derived growth factor and its effect on DNA synthesis in human decidual cells

To clarify the biological significance of platelet-derived growth factor (PDGF) in human decidual cell function, which is important for the maintenance of pregnancy, we investigated gene expression of the PDGF subunits, PDGF-A and PDGF-B, specific binding of the PDGF isoform, and the effect of PDGF dimers on DNA synthesis in human decidual cells. We detected in decidua from early pregnancy the expected DNA bands of PDGF-A and PDGF-B by reverse transcriptase-polymerase chain reaction (RT-PCR) as well as mRNAs of each PDGF subunit by Northern blot hybridization, demonstrating that both PDGF subunits exist in this tissue. Scatchard plot analysis showed that decidual cells had both PDGF-alpha and PDGF-beta receptors. PDGF-AA, -AB and -BB stimulated [3H]-thymidine incorporation in cultured decidual cells in a dose-dependent manner. These results indicate the importance of PDGF in human decidua.

Optical inactivation of synaptic AMPA receptors erases fear memory

The synaptic delivery of neurotransmitter receptors, such as GluA1 AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, mediates important processes in cognitive function, including memory acquisition and retention. Understanding the roles of these receptors has been hampered by the lack of a method to inactivate them in vivo with high spatiotemporal precision. We developed a technique to inactivate synaptic GluA1 AMPA receptors in vivo using chromophore-assisted light inactivation (CALI). We raised a monoclonal antibody specific for the extracellular domain of GluA1 that induced effective CALI when conjugated with a photosensitizer (eosin). Mice that had been injected in the CA1 hippocampal region with the antibody conjugate underwent a fear memory task. Exposing the hippocampus to green light using an implanted cannula erased acquired fear memory in the animals by inactivation of synaptic GluA1. Our optical technique for inactivating synaptic proteins will enable elucidation of their physiological roles in cognition.

Estrous Cycle-Dependent Phasic Changes in the Stoichiometry of Hippocampal Synaptic AMPA Receptors in Rats.

Cognitive function can be affected by the estrous cycle. However, the effect of the estrous cycle on synaptic functions is poorly understood. Here we show that in female rats, inhibitory-avoidance (IA) task (hippocampus-dependent contextual fear-learning task) drives GluA2-lacking Ca2+-permeable AMPA receptors (CP-AMPARs) into the hippocampal CA3-CA1 synapses during all periods of the estrous cycle except the proestrous period, when estrogen levels are high. In addition, IA task failed to drive CP-AMPARs into the CA3-CA1 synapses of ovariectomized rats only when estrogen was present. Thus, changes in the stoichiometry of AMPA receptors during learning depend on estrogen levels. Furthermore, the induction of long-term potentiation (LTP) after IA task was prevented during the proestrous period, while intact LTP is still expressed after IA task during other period of the estrous cycle. Consistent with this finding, rats conditioned by IA training failed to acquire hippocampus-dependent Y-maze task during the proestrous period. On the other hand, during other estrous period, rats were able to learn Y-maze task after IA conditioning. These results suggest that high estrogen levels prevent the IA learning-induced delivery of CP-AMPARs into hippocampal CA3-CA1 synapses and limit synaptic plasticity after IA task, thus preventing the acquisition of additional learning.

Nicotine inhibition of pulsatile GnRH secretion is mediated by GABAA receptor system in the cultured rat embryonic olfactory placode

In past work, we suggested that nicotine inhibition of in vivo pulsatile LH release is not mediated by opiate receptors known to be involved in the inhibition of LH release. In the present study, we examined whether nicotine inhibits the pulsatile gonadotropin-releasing hormone (GnRH) release, and whether this inhibition of GnRH release by nicotine is mediated by the GABA receptor system, by checking in vitro pulsatile GnRH release from cultured GnRH neurons obtained from olfactory placodes of rat embryos at E13.5. The mean interpulse interval of pulsatile GnRH release into the medium was 34.2+/-2.0 min in the control period and increased to 95.3+/-19.0 min (n=6) in the period of nicotine treatment at a concentration of 500 nM, showing an inhibitory effect of nicotine on pulsatile GnRH release. The GABA(A) receptor antagonist bicuculline used alone at a concentration of 20 microM caused no significant changes in the pulsatile GnRH release, but when used in combination with 500 nM of nicotine, bicuculline blocked the nicotine inhibition of GnRH release. In a separate experiment, nicotine treatment at a concentration of 500 nM significantly increased GABA release. These results suggest that, in the cultured embryonic olfactory placode, nicotine stimulates GABA release, which then inhibits GnRH release through GABA(A) receptor system.

Biological action of keratinocyte growth factor in BeWo cells, a human choriocarcinoma cell line

Previously, we reported that the expression of keratinocyte growth factor (KGF) is enhanced in secretory phase endometrial and decidual cells in early pregnancy as compared with the expression of KGF in proliferative phase endometrial cells, in humans. In order to clarify the role of KGF in embryo-endometrial interaction, we analyzed the in vitro effect of KGF on the human chorionic gonadotropin (hCG) secretion and on DNA synthesis in chorionic villi which are in close contact with the endometrium/decidua in the early stage of pregnancy. In this study, we used the BeWo cell line, a human choriocarcinoma cell line that possesses the biological features of secreting various placental hormones including hCG. Furthermore, we investigated the expression of KGF receptor (KGF-R) in these cells. KGF significantly stimulated hCG secretion in cultured BeWo cells but did not affect [3H]-thymidine incorporation. KGF-R mRNA was detected in BeWo cells by reverse transcriptase-polymerase chain reaction. These results suggest that the expression of KGF, which is induced in endometrial/decidual cells by progesterone, plays an important role in the embryo-endometrial/ decidual interaction by stimulating hCG secretion rather than affecting cell proliferation.

Gonadotropin-releasing hormone (GnRH) surge generator in female rats.

Publisher Summary In rats, gonadotropin-releasing hormone (GnRH) expressing neurons, which control luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretions from the anterior pituitary, are mainly located in the preoptic area (POA), but some GnRH-I neurons are also found in the mediobasal hypothalamus (MBH) in rats. One of most remarkable characteristics of GnRH neurons is that they do not originate from the hypothalamus but from the olfactory placode and migrate toward the hypothalamus during developmental stages. Another interesting characteristic is their secretion patterns. Secretion of LH, which is assumed to reflect GnRH secretion from the hypothalamus into the portal vein, shows two remarkably different patterns. The chapter has hypothesized two patterns of GnRH secretion that are controlled by different neural mechanisms: (1) the GnRH pulse generator, previously known as the tonic center, and (2) the GnRH surge generator, previously known as the phasic or the cyclic center. This chapter focuses on the putative GnRH surge generator of female rats.

Role of gamma-aminobutyric acid neurons in the release of gonadotropin-releasing hormone in cultured rat embryonic olfactory placodes.

We recently established a primary cell culture system of gonadotropin-releasing hormone (GnRH) neurons originating from olfactory placodes of rat embryos at E13.5 and showed that cultured olfactory placodes released GnRH into the medium in a pulsatile fashion with an interpulse interval of about 30 min. Since the reported presence of γ-aminobutyric acid (GABA) neurons in the culture of rat olfactory placode raises questions as to the role played by these GABA neurons in the GnRH pulse generation, we immunostained GnRH neurons and GABA neurons in this culture system to examine the interrelationship between both types of neurons, and determined the effects of GABA and the GABAA receptor antagonist, bicuculline, on GnRH release. The immunohistochemical study showed that GnRH neurons received fiber terminals from GABA neurons. GnRH neurons in culture released GnRH into the medium at intervals of 30–40 min, confirming our previous study. Treatment with 20 µM GABA prolonged the interpulse interval and decreased the amplitude of GnRH pulses. Bicuculline administered at 20 µM did not affect either parameter, but 50 µM bicuculline elevated the mean GnRH level, although it did not affect either the interpulse interval or the amplitude of GnRH pulses. In addition, 50 µM bicuculline increased the mean trough levels of GnRH pulses, although 20 µM bicuculline did not. In light of the in vivo studies performed previously, we suggest that the GnRH pulse generator, which probably consists of a small population of GnRH neurons in the culture, does not involve GABA neurons to generate the pulsatile GnRH release, although it may be responsive to the inhibitory transmitter GABA. We also found that there may be another population of GnRH neurons in the culture whose activity is strongly suppressed by the tonic inhibition of GABA neurons. Although it is speculative, these latter GnRH neurons may be responsible for the surge of GnRH release.

Gene expression of transforming growth factor-α in human endometrium during decidualization

AbstractPurpose: We previously reported the gene expression of epidermal growth factor (EGF) in the process of decidualization in the human endometrium. This study was undertaken to investigate the biological significance of transforming growth factor-α (TGF-α), which shares a significant sequence homology with EGF, in the regulation of decidualization. Methods: The gene encoding TGF-α was analyzed by Northern blot hybridization in nonpregnant human endometria and decidua from 6 to 8 weeks of gestation as well as in cultured stromal cells with or without medroxyprogesterone acetate. Results: No transcript was detected in proliferative and secretory endometrium, whereas a transcript of 4.8 kb, which was in agreement with the size of human prepro-TGF-α mRNA previously reported, was clearly detected in decidua. Transcript of 4.8 kb was also detected in vitroin medroxyprogesterone acetate (MPA)-induced decidual cells, while no TGF-α transcript was found in endometrial stromal cells cultured without MPA. Conclusions: These results suggest that the gene expression of TGF-α in uterine stromal cells is enhanced by stimulation from sex steroids and that TGF-α, like EGF, functions as one of the regulatory factors for decidualization in the human uterus.