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      Gamma‐aminobutyric acid A receptor agonist, muscimol, increases KiSS‐1 gene expression in hypothalamic cell models

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          Abstract

          Purpose

          Accumulating evidence indicates that hypothalamic kisspeptin plays a pivotal role in the regulation of the hypothalamic–pituitary–gonadal ( HPG) axis. In this study, the direct action of the gamma‐aminobutyric acid ( GABA) A receptor agonist on kisspeptin‐expressing neuronal cells was examined.

          Methods

          A hypothalamic cell model of rat hypothalamic cell line R8 ( rHypoE8) cells and primary cultures of neuronal cells from fetal rat brains were stimulated with a potent and selective GABA A receptor agonist, muscimol, to determine the expression of the Ki SS‐1 gene.

          Results

          Stimulation of the rHypoE8 cells with muscimol significantly increased the level of Ki SS‐1 messenger (m) RNA expression. The ability of muscimol to increase the level of Ki SS‐1 mRNA also was observed in the primary cultures of the neuronal cells from the fetal rat brains. The muscimol‐induced increase in Ki SS‐1 mRNA expression was completely inhibited in the presence of the GABA A receptor antagonist. Although muscimol increased the expression of Ki SS‐1, the natural compound, GABA, failed to induce the expression of Ki SS‐1 in the rHypoE8 cells. Muscimol did not modulate gonadotropin‐releasing hormone expression in either the rHypoE8 cells or the primary cultures of the fetal rat brains.

          Conclusions

          This study's observations suggest that the activation of the GABA A receptor modulates the HPG axis by increasing kisspeptin expression in the hypothalamic neurons.

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          Most cited references 31

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          Real-time PCR for mRNA quantitation

          Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range, boasts tremendous sensitivity, can be highly sequence-specific, has little to no post-amplification processing, and is amenable to increasing sample throughput. However, optimal benefit from these advantages requires a clear understanding of the many options available for running a real-time PCR experiment. Starting with the theory behind real-time PCR, this review discusses the key components of a real-time PCR experiment, including one-step or two-step PCR, absolute versus relative quantitation, mathematical models available for relative quantitation and amplification efficiency calculations, types of normalization or data correction, and detection chemistries. In addition, the many causes of variation as well as methods to calculate intra- and inter-assay variation are addressed.
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            Is there more to GABA than synaptic inhibition?

            In the mature brain, GABA (gamma-aminobutyric acid) functions primarily as an inhibitory neurotransmitter. But it can also act as a trophic factor during nervous system development to influence events such as proliferation, migration, differentiation, synapse maturation and cell death. GABA mediates these processes by the activation of traditional ionotropic and metabotropic receptors, and probably by both synaptic and non-synaptic mechanisms. However, the functional properties of GABA receptor signalling in the immature brain are significantly different from, and in some ways opposite to, those found in the adult brain. The unique features of the early-appearing GABA signalling systems might help to explain how GABA acts as a developmental signal.
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              GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain.

              GABA(A) receptors are ligand-operated chloride channels assembled from five subunits in a heteropentameric manner. Using immunocytochemistry, we investigated the distribution of GABA(A) receptor subunits deriving from 13 different genes (alpha1-alpha6, beta1-beta3, gamma1-gamma3 and delta) in the adult rat brain. Subunit alpha1-, beta1-, beta2-, beta3- and gamma2-immunoreactivities were found throughout the brain, although differences in their distribution were observed. Subunit alpha2-, alpha3-, alpha4-, alpha5-, alpha6-, gamma1- and delta-immunoreactivities were more confined to certain brain areas. Thus, alpha2-subunit-immunoreactivity was preferentially located in forebrain areas and the cerebellum. Subunit alpha6-immunoreactivity was only present in granule cells of the cerebellum and the cochlear nucleus, and subunit gamma1-immunoreactivity was preferentially located in the central and medial amygdaloid nuclei, in pallidal areas, the substantia nigra pars reticulata and the inferior olive. The alpha5-subunit-immunoreactivity was strongest in Ammon's horn, the olfactory bulb and hypothalamus. In contrast, alpha4-subunit-immunoreactivity was detected in the thalamus, dentate gyrus, olfactory tubercle and basal ganglia. Subunit alpha3-immunoreactivity was observed in the glomerular and external plexiform layers of the olfactory bulb, in the inner layers of the cerebral cortex, the reticular thalamic nucleus, the zonal and superficial layers of the superior colliculus, the amygdala and cranial nerve nuclei. Only faint subunit gamma3-immunoreactivity was detected in most areas; it was darkest in midbrain and pontine nuclei. Subunit delta-immunoreactivity was frequently co-distributed with alpha4 subunit-immunoreactivity, e.g. in the thalamus, striatum, outer layers of the cortex and dentate molecular layer. Striking examples of complementary distribution of certain subunit-immunoreactivities were observed. Thus, subunit alpha2-, alpha4-, beta1-, beta3- and delta-immunoreactivities were considerably more concentrated in the neostriatum than in the pallidum and entopeduncular nucleus. In contrast, labeling for the alpha1-, beta2-, gamma1- and gamma2-subunits prevailed in the pallidum compared to the striatum. With the exception of the reticular thalamic nucleus, which was prominently stained for subunits alpha3, beta1, beta3 and gamma2, most thalamic nuclei were rich in alpha1-, alpha4-, beta2- and delta-immunoreactivities. Whereas the dorsal lateral geniculate nucleus was strongly immunoreactive for subunits alpha4, beta2 and delta, the ventral lateral geniculate nucleus was predominantly labeled for subunits alpha2, alpha3, beta1, beta3 and gamma2; subunit alpha1- and alpha5-immunoreactivities were about equally distributed in both areas. In most hypothalamic areas, immunoreactivities for subunits alpha1, alpha2, beta1, beta2 and beta3 were observed. In the supraoptic nucleus, staining of conspicuous dendritic networks with subunit alpha1, alpha2, beta2, and gamma2 antibodies was contrasted by perykarya labeled for alpha5-, beta1- and delta-immunoreactivities. Among all brain regions, the median emminence was most heavily labeled for subunit beta2-immunoreactivity. In most pontine and cranial nerve nuclei and in the medulla, only subunit alpha1-, beta2- and gamma2-immunoreactivities were strong, whereas the inferior olive was significantly labeled only for subunits beta1, gamma1 and gamma2. In this study, a highly heterogeneous distribution of 13 different GABA(A) receptor subunit-immunoreactivities was observed. This distribution and the apparently typical patterns of co-distribution of these GABA(A) receptor subunits support the assumption of multiple, differently assembled GABA(A) receptor subtypes and their heterogeneous distribution within the adult rat brain.
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                Author and article information

                Contributors
                kanasaki@med.shimane-u.ac.jp
                Journal
                Reprod Med Biol
                Reprod. Med. Biol
                10.1111/(ISSN)1447-0578
                RMB2
                Reproductive Medicine and Biology
                John Wiley and Sons Inc. (Hoboken )
                1445-5781
                1447-0578
                04 October 2017
                October 2017
                : 16
                : 4 ( doiID: 10.1111/rmb2.2017.16.issue-4 )
                : 386-391
                Affiliations
                [ 1 ] Department of Obstetrics and Gynecology Shimane University School of Medicine Izumo Japan
                Author notes
                [* ] Correspondence

                Haruhiko Kanasaki, Department of Obstetrics and Gynecology, School of Medicine, Shimane University, Izumo, Japan.

                Email: kanasaki@ 123456med.shimane-u.ac.jp

                Article
                RMB212061
                10.1002/rmb2.12061
                5715903
                © 2017 The Authors. Reproductive Medicine and Biology published by John Wiley & Sons Australia, Ltd on behalf of Japan Society for Reproductive Medicine.

                This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

                Page count
                Figures: 3, Tables: 0, Pages: 6, Words: 4068
                Product
                Funding
                Funded by: Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan,
                Categories
                Original Article
                Original Articles
                Custom metadata
                2.0
                rmb212061
                October 2017
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.2.7 mode:remove_FC converted:04.12.2017

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