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Sample records for cells express gaba

  1. Guinea Pig Horizontal Cells Express GABA, the GABA-Synthesizing Enzyme GAD65, and the GABA Vesicular Transporter

    PubMed Central

    Guo, Chenying; Hirano, Arlene A.; Stella, Salvatore L.; Bitzer, Michaela; Brecha, Nicholas C.

    2013-01-01

    γ-Aminobutyric acid (GABA) is likely expressed in horizontal cells of all species, although conflicting physiological findings have led to considerable controversy regarding its role as a transmitter in the outer retina. This study has evaluated key components of the GABA system in the outer retina of guinea pig, an emerging retinal model system. The presence of GABA, its rate-limiting synthetic enzyme glutamic acid decarboxylase (GAD65 and GAD67 isoforms), the plasma membrane GABA transporters (GAT-1 and GAT-3), and the vesicular GABA transporter (VGAT) was evaluated by using immunohistochemistry with well-characterized antibodies. The presence of GAD65 mRNA was also evaluated by using laser capture microdissection and reverse transcriptase-polymerase chain reaction. Specific GABA, GAD65, and VGAT immunostaining was localized to horizontal cell bodies, as well as to their processes and tips in the outer plexiform layer. Furthermore, immunostaining of retinal whole mounts and acutely dissociated retinas showed GAD65 and VGAT immunoreactivity in both A-type and B-type horizontal cells. However, these cells did not contain GAD67, GAT-1, or GAT-3 immunoreactivity. GAD65 mRNA was detected in horizontal cells, and sequencing of the amplified GAD65 fragment showed approximately 85% identity with other mammalian GAD65 mRNAs. These studies demonstrate the presence of GABA, GAD65, and VGAT in horizontal cells of the guinea pig retina, and support the idea that GABA is synthesized from GAD65, taken up into synaptic vesicles by VGAT, and likely released by a vesicular mechanism from horizontal cells. PMID:20235161

  2. Acetylcholine induces GABA release onto rod bipolar cells through heteromeric nicotinic receptors expressed in A17 amacrine cells

    PubMed Central

    Elgueta, Claudio; Vielma, Alex H.; Palacios, Adrian G.; Schmachtenberg, Oliver

    2015-01-01

    Acetylcholine (ACh) is a major retinal neurotransmitter that modulates visual processing through a large repertoire of cholinergic receptors expressed on different retinal cell types. ACh is released from starburst amacrine cells (SACs) under scotopic conditions, but its effects on cells of the rod pathway have not been investigated. Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB) cells. GABA was released from A17 amacrine cells and activated postsynaptic GABAA and GABAC receptors in RB cells. The sensitivity of ACh-induced currents to nicotinic ACh receptor (nAChR) antagonists (TMPH ~ mecamylamine > erysodine > DhβE > MLA) together with the differential potency of specific agonists to mimic ACh responses (cytisine >> RJR2403 ~ choline), suggest that A17 cells express heteromeric nAChRs containing the β4 subunit. Activation of nAChRs induced GABA release after Ca2+ accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca2+ stores. Inhibition of acetylcholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells. Moreover, injection of neostigmine or cytisine reduced the b-wave of the scotopic flash electroretinogram (ERG), suggesting that cholinergic modulation of GABA release controls RB cell activity in vivo. These results describe a novel regulatory mechanism of RB cell inhibition and complement our understanding of the neuromodulatory control of retinal signal processing. PMID:25709566

  3. Etomidate, propofol and the neurosteroid THDOC increase the GABA efficacy of recombinant alpha4beta3delta and alpha4beta3 GABA A receptors expressed in HEK cells.

    PubMed

    Meera, Pratap; Olsen, Richard W; Otis, Thomas S; Wallner, Martin

    2009-01-01

    General anesthetics, once thought to exert their effects through non-specific membrane effects, have highly specific ion channel targets that can silence neuronal populations in the nervous system, thereby causing unconsciousness and immobility, characteristic of general anesthesia. Inhibitory GABA(A) receptors (GABA(A)Rs), particularly highly GABA-sensitive extrasynaptic receptor subtypes that give rise to sustained inhibitory currents, are uniquely sensitive to GABA(A)R-active anesthetics. A prominent population of extrasynaptic GABA(A)Rs is made up of alpha4, beta2 or beta3, and delta subunits. Considering the demonstrated importance of GABA receptor beta3 subunits for in vivo anesthetic effects of etomidate and propofol, we decided to investigate the effects of GABA anesthetics on "extrasynaptic" alpha4beta3delta and also binary alpha4beta3 receptors expressed in human embryonic kidney (HEK) cells. Consistent with previous work on similar receptor subtypes we show that maximal GABA currents through "extrasynaptic" alpha4beta3delta receptors, receptors defined by sensitivity to EtOH (30mM) and the beta-carboline beta-CCE (1microM), are enhanced by the GABA(A)R-active anesthetics etomidate, propofol, and the neurosteroid anesthetic THDOC. Furthermore, we show that receptors formed by alpha4beta3 subunits alone also show high GABA sensitivity and that saturating GABA responses of alpha4beta3 receptors are increased to the same extent by etomidate, propofol, and THDOC as are alpha4beta3delta receptors. Therefore, both alpha4beta3 and alpha4beta3delta receptors show low GABA efficacy, and GABA is also a partial agonist on certain binary alphabeta receptor subtypes. Increasing GABA efficacy on alpha4/6beta3delta and alpha4beta3 receptors is likely to make an important contribution to the anesthetic effects of etomidate, propofol and the neurosteroid THDOC.

  4. The effect of GABA stimulation on GABAA receptor subunit protein and mRNA expression in rat cultured cerebellar granule cells.

    PubMed Central

    Platt, K. P.; Zwartjes, R. E.; Bristow, D. R.

    1996-01-01

    1. After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM gamma-aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor alpha 1, alpha 6 and beta 2,3 subunit protein expression and alpha 1 and alpha 6 subunit steady-state mRNA levels, was examined using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. 2. GABA exposure for 2 days decreased alpha 1 (35 +/- 10%, mean +/- s.e.mean), beta 2,3 (21 +/- 9%) and alpha 6 (28 +/- 10%) subunit protein expression compared to control levels. The GABA-mediated reduction in alpha 1 subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 microM). 3. GABA exposure for 8 days increased alpha 1 (26 +/- 10%, mean +/- s.e.mean) and beta 2,3 (56 +/- 23%) subunit protein expression over control levels, whereas alpha 6 subunit protein expression remained below control levels (by 38 +/- 10%). However, after 10 days GABA exposure, alpha 6 subunit protein expression was also increased over control levels by 65 +/- 29% (mean +/- s.e.mean). 4. GABA exposure did not change the alpha 1 or alpha 6 subunit steady-state mRNA levels over and 8 day period, nor did it alter the expression of cyclophilin mRNA over 1-8 days. 5. These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi-phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve post-transcriptional and/or post-translational processes. Images Figure 1 Figure 3 Figure 4 PMID:8968548

  5. Subcellular localization and complements of GABA(A) and GABA(C) receptors on bullfrog retinal bipolar cells.

    PubMed

    Du, J L; Yang, X L

    2000-08-01

    gamma-Aminobutyric acid (GABA) receptors on retinal bipolar cells (BCs) are highly relevant to spatial and temporal integration of visual signals in the outer and inner retina. In the present work, subcellular localization and complements of GABA(A) and GABA(C) receptors on BCs were investigated by whole cell recordings and local drug application via multi-barreled puff pipettes in the bullfrog retinal slice preparation. Four types of the BCs (types 1-4) were identified morphologically by injection of Lucifer yellow. According to the ramification levels of the axon terminals and the responses of these cells to glutamate (or kainate) applied at their dendrites, types 1 and 2 of BCs were supposed to be OFF type, whereas types 3 and 4 of BCs might be ON type. Bicuculline (BIC), a GABA(A) receptor antagonist, and imidazole-4-acetic acid (I4AA), a GABA(C) receptor antagonist, were used to distinguish GABA receptor-mediated responses. In all BCs tested, not only the axon terminals but also the dendrites showed high GABA sensitivity mediated by both GABA(A) and GABA(C) receptors. Subcellular localization and complements of GABA(A) and GABA(C) receptors at the dendrites and axon terminals were highly related to the dichotomy of OFF and ON BCs. In the case of OFF BCs, GABA(A) receptors were rather evenly distributed at the dendrites and axon terminals, but GABA(C) receptors were predominantly expressed at the axon terminals. Moreover, the relative contribution of GABA(C) receptors to the axon terminals was prevalent over that of GABA(A) receptors, while the situation was reversed at the dendrites. In the case of ON BCs, GABA(A) and GABA(C) receptors both preferred to be expressed at the axon terminals; relative contributions of these two GABA receptor subtypes to both the sites were comparable, while GABA(C) receptors were much less expressed than GABA(A) receptors. GABA(A), but not GABA(C) receptors, were expressed clusteringly at axons of a population of BCs. In a

  6. Expression levels of the α4 subunit of the GABAA receptor in differentiated neuroblastoma cells are correlated with GABA-gated current

    PubMed Central

    Zhou, Xiangping; Smith, Sheryl S.

    2009-01-01

    The α4 subunit of the GABAA receptor (GABAR) is capable of rapid plasticity, increased by chronic exposure to positive GABA modulators, such as the neurosteroid 3α-OH-5α[β]-pregnan-20-one (THP). Here, we show that 48 h exposure of differentiated neuroblastoma cells (IMR-32) to 100 nM THP increases α4 expression, without changing the current density or the concentration-response curve. Increased expression of α4-containing GABAR was verified by a relative insensitivity of GABA (EC20)-gated current to modulation by the benzodiazepine (BZ) lorazepam (0.01 – 100 μM), and potentiation of current by flumazenil and RO15-4513, characteristic of α4βγ2 pharmacology. In contrast to THP, compounds which decrease GABA-gated current, such as the BZ inverse agonist DMCM, the GABAR antagonist gabazine and the open channel blocker penicillin, decreased α4 expression after a 48 h exposure, without changing BZ responsiveness. However, pentobarbital, another positive GABA modulator, increased α4 expression, while the BZ antagonist flumazenil had no effect. In order to test whether changes in current were responsible for increased α4 expression, decreases in the Cl− driving force were produced by chronic exposure to the NKCC1 blocker bumetanide (10 μM). When applied under these conditions of reduced GABA-gated current, THP failed to increase α4 expression. The results of this study suggest that α4 expression is correlated with changes in GABA-gated current, rather than simply through ligand-receptor interactions. These findings have relevance for GABAR subunit plasticity produced by fluctuations in endogenous steroids across the menstrual cycle, when altered BZ sensitivity is reported. PMID:19285093

  7. Effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine by their transporters heterologously expressed in COS cells and in rat brain synaptosomes.

    PubMed

    Sugimura, M; Kitayama, S; Morita, K; Irifune, M; Takarada, T; Kawahara, M; Dohi, T

    2001-08-01

    Although the neurotransmitter uptake system is considered a possible target for the presynaptic action of anesthetic agents, observations are inconsistent concerning effects on the transporter and their clinical relevance. The present study examined the effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine in COS cells heterologously expressing the transporters for these neurotransmitters and in the rat brain synaptosomes. Halothane and isoflurane, but not thiamylal or thiopental, significantly inhibited uptake by COS cell systems of GABA, dopamine and glutamic acid in a concentration-dependent manner within clinically relevant ranges for anesthesia induced by these agents. Similarly, in synaptosomes halothane and isoflurane but not thiopental significantly suppressed the uptake of GABA and glutamic acid, respectively. These results do not support the hypothesis that volatile and intravenous anesthetics exert their action via specific inhibition of GABA uptake to enhance inhibitory GABAergic neuronal activity. Rather, they suggest that presynaptic uptake systems for various neurotransmitters including GABA may be the molecular targets for volatile anesthetic agents.

  8. GABA Signaling and Neuroactive Steroids in Adrenal Medullary Chromaffin Cells

    PubMed Central

    Harada, Keita; Matsuoka, Hidetada; Fujihara, Hiroaki; Ueta, Yoichi; Yanagawa, Yuchio; Inoue, Masumi

    2016-01-01

    Gamma-aminobutyric acid (GABA) is produced not only in the brain, but also in endocrine cells by the two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67. In rat adrenal medullary chromaffin cells only GAD67 is expressed, and GABA is stored in large dense core vesicles (LDCVs), but not synaptic-like microvesicles (SLMVs). The α3β2/3γ2 complex represents the majority of GABAA receptors expressed in rat and guinea pig chromaffin cells, whereas PC12 cells, an immortalized rat chromaffin cell line, express the α1 subunit as well as the α3. The expression of α3, but not α1, in PC12 cells is enhanced by glucocorticoid activity, which may be mediated by both the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). GABA has two actions mediated by GABAA receptors in chromaffin cells: it induces catecholamine secretion by itself and produces an inhibition of synaptically evoked secretion by a shunt effect. Allopregnanolone, a neuroactive steroid which is secreted from the adrenal cortex, produces a marked facilitation of GABAA receptor channel activity. Since there are no GABAergic nerve fibers in the adrenal medulla, GABA may function as a para/autocrine factor in the chromaffin cells. This function of GABA may be facilitated by expression of the immature isoforms of GAD and GABAA receptors and the lack of expression of plasma membrane GABA transporters (GATs). In this review, we will consider how the para/autocrine function of GABA is achieved, focusing on the structural and molecular mechanisms for GABA signaling. PMID:27147972

  9. Characterization of the "sporadically lurking HAP1-immunoreactive (SLH) cells" in the hippocampus, with special reference to the expression of steroid receptors, GABA, and progenitor cell markers.

    PubMed

    Islam, Md N; Fujinaga, R; Yanai, A; Jahan, M R; Takeshita, Y; Kokubu, K; Shinoda, K

    2012-05-17

    Huntingtin-associated protein 1 (HAP1) is a neural huntingtin interactor that is widely expressed as a core molecule of the stigmoid body (a neurocytoplasmic inclusion) in the limbic and hypothalamic regions and has putative protective functions against some neurodegenerative diseases (HAP1 protection hypothesis). Although HAP1 has been reported to be intimately associated with several steroid receptors, HAP1-immunoreactive (HAP1-ir) cells remain to be identified in the hippocampus, which is one of the major steroidal targets. In this study, we determined the distribution of hippocampal HAP1-ir cells in light and fluorescence microscopy and characterized their morphological relationships with steroid receptors, markers of adult neurogenesis, and the GABAergic system in adult male and female Wistar rats. HAP1-ir cells, which were sporadically distributed particularly in the subgranular zone (SGZ) of the dentate gyrus and in the interface between the stratum lacunosum-moleculare and stratum radiatum of Ammon's horn, were identified as the "sporadically lurking HAP1-ir (SLH)" cells. The SLH cells showed no clear association with neural progenitor/proliferating or migrating cell markers of adult neurogenesis, such as Ki-67, proliferating cell nuclear antigen, doublecortin, and glial fibrillary acidic protein in the SGZ, whereas all the SLH cells expressed a neuronal specific nuclear protein (NeuN). More than 90% of the SLH cells expressed nuclear estrogen receptor (ER) α but neither ERβ nor the androgen receptor, whereas glucocorticoid receptor was differently stained in the SLH cells depending on the antibodies. More than 60% of them exhibited GABA immunoreactivity in the SGZ, suggestive of basket cells, but they were distinct from the ones expressing cholecystokinin or parvalbumin. We conclude that SLH cells, which should be stable against apoptosis due to putative HAP1 protectivity, might be involved in estrogen-dependent maturation, remodeling and activation of

  10. GABA promotes human β-cell proliferation and modulates glucose homeostasis.

    PubMed

    Purwana, Indri; Zheng, Juan; Li, Xiaoming; Deurloo, Marielle; Son, Dong Ok; Zhang, Zhaoyun; Liang, Christie; Shen, Eddie; Tadkase, Akshaya; Feng, Zhong-Ping; Li, Yiming; Hasilo, Craig; Paraskevas, Steven; Bortell, Rita; Greiner, Dale L; Atkinson, Mark; Prud'homme, Gerald J; Wang, Qinghua

    2014-12-01

    γ-Aminobutyric acid (GABA) exerts protective and regenerative effects on mouse islet β-cells. However, in humans it is unknown whether it can increase β-cell mass and improve glucose homeostasis. To address this question, we transplanted a suboptimal mass of human islets into immunodeficient NOD-scid-γ mice with streptozotocin-induced diabetes. GABA treatment increased grafted β-cell proliferation, while decreasing apoptosis, leading to enhanced β-cell mass. This was associated with increased circulating human insulin and reduced glucagon levels. Importantly, GABA administration lowered blood glucose levels and improved glucose excursion rates. We investigated GABA receptor expression and signaling mechanisms. In human islets, GABA activated a calcium-dependent signaling pathway through both GABA A receptor and GABA B receptor. This activated the phosphatidylinositol 3-kinase-Akt and CREB-IRS-2 signaling pathways that convey GABA signals responsible for β-cell proliferation and survival. Our findings suggest that GABA regulates human β-cell mass and may be beneficial for the treatment of diabetes or improvement of islet transplantation.

  11. GABA-shunt enzymes activity in GH3 cells with reduced level of PMCA2 or PMCA3 isoform

    SciTech Connect

    Kowalski, Antoni

    2011-08-12

    Highlights: {yields} Suppression of PMCA2 or PMCA3 slows down proliferation of GH3 cells. {yields} PMCA2 suppression lowers the activity of GABA-shunt enzymes. {yields} PMCA3 suppression increases the expression of glutamate decarboxylase 65. {yields} PMCA2 and PMCA3 function appears to be linked to regulation of GABA metabolism. -- Abstract: GABA ({gamma}-aminobutyric acid) is important neurotransmitter and regulator of endocrine functions. Its metabolism involves three enzymes: glutamate decarboxylase (GAD65 and GAD67), GABA aminotransferase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). As many cellular processes GABA turnover can depend on calcium homeostasis, which is maintained by plasma membrane calcium ATPases (PMCAs). In excitable cells PMCA2 and PMCA3 isoforms are particularly important. In this study we focused on GABA-metabolizing enzymes expression and activity in rat anterior pituitary GH3 cells with suppressed expression of PMCA2 or PMCA3. We observed that PMCA3-reduced cells have increased GAD65 expression. Suppression of PMCA2 caused a decrease in total GAD and GABA-T activity. These results indicate that PMCA2 and PMCA3 presence may be an important regulatory factor in GABA metabolism. Results suggest that PMCA2 and PMCA3 function is rather related to regulation of GABA synthesis and degradation than supplying cells with metabolites, which can be potentially energetic source.

  12. Comparative density of CCK- and PV-GABA cells within the cortex and hippocampus

    PubMed Central

    Whissell, Paul D.; Cajanding, Janine D.; Fogel, Nicole; Kim, Jun Chul

    2015-01-01

    Cholecystokinin (CCK)- and parvalbumin (PV)-expressing neurons constitute the two major populations of perisomatic GABAergic neurons in the cortex and the hippocampus. As CCK- and PV-GABA neurons differ in an array of morphological, biochemical and electrophysiological features, it has been proposed that they form distinct inhibitory ensembles which differentially contribute to network oscillations and behavior. However, the relationship and balance between CCK- and PV-GABA neurons in the inhibitory networks of the brain is currently unclear as the distribution of these cells has never been compared on a large scale. Here, we systemically investigated the distribution of CCK- and PV-GABA cells across a wide number of discrete forebrain regions using an intersectional genetic approach. Our analysis revealed several novel trends in the distribution of these cells. While PV-GABA cells were more abundant overall, CCK-GABA cells outnumbered PV-GABA cells in several subregions of the hippocampus, medial prefrontal cortex and ventrolateral temporal cortex. Interestingly, CCK-GABA cells were relatively more abundant in secondary/association areas of the cortex (V2, S2, M2, and AudD/AudV) than they were in corresponding primary areas (V1, S1, M1, and Aud1). The reverse trend was observed for PV-GABA cells. Our findings suggest that the balance between CCK- and PV-GABA cells in a given cortical region is related to the type of processing that area performs; inhibitory networks in the secondary cortex tend to favor the inclusion of CCK-GABA cells more than networks in the primary cortex. The intersectional genetic labeling approach employed in the current study expands upon the ability to study molecularly defined subsets of GABAergic neurons. This technique can be applied to the investigation of neuropathologies which involve disruptions to the GABAergic system, including schizophrenia, stress, maternal immune activation and autism. PMID:26441554

  13. Immature Responses to GABA in Fragile X Neurons Derived from Human Embryonic Stem Cells

    PubMed Central

    Telias, Michael; Segal, Menahem; Ben-Yosef, Dalit

    2016-01-01

    Fragile X Syndrome (FXS) is the most common form of inherited cognitive disability. However, functional deficiencies in FX neurons have been described so far almost exclusively in animal models. In a recent study we found several functional deficits in FX neurons differentiated in-vitro from human embryonic stem cells (hESCs), including their inability to fire repetitive action potentials, and their lack of synaptic activity. Here, we investigated the responses of such neurons to pulse application of the neurotransmitter GABA. We found two distinct types of responses to GABA and sensitivity to the GABA-A receptor antagonist bicuculline; type 1 (mature) characterized by non-desensitized responses to GABA as well as a high sensitivity to bicuculline, and type 2 (immature) which are desensitized to GABA and insensitive to bicuculline. Type 1 responses were age-dependent and dominant in mature WT neurons. In contrast, FX neurons expressed primarily type 2 phenotype. Expression analysis of GABA-A receptor subunits demonstrated that this bias in human FX neurons was associated with a significant alteration in the expression pattern of the GABA-A receptor subunits α2 and β2. Our results indicate that FMRP may play a role in the development of the GABAergic synapse during neurogenesis. This is the first demonstration of the lack of a mature response to GABA in human FX neurons and may explain the inappropriate synaptic functions in FXS. PMID:27242433

  14. Cloning of the. gamma. -aminobutyric acid (GABA). rho. sub 1 cDNA: A GABA receptor subunit highly expressed in the retina

    SciTech Connect

    Cutting, G.R.; Lu, Luo; Kasch, L.M.; Montrose-Rafizadeh, C.; Antonarakis, S.E.; Guggino, W.B.; Kazazian, H.H. Jr. ); O'Hara, B.F.; Donovan, D.M.; Shimada, Shoichi ); Uhl, G.R. Johns Hopkins Univ. School of Medicine, Baltimore, MD )

    1991-04-01

    Type A {gamma}-aminobutyric acid (GABA{sub A}) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. The authors have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence is 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABA{sub A} subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA {rho}{sub 1}, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family.

  15. A new role for GABA: inhibition of tumor cell migration.

    PubMed

    Ortega, Arturo

    2003-04-01

    GABA, the main inhibitory neurotransmitter in the vertebrate brain, participates outside the CNS in diverse functions such as platelet aggregation and the acrosomal reaction in spermatozoa. A recent study now demonstrates that GABA inhibits the migration of colon carcinoma cells, paving the way to the development of specific pharmacological agents that delay or inhibit invasion and metastasis of various cancer types.

  16. GABA and GAD expression in the X-organ sinus gland system of the Procambarus clarkii crayfish: inhibition mediated by GABA between X-organ neurons.

    PubMed

    Pérez-Polanco, Paola; Garduño, Julieta; Cebada, Jorge; Zarco, Natanael; Segovia, José; Lamas, Mónica; García, Ubaldo

    2011-09-01

    In crustaceans, the X-organ-sinus gland (XO-SG) neurosecretory system is formed of distinct populations of neurons that produce two families of neuropeptides: crustacean hyperglycemic hormone and adipokinetic hormone/red pigment-concentrating hormone. On the basis of electrophysiological evidence, it has been proposed that γ-aminobutyric acid (GABA) regulates both electrical and secretory activity of the XO-SG system. In this work we observed that depolarizing current pulses to neurons located in the external rim of the X-organ induced repetitive firing that suppressed the spontaneous firing of previously active X-organ neurons. Picrotoxin reversibly blocked this inhibitory effect suggesting that the GABA released from the stimulated neuron inhibited neighboring cells. Immunoperoxidase in X-organ serial sections showed co-localization of GABA and glutamic acid decarboxylase (GAD) including the aforementioned neurons. Immunofluorescence in whole mount preparations showed that two subpopulations of crustacean hyperglycemic hormone-containing neurons colocalized with GABA. The expression of GAD mRNA was determined in crayfish tissue and X-organ single cells by RT-PCR. Bioinformatics analysis shows, within the amplified region, 90.4% consensus and 41.9% identity at the amino acid level compared with Drosophila melanogaster and Caenorhabditis elegans. We suggest that crustacean hyperglycemic hormone-GABA-containing neurons can regulate the excitability of other X-organ neurons that produce different neurohormones. PMID:21626307

  17. Cell and Receptor Type-Specific Alterations in Markers of GABA Neurotransmission in the Prefrontal Cortex of Subjects with Schizophrenia

    PubMed Central

    Lewis, David A.; Hashimoto, Takanori; Morris, Harvey M.

    2010-01-01

    Impairments in cognitive control, such as those involved in working memory, are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC) in individuals with schizophrenia. This dysfunction appears to result, at least in part, from abnormalities in GABA-mediated neurotransmission. In this paper, we review recent findings indicating that the altered DLPFC circuitry in subjects with schizophrenia reflects changes in the expression of genes that encode selective presynaptic and postsynaptic components of GABA neurotransmission. Specifically, using a combination of methods, we found that subjects with schizophrenia exhibited expression deficits in GABA-related transcripts encoding presynaptic regulators of GABA neurotransmission, neuropeptide markers of specific subpopulations of GABA neurons, and certain subunits of the GABAA receptor. In particular, alterations in the expression of the neuropeptide somatostatin suggested that GABA neurotransmission is impaired in the Martinotti subset of GABA neurons that target the dendrites of pyramidal cells. In contrast, none of the GABA-related transcripts assessed to date were altered in the DLPFC of monkeys chronically exposed to antipsychotic medications, suggesting that the effects observed in the human studies reflect the disease process and not its treatment. In concert with previous findings, these data suggest that working memory dysfunction in schizophrenia may be attributable to altered GABA neurotransmission in specific DLPFC microcircuits. PMID:19073429

  18. Stable expression of the vesicular GABA transporter following photothrombotic infarct in rat brain.

    PubMed

    Frahm, C; Siegel, G; Grass, S; Witte, O W

    2006-07-01

    Before exocytotic release of the inhibitory neurotransmitter GABA, this amino acid has to be stored in synaptic vesicles. Accumulation of GABA in vesicles is achieved by a specific membrane-integrated transporter termed vesicular GABA transporter. This vesicular protein is mainly located at presynaptic terminals of GABAergic interneurons. In the present study we investigated the effects of focal ischemia on the expression of the vesicular GABA transporter. Vesicular GABA transporter mRNA and protein expression was examined after photothrombosis in different cortical and hippocampal brain regions of Wistar rats. In situ hybridization and quantitative real-time RT-PCR were performed to analyze vesicular GABA transporter mRNA. Both vesicular GABA transporter mRNA-stained perikarya and mRNA expression levels remained unaffected. Vesicular GABA transporter protein-containing synaptic terminals and somata were visualized by immunohistochemistry. The pattern of vesicular GABA transporter immunoreactivity as well as the protein expression level revealed by semiquantitative image analysis and by Western blot remained stable after stroke. The steady expression of vesicular GABA transporter mRNA and protein after photothrombosis indicates that the exocytotic release mechanism of GABA is not affected by ischemia.

  19. Localization of a GABA transporter to glial cells in the developing and adult olfactory pathway of the moth Manduca sexta1

    PubMed Central

    Oland, Lynne A; Gibson, Nicholas J; Tolbert, Leslie P

    2010-01-01

    Glial cells have several critical roles in the developing and adult olfactory (antennal) lobe of the moth Manduca sexta. Early in development, glial cells occupy discrete regions of the developing olfactory pathway and processes of GABAergic neurons extend into some of these regions. Because GABA is known to have developmental effects in a variety of systems, we explored the possibility that the glial cells express a GABA transporter that could regulate GABA levels to which olfactory neurons and glial cells are exposed. Using an antibody raised against a characterized high-affinity M. sexta GABA transporter with high sequence homology to known mammalian GABA transporters (Mbungu et al., 1995; Umesh and Gill, 2002), we found that the GABA transporter is localized to subsets of centrally derived glial cells during metamorphic adult development. The transporter persists into adulthood in a subset of the neuropil-associated glial cells, but its distribution pattern as determined by light- and electron-microscopic-level immunocytochemistry indicates that it could not serve to regulate GABA concentration in the synaptic cleft. Rather its role is more likely to regulate extracellular GABA levels within the glomerular neuropil. Expression in the sorting zone glial cells disappears after the period of olfactory receptor axon ingrowth, but may be important during ingrowth if GABA regulates axon growth. Glial cells take up GABA, and that uptake can be blocked by DABA. This is the first molecular evidence that the central glial cell population in this pathway is heterogeneous. PMID:20058309

  20. Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii.

    PubMed

    Jiménez-Vázquez, Eric N; Díaz-Velásquez, Clara E; Uribe, R M; Arias, Juan M; García, Ubaldo

    2016-02-01

    Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero- oligomeric receptors. Extensive research on the molecular structure of the γ-aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo-oligomeric GABA-activated Cl(-) channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo-oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA-activated Cl(-) channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo-oligomeric complex. To investigate the structure-function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo-oligomeric ionotropic GABAR that gates a bicuculline-insensitive Cl(-) current. The order of potency of the agonists was GABA > trans-4-amino-crotonic acid = cis-4-aminocrotonic acid > muscimol. These data support the notion that X-organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero-oligomeric and homo-oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X-organ neurons from crayfish eyestalk express the isolated pcGABAA β subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates.

  1. Cloning and expression of a rat brain GABA transporter

    SciTech Connect

    Guastella, J.; Czyzyk, L.; Davidson, N.; Lester, H.A. ); Nelson, N.; Nelson, H.; Miedel, M.C. ); Keynan, S.; Kanner, B.I. )

    1990-09-14

    A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter {gamma}-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated ({sup 3}H)GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium- and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.

  2. Actions of picrotoxinin analogues on an expressed, homo-oligomeric GABA receptor of Drosophila melanogaster.

    PubMed

    Shirai, Y; Hosie, A M; Buckingham, S D; Holyoke, C W; Baylis, H A; Sattelle, D B

    1995-04-01

    The actions of picrotoxinin and four of its analogues were tested on a Drosophila melanogaster homo-oligomeric GABA (gamma-aminobutyric acid) receptor formed when RDL (resistance to dieldrin) subunits were expressed in Xenopus oocytes. In agreement with previously reported studies on native insect GABA receptors and native expressed vertebrate GABA receptors, acetylation of the bridgehead hydroxyl group (picrotoxinin acetate) greatly reduced the activity of the molecule, but surprisingly, substitution with flourine at the same position also reduced the activity. Conversion of the terminal isopropenyl group to an acetyl (alpha-picrotoxinone) or hydration of the double bond (picrotin) also reduced activity, in agreement with findings for native insect and mammalian receptors. The present results suggest that interactions of convulsants with homo-oligomeric and multimeric GABA receptors are qualitatively similar. Thus, the RDL homo-oligomer exhibits a pharmacological profile for picrotoxinin analogues resembling that of native GABA receptors. PMID:7603613

  3. Expression of the γ-Aminobutyric Acid (GABA) Plasma Membrane Transporter-1 in Monkey and Human Retina

    PubMed Central

    Casini, Giovanni; Rickman, Dennis W.; Brecha, Nicholas C.

    2010-01-01

    Purpose To determine the expression pattern of the predominant γ-aminobutyric acid (GABA) plasma membrane transporter GAT-1 in Old World monkey (Macaca mulatta) and human retina. Methods GAT-1 was localized in retinal sections by using immunohistochemical techniques with fluorescence and confocal microscopy. Double-labeling studies were performed with the GAT-1 antibody using antibodies to GABA, vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH), and the bipolar cell marker Mab115A10. Results The pattern of GAT-1 immunostaining was similar in human and monkey retinas. Numerous small immunoreactive somata were in the inner nuclear layer (INL) and were present rarely in the inner plexiform layer (IPL) of all retinal regions. Medium GAT-1 somata were in the ganglion cell layer in the parafoveal and peripheral retinal regions. GAT-1 fibers were densely distributed throughout the IPL. Varicose processes, originating from both the IPL and somata in the INL, arborized in the outer plexiform layer (OPL), forming a sparse network in all retinal regions, except the fovea. Sparsely occurring GAT-1 processes were in the nerve fiber layer in parafoveal regions and near the optic nerve head but not in the optic nerve. In the INL, 99% of the GAT-1 somata contained GABA, and 66% of the GABA immunoreactive somata expressed GAT-1. GAT-1 immunoreactivity was in all VIP-containing cells, but it was absent in TH-immunoreactive amacrine cells and in Mab115A10 immunoreactive bipolar cells. Conclusions GAT-1 in primate retinas is expressed by amacrine and displaced amacrine cells. The predominant expression of GAT-1 in the inner retina is consistent with the idea that GABA transporters influence neurotransmission and thus participate in visual information processing in the retina. PMID:16565409

  4. Enhanced behavioral sensitivity to the competitive GABA agonist, gaboxadol, in transgenic mice over-expressing hippocampal extrasynaptic alpha6beta GABA(A) receptors.

    PubMed

    Saarelainen, Kati S; Ranna, Martin; Rabe, Holger; Sinkkonen, Saku T; Möykkynen, Tommi; Uusi-Oukari, Mikko; Linden, Anni-Maija; Lüddens, Hartmut; Korpi, Esa R

    2008-04-01

    The behavioral and functional significance of the extrasynaptic inhibitory GABA(A) receptors in the brain is still poorly known. We used a transgenic mouse line expressing the GABA(A) receptor alpha6 subunit gene in the forebrain under the Thy-1.2 promoter (Thy1alpha6) mice ectopically expressing alpha6 subunits especially in the hippocampus to study how extrasynaptically enriched alphabeta(gamma2)-type receptors alter animal behavior and receptor responses. In these mice extrasynaptic alpha6beta receptors make up about 10% of the hippocampal GABA(A) receptors resulting in imbalance between synaptic and extrasynaptic inhibition. The synthetic GABA-site competitive agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; 3 mg/kg) induced remarkable anxiolytic-like response in the light : dark exploration and elevated plus-maze tests in Thy1alpha6 mice, while being almost inactive in wild-type mice. The transgenic mice also lost quicker and for longer time their righting reflex after 25 mg/kg gaboxadol than wild-type mice. In hippocampal sections of Thy1alpha6 mice, the alpha6beta receptors could be visualized autoradiographically by interactions between gaboxadol and GABA via [(35)S]TBPS binding to the GABA(A) receptor ionophore. Gaboxadol inhibition of the binding could be partially prevented by GABA. Electrophysiology of recombinant GABA(A) receptors revealed that GABA was a partial agonist at alpha6beta3 and alpha6beta3delta receptors, but a full agonist at alpha6beta3gamma2 receptors when compared with gaboxadol. The results suggest strong behavioral effects via selective pharmacological activation of enriched extrasynaptic alphabeta GABA(A) receptors, and the mouse model represents an example of the functional consequences of altered balance between extrasynaptic and synaptic inhibition.

  5. Acupuncture improves locomotor function by enhancing GABA receptor expression in transient focal cerebral ischemia rats.

    PubMed

    Xu, Qian; Yang, Jing-Wen; Cao, Yan; Zhang, Li-Wen; Zeng, Xiang-Hong; Li, Fang; Du, Si-Qi; Wang, Lin-Peng; Liu, Cun-Zhi

    2015-02-19

    Stroke is the major cause of long-term disability among adults. Recent studies have found that GABAergic inhibitory neurotransmission plays a vital role in ameliorate locomotor damage after ischemic injury. Acupuncture has been widely used to improve locomotor function. However, the underlying mechanisms remain unclear. The present study is designed to investigate whether GABA and GABA receptors are involved in the mechanism underlying acupuncture treatment in rats with middle cerebral artery occlusion (MCAO). One week after acupuncture at JiaJi acupoint, the locomotor function and infarct volumes were tested. Then level of GABA and the expressions of GABAAγ2 and GABABR2 were assessed by high-performance liquid chromatography, immunofluorescence and immunohistochemistry, respectively. Compared with normal group, GABAAγ2 and GABABR2 expressions were decreased in striatum and spinal cord of the MCAO group. After acupuncture, the expressions of the two receptors were increased, but levels of GABA and trafficking protein, kinesin binding 1 (TRAK1), which plays a role in the intracellular transport of GABA receptors, were unchanged. The present study suggests that acupuncture could reverse locomotor function by modulating the expressions of GABA receptors in MCAO rats. PMID:25556683

  6. Connections between EM2-containing terminals and GABA/μ-opioid receptor co-expressing neurons in the rat spinal trigeminal caudal nucleus.

    PubMed

    Li, Meng-Ying; Wu, Zhen-Yu; Lu, Ya-Cheng; Yin, Jun-Bin; Wang, Jian; Zhang, Ting; Dong, Yu-Lin; Wang, Feng

    2014-01-01

    Endomorphin-2 (EM2) demonstrates a potent antinociceptive effect via the μ-opioid receptor (MOR). To provide morphological evidence for the pain control effect of EM2, the synaptic connections between EM2-immunoreactive (IR) axonal terminals and γ-amino butyric acid (GABA)/MOR co-expressing neurons in lamina II of the spinal trigeminal caudal nucleus (Vc) were investigated in the rat. Dense EM2-, MOR- and GABA-IR fibers and terminals were mainly observed in lamina II of the Vc. Within lamina II, GABA- and MOR-neuronal cell bodies were also encountered. The results of immunofluorescent histochemical triple-staining showed that approximately 14.2 or 18.9% of GABA-IR or MOR-IR neurons also showed MOR- or GABA-immunopositive staining in lamina II; approximately 45.2 and 36.1% of the GABA-IR and MOR-IR neurons, respectively, expressed FOS protein in their nuclei induced by injecting formalin into the left lower lip of the mouth. Most of the GABA/MOR, GABA/FOS, and MOR/FOS double-labeled neurons made close contacts with EM2-IR fibers and terminals. Immuno-electron microscopy confirmed that the EM2-IR terminals formed synapses with GABA-IR or MOR-IR dendritic processes and neuronal cell bodies in lamina II of the Vc. These results suggest that EM2 might participate in pain transmission and modulation by binding to MOR-IR and GABAergic inhibitory interneuron in lamina II of the Vc to exert inhibitory effect on the excitatory interneuron in lamina II and projection neurons in laminae I and III.

  7. GABA A receptor π subunit promotes apoptosis of HTR-8/SVneo trophoblastic cells: Implications in preeclampsia

    PubMed Central

    LU, JUNJIE; ZHANG, QIAN; TAN, DONGMEI; LUO, WENPING; ZHAO, HAI; MA, JING; LIANG, HAO; TAN, YI

    2016-01-01

    Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter through its receptors in the mature central nervous system. The GABA type A receptor π subunit (GABRP) has been identified in the tissues of the reproductive system, particularly in the uterus. In addition, we have previously detected GABRP expression in both human and mouse placentas. To examine the role of GABRP in trophoblastic cell invasion, we constructed a pIRES2-GABRP-EGFP plasmid which was used for the transfection of a human placental cell line derived from first trimester extravillous trophoblasts (HTR-8/SVneo). The number of invaded cells was decreased by GABRP overexpression. Notably, the decrease in the invasive cell number may be due to the increased apoptosis of the HTR-8/SVneo cells following GABRP transfection, which was further confirmed by flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Based on the increased apoptosis of trophoblastic cells in pregnancies complicated by preeclampsia (PE) and the fact that GABRP promotes the apoptosis of trophoblastic cells, we hypothesized that GABRP expression is increased in the placental tissues from patients with PE compared with that in the normal groups and this hypothesis was confirmed by RT-qPCR and immunohistochemical analysis. Taken together, these findings imply that GABRP plays an important role in placentation and this pathway may be a promising molecular target for the development of novel therapeutic strategies for PE. PMID:27221053

  8. Co-release of glutamate and GABA from single vesicles in GABAergic neurons exogenously expressing VGLUT3

    PubMed Central

    Zimmermann, Johannes; Herman, Melissa A.; Rosenmund, Christian

    2015-01-01

    The identity of the vesicle neurotransmitter transporter expressed by a neuron largely corresponds with the primary neurotransmitter that cell releases. However, the vesicular glutamate transporter subtype 3 (VGLUT3) is mainly expressed in non-glutamatergic neurons, including cholinergic, serotonergic, or GABAergic neurons. Though a functional role for glutamate release from these non-glutamatergic neurons has been demonstrated, the interplay between VGLUT3 and the neuron’s characteristic neurotransmitter transporter, particularly in the case of GABAergic neurons, at the synaptic and vesicular level is less clear. In this study, we explore how exogenous expression of VGLUT3 in striatal GABAergic neurons affects the packaging and release of glutamate and GABA in synaptic vesicles (SVs). We found that VGLUT3 expression in isolated, autaptic GABAergic neurons leads to action potential evoked release of glutamate. Under these conditions, glutamate and GABA could be packaged together in single vesicles release either spontaneously or asynchronously. However, the presence of glutamate in GABAergic vesicles did not affect uptake of GABA itself, suggesting a lack of synergy in vesicle filling for these transmitters. Finally, we found postsynaptic detection of glutamate released from GABAergic terminals difficult when bona fide glutamatergic synapses were present, suggesting that co-released glutamate cannot induce postsynaptic glutamate receptor clustering. PMID:26441632

  9. Ventral tegmental area dopamine and GABA neurons: Physiological properties and expression of mRNA for endocannabinoid biosynthetic elements

    PubMed Central

    Merrill, Collin B.; Friend, Lindsey N.; Newton, Scott T.; Hopkins, Zachary H.; Edwards, Jeffrey G.

    2015-01-01

    The ventral tegmental area (VTA) is involved in adaptive reward and motivation processing and is composed of dopamine (DA) and GABA neurons. Defining the elements regulating activity and synaptic plasticity of these cells is critical to understanding mechanisms of reward and addiction. While endocannabinoids (eCBs) that potentially contribute to addiction are known to be involved in synaptic plasticity mechanisms in the VTA, where they are produced is poorly understood. In this study, DA and GABAergic cells were identified using electrophysiology, cellular markers, and a transgenic mouse model that specifically labels GABA cells. Using single-cell RT-qPCR and immunohistochemistry, we investigated mRNA and proteins involved in eCB signaling such as diacylglycerol lipase α, N-acyl-phosphatidylethanolamine-specific phospholipase D, and 12-lipoxygenase, as well as type I metabotropic glutamate receptors (mGluRs). Our results demonstrate the first molecular evidence of colocalization of eCB biosynthetic enzyme and type I mGluR mRNA in VTA neurons. Further, these data reveal higher expression of mGluR1 in DA neurons, suggesting potential differences in eCB synthesis between DA and GABA neurons. These data collectively suggest that VTA GABAergic and DAergic cells have the potential to produce various eCBs implicated in altering neuronal activity or plasticity in adaptive motivational reward or addiction. PMID:26553597

  10. SSRI augmentation of antipsychotic alters expression of GABA(A) receptor and related genes in PMC of schizophrenia patients.

    PubMed

    Silver, Henry; Susser, Ehud; Danovich, Lena; Bilker, Warren; Youdim, Moussa; Goldin, Vladimir; Weinreb, Orly

    2011-06-01

    Clinical studies have shown that negative symptoms of schizophrenia unresponsive to antipsychotic given alone can improve after augmentation with SSRI antidepressant. Laboratory investigations into the mechanism of this synergism showed that co-administration of SSRI and antipsychotic produces changes in GABA(A) receptor and related systems, which differ from the effects of each drug alone. To examine the clinical relevance of these findings, the current study examined the effects of SSRI augmentation treatment on GABA(A) receptor and related systems in schizophrenia patients. Schizophrenia patients with high levels of negative symptoms unresponsive to antipsychotic treatment received add-on fluvoxamine (100 mg/d). Blood was taken before and 1, 3 and 6 wk after adding fluvoxamine and peripheral mononuclear cells (PMC) isolated. RNA encoding for GABA(A)β3, 5-HT2A, and 5-HT7 receptors, PKCβ2, and brain-derived neurotrophic factor (BDNF) was assayed with real-time RT-PCR. Plasma BDNF protein was assayed using ELISA. Clinical symptoms were assessed with validated rating scales. We found significant increase in mRNA encoding for GABA(A)β3 and 5-HT2A, 5-HT7 receptors and BDNF and a reduction in PKCβ2 mRNA. Plasma BDNF protein concentrations were increased. There were significant correlations among the genes. Clinical symptoms improved significantly. mRNA expression of PKCβ2, 5-HT2A and 5-HT7 showed significant associations with clinical symptoms. Combined SSRI+antipsychotic treatment is associated with changes in GABA(A) receptor and in related signalling systems in patients. These changes may be part of the mechanism of clinically effective drug action and may prove to be biomarkers of pharmacological response.

  11. Modulation of GABA receptors expressed in Xenopus oocytes by 13-L-hydroxylinoleic acid and food additives.

    PubMed

    Aoshima, H; Tenpaku, Y

    1997-12-01

    To study the effects of 13-L-hydroxylinoleic acid (LOH) and food additives on gamma-aminobutyric acid (GABA) receptors, ionotropic GABA receptors were expressed in Xenopus oocytes by injecting mRNAs prepared from rat whole brain. LOH, which was prepared by reduction of 13-L-hydroperoxylinoleic acid (LOOH), inhibited the response of GABA receptors in the presence of high concentrations of GABA. LOH also inhibited nicotinic acetylcholine, glycine, and kainate receptors, while it had little effect on NMDA receptors expressed in Xenopus oocytes. However, LOH potentiated the response of GABA receptors as well as LOOH in the presence of low concentrations of GABA, possibly increasing the affinity of GABA for the receptors, while linoleic acid did not. Since some modification of the compounds seemed to change their effects on GABA receptors, the responses of GABA receptors elicited by 10 microM GABA were measured in the presence of compounds with various kinds of functional groups or the structural isomers of pentanol. Potentiation of GABA receptors depended strongly on the species of functional groups and also depended on the structure of the isomers. Then effects of various kinds of food additives on GABA receptors were also examined; perfumes such as alcohols or esters potentiated the responses strongly, while hexylamine, nicotinamide, or caffeine inhibited the responses, mainly in a competitive manner, and vanillin inhibited the responses noncompetitively. These results suggest the possibility that production of LOOH and LOH, or intake of much of some food additives, modulates the neural transmission in the brain, especially through ionotropic GABA receptors and changes the frame of the human mind, as alcohol or tobacco does.

  12. Depolarizing effect of GABA in horizontal cells of the rabbit retina.

    PubMed

    Varela, Carolina; Rivera, Luis; Blanco, Román; De la Villa, Pedro

    2005-11-01

    Gamma-amino butyric acid (GABA) has been characterized as an inhibitory neurotransmitter acting through chloride mediated channels in the adult nervous system. Using gramicidin-perforated patch clamp recordings from horizontal cells dissociated from the retinas of adult rabbits, we found that GABA is able to induce cell depolarization. Ionic currents induced by GABA in dissociated horizontal cells showed a reversal potential close to -30 mV. This value is more positive than the resting potential of these cells (ca. -70 mV). Therefore, according to the Nernst equation, the intracellular chloride concentration in horizontal cells was estimated to be of 44 mM. The depolarizing effect of GABA at the dendrites of horizontal cells may serve to shape the center-surround organization of the receptive fields in retinal cells, thereby securing the shape discrimination of visual input.

  13. Constitutive GABA expression via a recombinant adeno-associated virus consistently attenuates neuropathic pain.

    PubMed

    Lee, Boyoung; Kim, Jaehyung; Kim, Sung Jin; Lee, Heuiran; Chang, Jin Woo

    2007-06-15

    Peripheral neuropathic pain is a common clinical problem with few existing treatments. Previously, we constructed rAAV bearing GAD65 and demonstrated that GAD65 and GABA can be constitutively produced in the CNS. To investigate the beneficial effects of GAD65 produced by rAAV and resulting GABA release in peripheral neuropathic pain, we established a neuropathic pain rat model. The direct administration of rAAV-GAD65 to dorsal root ganglion induced constitutive GAD65 expression, which was readily detected by immunohistochemistry. Both allodynic and hyperalgeic behavior tests suggested that neuropathic pain was noticeably reduced, along with the transgenic GAD65 expression. Moreover, the magnitude of pain relief was maintained during the entire experimental period. Concomitantly, the significant enhancement in GABA release following transgenic GAD65 expression was identified in vivo. Taken all together, these results provide evidence that persistent GAD65 and subsequent GABA expression in DRGs via rAAV effectively attenuates peripheral neuropathic pain for long period of time.

  14. Genetic differences in the ethanol sensitivity of GABA sub A receptors expressed in Xenopus oocytes

    SciTech Connect

    Wafford, K.A.; Burnett, D.M.; Dunwiddie, T.V.; Harris, R.A. )

    1990-07-20

    Animal lines selected for differences in drug sensitivity can be used to help determine the molecular basis of drug action. Long-sleep (LS) and short-sleep (SS) mice differ markedly in their genetic sensitivity to ethanol. To investigate the molecular basis for this difference, mRNA from brains of LS and SS mice was expressed in Xenopus oocytes and the ethanol sensitivity of gamma-aminobutyric acid A (GABA{sub A})- and N-methyl D-aspartate (NMDA) - activated ion channels was tested. Ethanol facilitated GABA responses in oocytes injected with mRNA from LS mice but antagonized responses in oocytes injected with mRNA from SS animals. Ethanol inhibited NMDA responses equally in the two lines. Thus, genes coding for the GABA{sub A} receptor or associated proteins may be critical determinants of individual differences in ethanol sensitivity.

  15. Conserved Regional Patterns of GABA-Related Transcript Expression in the Neocortex of Subjects With Schizophrenia

    PubMed Central

    Hashimoto, Takanori; Bazmi, H. Holly; Mirnics, Karoly; Wu, Qiang; Sampson, Allan R.; Lewis, David A.

    2010-01-01

    Objective Individuals with schizophrenia exhibit disturbances in a number of cognitive, affective, sensory, and motor functions that depend on the circuitry of different cortical areas. The cognitive deficits associated with dysfunction of the dorsolateral prefrontal cortex result, at least in part, from abnormalities in GABA neurotransmission, as reflected in a specific pattern of altered expression of GABA-related genes. Consequently, the authors sought to determine whether this pattern of altered gene expression is restricted to the dorsolateral prefrontal cortex or could also contribute to the dysfunction of other cortical areas in subjects with schizophrenia. Method Real-time quantitative polymerase chain reaction was used to assess the levels of eight GABA-related transcripts in four cortical areas (dorsolateral prefrontal cortex, anterior cingulate cortex, and primary motor and primary visual cortices) of subjects (N=12) with schizophrenia and matched normal comparison subjects. Results Expression levels of seven transcripts were lower in subjects with schizophrenia, with the magnitude of reduction for each transcript comparable across the four areas. The largest reductions were detected for mRNA encoding somatostatin and parvalbumin, followed by moderate decreases in mRNA expression for the 67-kilodalton isoform of glutamic acid decarboxylase, the GABA membrane transporter GAT-1, and the α1 and δ subunits of GABAA receptors. In contrast, the expression of calretinin mRNA did not differ between the subject groups in any of the four areas. Conclusions Because the areas examined represent the major functional domains (e.g., association, limbic, motor, and sensory) of the cerebral cortex, our findings suggest that a conserved set of molecular alterations affecting GABA neurotransmission contribute to the pathophysiology of different clinical features of schizophrenia. PMID:18281411

  16. Overexpression of Glutamate Decarboxylase in Mesenchymal Stem Cells Enhances Their Immunosuppressive Properties and Increases GABA and Nitric Oxide Levels

    PubMed Central

    González, Marisol; Vilches, Rodrigo; Rojas, Pablo; Vásquez, Manuel; Kurte, Mónica; Vega-Letter, Ana María; Carrión, Flavio; Figueroa, Fernando; Rojas, Patricio; Irarrázabal, Carlos

    2016-01-01

    The neurotransmitter GABA has been recently identified as a potent immunosuppressive agent that targets both innate and adaptive immune systems and prevents disease progression of several autoimmunity models. Mesenchymal stem cells (MSCs) are self-renewing progenitor cells that differentiate into various cell types under specific conditions, including neurons. In addition, MSC possess strong immunosuppressive capabilities. Upon cytokine priming, undifferentiated MSC suppress T-cell proliferation via cell-to-cell contact mechanisms and the secretion of soluble factors like nitric oxide, prostaglandin E2 and IDO. Although MSC and MSC-derived neuron-like cells express some GABAergic markers in vitro, the role for GABAergic signaling in MSC-mediated immunosuppression remains completely unexplored. Here, we demonstrate that pro-inflammatory cytokines selectively regulate GAD-67 expression in murine bone marrow-MSC. However, expression of GAD-65 is required for maximal GABA release by MSC. Gain of function experiments using GAD-67 and GAD-65 co-expression demonstrates that GAD increases immunosuppressive function in the absence of pro-inflammatory licensing. Moreover, GAD expression in MSC evokes an increase in both GABA and NO levels in the supernatants of co-cultured MSC with activated splenocytes. Notably, the increase in NO levels by GAD expression was not observed in cultures of isolated MSC expressing GAD, suggesting crosstalk between these two pathways in the setting of immunosuppression. These results indicate that GAD expression increases MSC-mediated immunosuppression via secretion of immunosuppressive agents. Our findings may help reconsider GABAergic activation in MSC for immunological disorders. PMID:27662193

  17. Role of glutamic acid decarboxylase 67 in regulating cortical parvalbumin and GABA membrane transporter 1 expression: Implications for schizophrenia

    PubMed Central

    Curley, Allison A.; Eggan, Stephen M.; Lazarus, Matt S.; Huang, Z. Josh; Volk, David W.; Lewis, David A.

    2012-01-01

    Markers of GABA neurotransmission are altered in multiple regions of the neocortex in individuals with schizophrenia. Lower levels of glutamic acid decarboxylase 67 (GAD67) mRNA and protein, which is responsible for most cortical GABA synthesis, are accompanied by lower levels of GABA membrane transporter 1 (GAT1) mRNA. These alterations are thought to be most prominent in the parvalbumin (PV)-containing subclass of interneurons, which also contain lower levels of PV mRNA. Since GAT1 and PV each reduce the availability of GABA at postsynaptic receptors, lower levels of GAT1 and PV mRNAs have been hypothesized to represent compensatory responses to an upstream reduction in cortical GABA synthesis in schizophrenia. However, such cause-and-effect hypotheses cannot be directly tested in a human illness. Consequently, we used two mouse models with reduced GAD67 expression specifically in PV neurons (PVGAD67+/−) or in all interneurons (GABAGAD67+/−) and quantified GAD67, GAT1 and PV mRNA levels using methods identical to those employed in studies of schizophrenia. Cortical levels of PV or GAT1 mRNAs were not altered in PVGAD67+/− mice during postnatal development or in adulthood. Furthermore, cellular analyses confirmed the predicted reduction in GAD67 mRNA, but failed to show a deficit in PV mRNA in these animals. Levels of PV and GAT1 mRNAs were also unaltered in GABAGAD67+/− mice. Thus, mouse lines with cortical reductions in GAD67 mRNA that match or exceed those present in schizophrenia, and that differ in the developmental timing and cell typespecificity of the GAD67 deficit, failed to provide proof-of-concept evidence that lower PV and GAT1 expression in schizophrenia are a consequence of lower GAD67 expression. Together, these findings suggest that the correlated decrements in cortical GAD67, PV and GAT1 mRNAs in schizophrenia may be a common consequence of some other upstream factor. PMID:23103418

  18. Effect of low doses of progesterone in the expression of the GABA(A) receptor α4 subunit and procaspase-3 in the hypothalamus of female rats.

    PubMed

    Arbo, Bruno D; Andrade, Susie; Osterkamp, Gabriela; Gomez, Rosane; Ribeiro, Maria Flávia M

    2014-08-01

    Progesterone is a steroid which regulates neural function, thereby modulating neurotransmission, cell survival, and behavior. Previous studies by our group have shown that chronic administration of low doses of progesterone in diestrus II female rats has an antidepressant-like effect in the forced swimming test (FST). Depression is associated with the several neurotransmitters systems, including GABA and serotonin, and with neurodegeneration and cell death in some brain circuits. The aim of this study was to verify the effect of progesterone on the protein expression of the GABA(A) receptor α4 subunit, serotonin transporter (SERT), Akt, extracellular signal-regulated kinase (Erk), and caspase-3 in the hypothalamus of diestrus II female rats exposed to the FST. Female rats were treated with a daily injection of progesterone (0.4 mg/kg) or vehicle, during two complete oestrous cycles. On the day of the experiment, the animals were euthanized 30 min after the FST, the hypothalamus was dissected and protein expression of GABA(A) receptor α4 subunit, SERT, Akt, Erk, and caspase-3 was evaluated. Progesterone increased the expression of GABA(A) receptor α4 subunit but did not change the expression of SERT. Progesterone decreased the expression of procaspase-3 in the hypothalamus without changing the activation of Akt and Erk in this structure. In summary, our results suggest that progesterone acts to increase the expression of the GABA(A) receptor α4 subunit and decrease the expression of procaspase-3 in the hypothalamus of female rats. Such effects may be involved in the antidepressant-like effect of progesterone in female rats exposed to the FST.

  19. Targeted Deletion of Vesicular GABA Transporter from Retinal Horizontal Cells Eliminates Feedback Modulation of Photoreceptor Calcium Channels123

    PubMed Central

    Liu, Xue; Boulter, Jim; Grove, James; Pérez de Sevilla Müller, Luis; Barnes, Steven; Brecha, Nicholas C.

    2016-01-01

    Abstract The cellular mechanisms underlying feedback signaling from horizontal cells to photoreceptors, which are important for the formation of receptive field surrounds of early visual neurons, remain unsettled. Mammalian horizontal cells express a complement of synaptic proteins that are necessary and sufficient for calcium-dependent exocytosis of inhibitory neurotransmitters at their contacts with photoreceptor terminals, suggesting that they are capable of releasing GABA via vesicular release. To test whether horizontal cell vesicular release is involved in feedback signaling, we perturbed inhibitory neurotransmission in these cells by targeted deletion of the vesicular GABA transporter (VGAT), the protein responsible for the uptake of inhibitory transmitter by synaptic vesicles. To manipulate horizontal cells selectively, an iCre mouse line with Cre recombinase expression controlled by connexin57 (Cx57) regulatory elements was generated. In Cx57-iCre mouse retina, only horizontal cells expressed Cre protein, and its expression occurred in all retinal regions. After crossing with a VGATflox/flox mouse line, VGAT was selectively eliminated from horizontal cells, which was confirmed immunohistochemically. Voltage-gated ion channel currents in horizontal cells of Cx57-VGAT−/− mice were the same as Cx57-VGAT+/+ controls, as were the cell responses to the ionotropic glutamate receptor agonist kainate, but the response to the GABAA receptor agonist muscimol in Cx57-VGAT−/− mice was larger. In contrast, the feedback inhibition of photoreceptor calcium channels, which in control animals is induced by horizontal cell depolarization, was completely absent in Cx57-VGAT−/− mice. The results suggest that vesicular release of GABA from horizontal cells is required for feedback inhibition of photoreceptors. PMID:27022629

  20. Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones

    PubMed Central

    James, Owain T; Livesey, Matthew R; Qiu, Jing; Dando, Owen; Bilican, Bilada; Haghi, Ghazal; Rajan, Rinku; Burr, Karen; Hardingham, Giles E; Chandran, Siddharthan; Kind, Peter C; Wyllie, David J A

    2014-01-01

    We have assessed, using whole-cell patch-clamp recording and RNA-sequencing (RNA-seq), the properties and composition of GABAA receptors (GABAARs) and strychnine-sensitive glycine receptors (GlyRs) expressed by excitatory cortical neurons derived from human embryonic stem cells (hECNs). The agonists GABA and muscimol gave EC50 values of 278 μm and 182 μm, respectively, and the presence of a GABAAR population displaying low agonist potencies is supported by strong RNA-seq signals for α2 and α3 subunits. GABAAR-mediated currents, evoked by EC50 concentrations of GABA, were blocked by bicuculline and picrotoxin with IC50 values of 2.7 and 5.1 μm, respectively. hECN GABAARs are predominantly γ subunit-containing as assessed by the sensitivity of GABA-evoked currents to diazepam and insensitivity to Zn2+, together with the weak direct agonist action of gaboxadol; RNA-seq indicated a predominant expression of the γ2 subunit. Potentiation of GABA-evoked currents by propofol and etomidate and the lack of inhibition of currents by salicylidine salycylhydrazide (SCS) indicate expression of the β2 or β3 subunit, with RNA-seq analysis indicating strong expression of β3 in hECN GABAARs. Taken together our data support the notion that hECN GABAARs have an α2/3β3γ2 subunit composition – a composition that also predominates in immature rodent cortex. GlyRs expressed by hECNs were activated by glycine with an EC50 of 167 μm. Glycine-evoked (500 μm) currents were blocked by strychnine (IC50 = 630 nm) and picrotoxin (IC50 = 197 μm), where the latter is suggestive of a population of heteromeric receptors. RNA-seq indicates GlyRs are likely to be composed of α2 and β subunits. PMID:25172951

  1. Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones.

    PubMed

    James, Owain T; Livesey, Matthew R; Qiu, Jing; Dando, Owen; Bilican, Bilada; Haghi, Ghazal; Rajan, Rinku; Burr, Karen; Hardingham, Giles E; Chandran, Siddharthan; Kind, Peter C; Wyllie, David J A

    2014-10-01

    We have assessed, using whole-cell patch-clamp recording and RNA-sequencing (RNA-seq), the properties and composition of GABAA receptors (GABAARs) and strychnine-sensitive glycine receptors (GlyRs) expressed by excitatory cortical neurons derived from human embryonic stem cells (hECNs). The agonists GABA and muscimol gave EC50 values of 278 μm and 182 μm, respectively, and the presence of a GABAAR population displaying low agonist potencies is supported by strong RNA-seq signals for α2 and α3 subunits. GABAAR-mediated currents, evoked by EC50 concentrations of GABA, were blocked by bicuculline and picrotoxin with IC50 values of 2.7 and 5.1 μm, respectively. hECN GABAARs are predominantly γ subunit-containing as assessed by the sensitivity of GABA-evoked currents to diazepam and insensitivity to Zn(2+), together with the weak direct agonist action of gaboxadol; RNA-seq indicated a predominant expression of the γ2 subunit. Potentiation of GABA-evoked currents by propofol and etomidate and the lack of inhibition of currents by salicylidine salycylhydrazide (SCS) indicate expression of the β2 or β3 subunit, with RNA-seq analysis indicating strong expression of β3 in hECN GABAARs. Taken together our data support the notion that hECN GABAARs have an α2/3β3γ2 subunit composition - a composition that also predominates in immature rodent cortex. GlyRs expressed by hECNs were activated by glycine with an EC50 of 167 μm. Glycine-evoked (500 μm) currents were blocked by strychnine (IC50 = 630 nm) and picrotoxin (IC50 = 197 μm), where the latter is suggestive of a population of heteromeric receptors. RNA-seq indicates GlyRs are likely to be composed of α2 and β subunits.

  2. Structure, function, and plasticity of GABA transporters

    PubMed Central

    Scimemi, Annalisa

    2014-01-01

    GABA transporters belong to a large family of neurotransmitter:sodium symporters. They are widely expressed throughout the brain, with different levels of expression in different brain regions. GABA transporters are present in neurons and in astrocytes and their activity is crucial to regulate the extracellular concentration of GABA under basal conditions and during ongoing synaptic events. Numerous efforts have been devoted to determine the structural and functional properties of GABA transporters. There is also evidence that the expression of GABA transporters on the cell membrane and their lateral mobility can be modulated by different intracellular signaling cascades. The strength of individual synaptic contacts and the activity of entire neuronal networks may be finely tuned by altering the density, distribution and diffusion rate of GABA transporters within the cell membrane. These findings are intriguing because they suggest the existence of complex regulatory systems that control the plasticity of GABAergic transmission in the brain. Here we review the current knowledge on the structural and functional properties of GABA transporters and highlight the molecular mechanisms that alter the expression and mobility of GABA transporters at central synapses. PMID:24987330

  3. Activation of the Tonic GABAC Receptor Current in Retinal Bipolar Cell Terminals by Nonvesicular GABA Release

    PubMed Central

    Jones, S. M.; Palmer, M. J.

    2009-01-01

    Within the second synaptic layer of the retina, bipolar cell (BC) output to ganglion cells is regulated by inhibitory input to BC axon terminals. GABAA receptors (GABAARs) mediate rapid synaptic currents in BC terminals, whereas GABAC receptors (GABACRs) mediate slow evoked currents and a tonic current, which is strongly regulated by GAT-1 GABA transporters. We have used voltage-clamp recordings from BC terminals in goldfish retinal slices to determine the source of GABA for activation of these currents. Inhibition of vesicular release with concanamycin A or tetanus toxin significantly inhibited GABAAR inhibitory postsynaptic currents and glutamate-evoked GABAAR and GABACR currents but did not reduce the tonic GABACR current, which was also not dependent on extracellular Ca2+. The tonic current was strongly potentiated by inhibition of GABA transaminase, under both normal and Ca2+-free conditions, and was activated by exogenous taurine; however inhibition of taurine transport had little effect. The tonic current was unaffected by GAT-2/3 inhibition and was potentiated by GAT-1 inhibition even in the absence of vesicular release, indicating that it is unlikely to be evoked by reversal of GABA transporters or by ambient GABA. In addition, GABA release does not appear to occur via hemichannels or P2X7 receptors. BC terminals therefore exhibit two forms of GABACR-mediated inhibition, activated by vesicular and by nonvesicular GABA release, which are likely to have distinct functions in visual signal processing. The tonic GABACR current in BC terminals exhibits similar properties to tonic GABAAR and glutamate receptor currents in the brain. PMID:19494193

  4. Allosteric uncoupling and up-regulation of benzodiazepine and GABA recognition sites following chronic diazepam treatment of HEK 293 cells stably transfected with alpha1beta2gamma2S subunits of GABA (A) receptors.

    PubMed

    Pericić, Danka; Strac, Dubravka Svob; Jembrek, Maja Jazvinsćak; Vlainić, Josipa

    2007-05-01

    Benzodiazepines are drugs known to produce tolerance and dependence and also to be abused and co-abused. The aim of this study was to further explore the mechanisms that underlie adaptive changes in GABA(A) receptors following prolonged exposure to these drugs. Human embryonic kidney (HEK 293) cells stably expressing recombinant alpha1beta2gamma2s GABA(A) receptors were exposed for 72 h to a high concentration of diazepam (50 microM) in the absence or presence of other drugs. Radioligand binding studies were used to determine the parameters of [(3)H]flunitrazepam and [(3)H]muscimol binding sites and allosteric interactions between these sites. Prolonged treatment with diazepam increased the maximum number (B (max)) of [(3)H]flunitrazepam and [(3)H]muscimol binding sites in the membranes, and of [(3)H]muscimol binding sites on the surface of HEK 293 cells. There was no change in the affinity (K (d)) of binding sites. The diazepam-induced increase in the B (max) value of [(3)H]flunitrazepam binding sites was reduced by two GABA(A) receptor antagonists, gabazine (1 and 10 microM) and picrotoxin (100 microM). In addition, it was reduced by cycloheximide (5 microg/ml), a protein synthesis inhibitor, and actinomycin D (7.5 microg/ml), an RNA synthesis inhibitor. Flumazenil (5 microM), the antagonist of benzodiazepine binding sites, also up-regulated [(3)H]flunitrazepam recognition sites. Simultaneous treatment with diazepam and flumazenil failed to produce an additive up-regulation. GABA (1 nM - 1 mM)-induced potentiation of [(3)H]flunitrazepam binding to membranes obtained from diazepam (50 microM)-pretreated cells was markedly reduced, suggesting functional uncoupling between GABA and benzodiazepine binding sites. The results suggest that diazepam up-regulated benzodiazepine binding sites on stably expressed GABA(A) receptors by stimulating their synthesis at both the transcriptional and translational levels. A comparable increase of [(3)H]muscimol binding sites

  5. Gamma aminobutyric acid B and 5-hydroxy tryptamine 2A receptors functional regulation during enhanced liver cell proliferation by GABA and 5-HT chitosan nanoparticles treatment.

    PubMed

    Shilpa, Joy; Pretty, Mary Abraham; Anitha, Malat; Paulose, Cheramadathikudyil Skaria

    2013-09-01

    Liver is one of the major organs in vertebrates and hepatocytes are damaged by many factors. The liver cell maintenance and multiplication after injury and treatment gained immense interest. The present study investigated the role of Gamma aminobutyric acid (GABA) and serotonin or 5-hydroxytryptamine (5-HT) coupled with chitosan nanoparticles in the functional regulation of Gamma aminobutyric acid B and 5-hydroxy tryptamine 2A receptors mediated cell signaling mechanisms, extend of DNA methylation and superoxide dismutase activity during enhanced liver cell proliferation. Liver injury was achieved by partial hepatectomy of male Wistar rats and the GABA and 5-HT chitosan nanoparticles treatments were given intraperitoneally. The experimental groups were sham operated control (C), partially hepatectomised rats with no treatment (PHNT), partially hepatectomised rats with GABA chitosan nanoparticle (GCNP), 5-HT chitosan nanoparticle (SCNP) and a combination of GABA and 5-HT chitosan nanoparticle (GSCNP) treatments. In GABA and 5-HT chitosan nanoparticle treated group there was a significant decrease (P<0.001) in the receptor expression of Gamma aminobutyric acid B and a significant increase (P<0.001) in the receptor expression of 5-hydroxy tryptamine 2A when compared to PHNT. The cyclic adenosine monophosphate content and its regulatory protein, presence of methylated DNA and superoxide dismutase activity were decreased in GCNP, SCNP and GSCNP when compared to PHNT. The Gamma aminobutyric acid B and 5-hydroxy tryptamine 2A receptors coupled signaling elements played an important role in GABA and 5-HT chitosan nanoparticles induced liver cell proliferation which has therapeutic significance in liver disease management.

  6. Toward dissecting the etiology of schizophrenia: HDAC1 and DAXX regulate GAD67 expression in an in vitro hippocampal GABA neuron model

    PubMed Central

    Subburaju, S; Coleman, A J; Ruzicka, W B; Benes, F M

    2016-01-01

    Schizophrenia (SZ) is associated with GABA neuron dysfunction in the hippocampus, particularly the stratum oriens of sector CA3/2. A gene expression profile analysis of human postmortem hippocampal tissue followed by a network association analysis had shown a number of genes differentially regulated in SZ, including the epigenetic factors HDAC1 and DAXX. To characterize the contribution of these factors to the developmental perturbation hypothesized to underlie SZ, lentiviral vectors carrying short hairpin RNA interference (shRNAi) for HDAC1 and DAXX were used. In the hippocampal GABA neuron culture model, HiB5, transduction with HDAC1 shRNAi showed a 40% inhibition of HDAC1 mRNA and a 60% inhibition of HDAC1 protein. GAD67, a enzyme associated with GABA synthesis, was increased twofold (mRNA); the protein showed a 35% increase. The expression of DAXX, a co-repressor of HDAC1, was not influenced by HDAC1 inhibition. Transduction of HiB5 cells with DAXX shRNAi resulted in a 30% inhibition of DAXX mRNA that translated into a 90% inhibition of DAXX protein. GAD1 mRNA was upregulated fourfold, while its protein increased by ~30%. HDAC1 expression was not altered by inhibition of DAXX. However, a physical interaction between HDAC1 and DAXX was demonstrated by co-immunoprecipitation. Inhibition of HDAC1 or DAXX increased expression of egr-1, transcription factor that had previously been shown to regulate the GAD67 promoter. Our in vitro results point to a key role of both HDAC1 and DAXX in the regulation of GAD67 in GABAergic HiB5 cells, strongly suggesting that these epigenetic/transcription factors contribute to mechanisms underlying GABA cell dysfunction in SZ. PMID:26812044

  7. Toward dissecting the etiology of schizophrenia: HDAC1 and DAXX regulate GAD67 expression in an in vitro hippocampal GABA neuron model.

    PubMed

    Subburaju, S; Coleman, A J; Ruzicka, W B; Benes, F M

    2016-01-26

    Schizophrenia (SZ) is associated with GABA neuron dysfunction in the hippocampus, particularly the stratum oriens of sector CA3/2. A gene expression profile analysis of human postmortem hippocampal tissue followed by a network association analysis had shown a number of genes differentially regulated in SZ, including the epigenetic factors HDAC1 and DAXX. To characterize the contribution of these factors to the developmental perturbation hypothesized to underlie SZ, lentiviral vectors carrying short hairpin RNA interference (shRNAi) for HDAC1 and DAXX were used. In the hippocampal GABA neuron culture model, HiB5, transduction with HDAC1 shRNAi showed a 40% inhibition of HDAC1 mRNA and a 60% inhibition of HDAC1 protein. GAD67, a enzyme associated with GABA synthesis, was increased twofold (mRNA); the protein showed a 35% increase. The expression of DAXX, a co-repressor of HDAC1, was not influenced by HDAC1 inhibition. Transduction of HiB5 cells with DAXX shRNAi resulted in a 30% inhibition of DAXX mRNA that translated into a 90% inhibition of DAXX protein. GAD1 mRNA was upregulated fourfold, while its protein increased by ~30%. HDAC1 expression was not altered by inhibition of DAXX. However, a physical interaction between HDAC1 and DAXX was demonstrated by co-immunoprecipitation. Inhibition of HDAC1 or DAXX increased expression of egr-1, transcription factor that had previously been shown to regulate the GAD67 promoter. Our in vitro results point to a key role of both HDAC1 and DAXX in the regulation of GAD67 in GABAergic HiB5 cells, strongly suggesting that these epigenetic/transcription factors contribute to mechanisms underlying GABA cell dysfunction in SZ.

  8. Toward dissecting the etiology of schizophrenia: HDAC1 and DAXX regulate GAD67 expression in an in vitro hippocampal GABA neuron model.

    PubMed

    Subburaju, S; Coleman, A J; Ruzicka, W B; Benes, F M

    2016-01-01

    Schizophrenia (SZ) is associated with GABA neuron dysfunction in the hippocampus, particularly the stratum oriens of sector CA3/2. A gene expression profile analysis of human postmortem hippocampal tissue followed by a network association analysis had shown a number of genes differentially regulated in SZ, including the epigenetic factors HDAC1 and DAXX. To characterize the contribution of these factors to the developmental perturbation hypothesized to underlie SZ, lentiviral vectors carrying short hairpin RNA interference (shRNAi) for HDAC1 and DAXX were used. In the hippocampal GABA neuron culture model, HiB5, transduction with HDAC1 shRNAi showed a 40% inhibition of HDAC1 mRNA and a 60% inhibition of HDAC1 protein. GAD67, a enzyme associated with GABA synthesis, was increased twofold (mRNA); the protein showed a 35% increase. The expression of DAXX, a co-repressor of HDAC1, was not influenced by HDAC1 inhibition. Transduction of HiB5 cells with DAXX shRNAi resulted in a 30% inhibition of DAXX mRNA that translated into a 90% inhibition of DAXX protein. GAD1 mRNA was upregulated fourfold, while its protein increased by ~30%. HDAC1 expression was not altered by inhibition of DAXX. However, a physical interaction between HDAC1 and DAXX was demonstrated by co-immunoprecipitation. Inhibition of HDAC1 or DAXX increased expression of egr-1, transcription factor that had previously been shown to regulate the GAD67 promoter. Our in vitro results point to a key role of both HDAC1 and DAXX in the regulation of GAD67 in GABAergic HiB5 cells, strongly suggesting that these epigenetic/transcription factors contribute to mechanisms underlying GABA cell dysfunction in SZ. PMID:26812044

  9. Expression of spinal cord GABA transporter 1 in morphine-tolerant male Wistar rats.

    PubMed

    Shokoofeh, Siroosi; Homa, Manaheji; Leila, Dargahi; Samira, Daniali

    2015-11-15

    Chronic morphine exposure produces morphine tolerance. One of the mechanisms of morphine tolerance involves γ-aminobutric acid (GABA), whose level is regulated by GABA transporter 1 (GAT-1). The aim of this study was to investigate the expression of GAT-1 in the spinal cord during morphine treatment. Morphine was administrated to rats via drinking water for 21 days. On day 21, a single dose of morphine (10mg/kg) was injected, followed by the administration of 5% formalin after 30 min. Expression of GAT-1 in the lumbar spinal cord during morphine treatment was analyzed by Western blotting and immunohistochemistry assay. In another set of experiments, a morphine-tolerant group was treated with a GAT-1 inhibitor, ethyl nipecotate (60 mg/kg), 5 min prior to the formalin test. To assess a possible analgesic effect of the GAT-1 inhibitor, a non-tolerant group was injected only with ethyl nipecotate 5 min prior to the formalin test. Our results indicated that a chronic consumption of morphine led to morphine tolerance. Morphine tolerance was also concomitant with GAT-1 up-regulation in the lumbar spinal cord. The GAT-1 inhibitor ethyl nipecotate improved the antinociceptive effect of morphine in the morphine-tolerant group. Ethyl nipecotate also had an antinociceptive effect on the non-tolerant group. Thus, our data suggest that GAT-1 overexpression in the spinal cord plays an important role in morphine tolerance.

  10. Neuronal gamma-aminobutyric acid (GABA) type A receptors undergo cognate ligand chaperoning in the endoplasmic reticulum by endogenous GABA

    PubMed Central

    Wang, Ping; Eshaq, Randa S.; Meshul, Charles K.; Moore, Cynthia; Hood, Rebecca L.; Leidenheimer, Nancy J.

    2015-01-01

    GABAA receptors mediate fast inhibitory neurotransmission in the brain. Dysfunction of these receptors is associated with various psychiatric/neurological disorders and drugs targeting this receptor are widely used therapeutic agents. Both the efficacy and plasticity of GABAA receptor-mediated neurotransmission depends on the number of surface GABAA receptors. An understudied aspect of receptor cell surface expression is the post-translational regulation of receptor biogenesis within the endoplasmic reticulum (ER). We have previously shown that exogenous GABA can act as a ligand chaperone of recombinant GABAA receptors in the early secretory pathway leading us to now investigate whether endogenous GABA facilitates the biogenesis of GABAA receptors in primary cerebral cortical cultures. In immunofluorescence labeling experiments, we have determined that neurons expressing surface GABAA receptors contain both GABA and its degradative enzyme GABA transaminase (GABA-T). Treatment of neurons with GABA-T inhibitors, a treatment known to increase intracellular GABA levels, decreases the interaction of the receptor with the ER quality control protein calnexin, concomittantly increasing receptor forward-trafficking and plasma membrane insertion. The effect of GABA-T inhibition on the receptor/calnexin interaction is not due to the activation of surface GABAA or GABAB receptors. Consistent with our hypothesis that GABA acts as a cognate ligand chaperone in the ER, immunogold-labeling of rodent brain slices reveals the presence of GABA within the rough ER. The density of this labeling is similar to that present in mitochondria, the organelle in which GABA is degraded. Lastly, the effect of GABA-T inhibition on the receptor/calnexin interaction was prevented by pretreatment with a GABA transporter inhibitor. Together, these data indicate that endogenous GABA acts in the rough ER as a cognate ligand chaperone to facilitate the biogenesis of neuronal GABAA receptors. PMID

  11. Heterogeneity in expression of functional ionotropic glutamate and GABA receptors in astrocytes across brain regions: insights from the thalamus

    PubMed Central

    Höft, Simon; Griemsmann, Stephanie; Seifert, Gerald; Steinhäuser, Christian

    2014-01-01

    Astrocytes may express ionotropic glutamate and gamma-aminobutyric acid (GABA) receptors, which allow them to sense and to respond to neuronal activity. However, so far the properties of astrocytes have been studied only in a few brain regions. Here, we provide the first detailed receptor analysis of astrocytes in the murine ventrobasal thalamus and compare the properties with those in other regions. To improve voltage-clamp control and avoid indirect effects during drug applications, freshly isolated astrocytes were employed. Two sub-populations of astrocytes were found, expressing or lacking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. AMPA receptor-bearing astrocytes displayed a lower Kir current density than cells lacking the receptors. In contrast, all cells expressed GABAA receptors. Single-cell RT-PCR was employed to identify the receptor subunits in thalamic astrocytes. Our findings add to the emerging evidence of functional heterogeneity of astrocytes, the impact of which still remains to be defined. PMID:25225096

  12. The central nervous system convulsant pentylenetetrazole stimulates gamma-aminobutyric acid (GABA)-activated current in picrotoxin-resistant GABA(A) receptors in HEK293 cells.

    PubMed

    Dibas, M I; Dillon, G H

    2000-05-19

    We tested the ability of the central nervous system convulsant pentylenetetrazole (PTZ) to inhibit gamma-aminobutyric acid (GABA)-gated current in receptors expressing a mutation that rendered them resistant to picrotoxin. Consistent with previous reports, receptors expressing beta2(T246F), along with alpha3 and gamma2 subunits, resulted in a greatly diminished sensitivity to picrotoxin. Sensitivity to PTZ was completely abolished in the mutant receptor, confirming the hypothesis that PTZ acts at the picrotoxin site. Quite unexpected, however, was our finding that PTZ elicited marked stimulation (up to 400% of control) in the mutated receptors. This stimulatory effect was not mediated via an interaction with the benzodiazepine site, as preincubation with the benzodiazepine antagonist flumazenil did not block the PTZ-induced stimulation. Our results reveal the existence of a novel stimulatory domain of PTZ in GABA(A) receptors.

  13. EXP-1 is an excitatory GABA-gated cation channel.

    PubMed

    Beg, Asim A; Jorgensen, Erik M

    2003-11-01

    Gamma-aminobutyric acid (GABA) mediates fast inhibitory neurotransmission by activating anion-selective ligand-gated ion channels. Although electrophysiological studies indicate that GABA may activate cation-selective ligand-gated ion channels in some cell types, such a channel has never been characterized at the molecular level. Here we show that GABA mediates enteric muscle contraction in the nematode Caenorhabditis elegans via the EXP-1 receptor, a cation-selective ligand-gated ion channel. The EXP-1 protein resembles ionotropic GABA receptor subunits in almost all domains. In the pore-forming domain of EXP-1, however, the residues that confer anion selectivity are exchanged for those that specify cation selectivity. When expressed in Xenopus laevis oocytes, EXP-1 forms a GABA receptor that is permeable to cations and not anions. We conclude that some of the excitatory functions assigned to GABA are mediated by cation channels rather than by anion channels.

  14. Stress metabolism in green coffee beans (Coffea arabica L.): expression of dehydrins and accumulation of GABA during drying.

    PubMed

    Kramer, Daniela; Breitenstein, Björn; Kleinwächter, Maik; Selmar, Dirk

    2010-04-01

    In order to produce tradeable standard green coffee, processed beans must be dried. The drying procedure affects the abundance of relevant aroma substances, e.g. carbohydrates. Using molecular tools, the corresponding metabolic basis is analyzed. A decrease in water potential of the still living coffee seeds induces massive drought stress responses. As a marker for these stress reactions, accumulation of a general stress metabolite, GABA (gamma-aminobutyric acid), and associated gene expression of drought stress-associated dehydrins were monitored. The results of this study indicate that metabolism in drying coffee beans is quite complex since several events trigger accumulation of GABA. The first peak of GABA accumulation during drying is correlated with expression of isocitrate lyase and thus with ongoing germination processes in coffee seeds. Two subsequent peaks of GABA accumulation correspond to maxima of dehydrin gene expression and are thought to be induced directly by drought stress in the embryo and endosperm tissue, respectively. Apart from the significance for understanding basic seed physiology, metabolic changes in coffee seeds during processing provide valuable information for understanding the role and effect of the steps of green coffee processing on the quality of the resulting coffee.

  15. Molecular cloning and expression analysis of GABA(A) receptor-associated protein (GABARAP) from small abalone, Haliotis diversicolor.

    PubMed

    Bai, Rongyao; You, Weiwei; Chen, Jun; Huang, Heqing; Ke, Caihuan

    2012-10-01

    GABA(A) receptor-associated protein (GABARAP), a multifunctional protein participating in autophagy process, is evolutionarily conserved and involves in innate immunity in eukaryotic cells, but currently there is no research on the relationship between GABARAP and innate immunity in mollusc. In the present study, the GABARAP full-length cDNA and its genomic DNA were firstly cloned from small abalone (Haliotis diversicolor), which was named as saGABARAP. Its full-length cDNA is 963 bp with a 354 bp open reading frame encoding a protein of 117 aa, a 276 bp 5'-UTR, and a 333 bp 3'-UTR including a poly(A) tail, two typical polyadenylation signals (AATAA) and two RNA instability motifs (ATTTA). The deduced protein has an estimated molecular weight of 13.9 kDa and a predicted PI of 8.73. Its genomic DNA comprises 4352 bp, containing three exons and two introns. Quantitative real-time PCR analysis revealed that saGABARAP was constitutively expressed in all examined tissues, with the highest expression level in hepatopancreas, and was upregulated in hepatopancreas and hemocytes after bacterial challenge. In addition, saGABARAP was ubiquitously expressed at all examined embryonic and larval development stages. These results suggested that saGABARAP could respond to bacteria challenge and may play a vital role in the adult innate immune system against pathogens and the development process of abalone embryo and larvae.

  16. Effect of diphenylhydantoin on gamma aminobutyric acid (GABA) and succinate activity in rat Purkinje cells.

    PubMed Central

    Hitchcock, E; Gabra-Sanders, T

    1977-01-01

    A study has been made of the effect of diphenylhydantoin (DPH) upon the levels of gamma aminobutyric acid (GABA) and succinic dehydrogenase in rat Purkinje cells. DPH was administered over 26 days in chronic experiments using controls receiving the same injection vehicle without DPH. Animals in this group received daily 1.25 mg/kg body weight, 12.5 mg/kg body weight, and 50 mg/kg body weight DPH. Acute experiments were carried out over the course of not more than four days, three groups of animals receiving 75 mg/kg body weight, 87.5 mg/kg body weight, and 100 mg/kg body weight DPH. No effect upon succinic dehydrogenase could be demonstrated at any dose level. There was a significant progressive loss of GABA with increasing dosage of DPH. Images PMID:903771

  17. Neuroprotective or neurotoxic effects of 4-aminopyridine mediated by KChIP1 regulation through adjustment of Kv 4.3 potassium channels expression and GABA-mediated transmission in primary hippocampal cells.

    PubMed

    Del Pino, Javier; Frejo, María Teresa; Baselga, María José Anadon; Capo, Miguel Andrés; Moyano, Paula; García, José Manuel; Díaz, María Jesús

    2015-07-01

    4-Aminopyridine (4-AP) is a potassium channel blocker used for the treatment of neuromuscular disorders. Otherwise, it has been described to produce a large number of adverse effects among them cell death mediated mainly by blockage of K(+) channels. However, a protective effect against cell death has also been described. On the other hand, Kv channel interacting protein 1 (KChIP1) is a neuronal calcium sensor protein that is predominantly expressed at GABAergic synapses and it has been related with modulation of K(+) channels, GABAergic transmission and cell death. According to this KChIP1 could play a key role in the protective or toxic effects induced by 4-AP. We evaluated, in wild type and KChIP1 silenced primary hippocampal neurons, the effect of 4-AP (0.25μM to 2mM) with or without semicarbazide (0.3M) co-treatment after 24h and after 14 days 4-AP alone exposure on cell viability, the effect of 4-AP (0.25μM to 2mM) on KChIP1 and Kv 4.3 potassium channels gene expression and GABAergic transmission after 24h treatment or after 14 days exposure to 4-AP (0.25μM to1μM). 4-AP induced cell death after 24h (from 1mM) and after 14 days treatment. We observed that 4-AP modulates KChIP1 which regulate Kv 4.3 channels expression and GABAergic transmission. Our study suggests that KChIP1 is a key gene that has a protective effect up to certain concentration after short-term treatment with 4-AP against induced cell injury; but this protection is erased after long term exposure, due to KChIP1 down-regulation predisposing cell to 4-AP induced damages. These data might help to explain protective and toxic effects observed after overdose and long term exposure.

  18. The Role of Genetic Sex in Affect Regulation and Expression of GABA-Related Genes Across Species

    PubMed Central

    Seney, Marianne L.; Chang, Lun-Ching; Oh, Hyunjung; Wang, Xingbin; Tseng, George C.; Lewis, David A.; Sibille, Etienne

    2013-01-01

    Although circulating hormones and inhibitory gamma-aminobutyric acid (GABA)-related factors are known to affect mood, considerable knowledge gaps persist for biological mechanisms underlying the female bias in mood disorders. Here, we combine human and mouse studies to investigate sexual dimorphism in the GABA system in the context of major depressive disorder (MDD) and then use a genetic model to dissect the role of sex-related factors in GABA-related gene expression and anxiety-/depressive-like behaviors in mice. First, using meta-analysis of gene array data in human postmortem brain (N = 51 MDD subjects, 50 controls), we show that the previously reported down-regulation in MDD of somatostatin (SST), a marker of a GABA neuron subtype, is significantly greater in women with MDD. Second, using gene co-expression network analysis in control human subjects (N = 214; two frontal cortex regions) and expression quantitative trait loci mapping (N = 170 subjects), we show that expression of SST and the GABA-synthesizing enzymes glutamate decarboxylase 67 (GAD67) and GAD65 are tightly co-regulated and influenced by X-chromosome genetic polymorphisms. Third, using a rodent genetic model [Four Core Genotypes (FCG) mice], in which genetic and gonadal sex are artificially dissociated (N ≥ 12/group), we show that genetic sex (i.e., X/Y-chromosome) influences both gene expression (lower Sst, Gad67, Gad65 in XY mice) and anxiety-like behaviors (higher in XY mice). This suggests that in an intact male animal, the observed behavior represents the outcomes of male genetic sex increasing and male-like testosterone decreasing anxiety-like behaviors. Gonadal sex was the only factor influencing depressive-like behavior (gonadal males < gonadal females). Collectively, these combined human and mouse studies provide mechanistic insight into sexual dimorphism in mood disorders, and specifically demonstrate an unexpected role of male-like factors (XY genetic sex) on

  19. A functional role for both γ-aminobutyric acid (GABA) transporter-1 and GABA transporter-3 in the modulation of extracellular GABA and GABAergic tonic conductances in the rat hippocampus

    PubMed Central

    Kersanté, Flavie; Rowley, Samuel C S; Pavlov, Ivan; Gutièrrez-Mecinas, María; Semyanov, Alexey; Reul, Johannes M H M; Walker, Matthew C; Linthorst, Astrid C E

    2013-01-01

    Tonic γ-aminobutyric acid (GABA)A receptor-mediated signalling controls neuronal network excitability in the hippocampus. Although the extracellular concentration of GABA (e[GABA]) is critical in determining tonic conductances, knowledge on how e[GABA] is regulated by different GABA transporters (GATs) in vivo is limited. Therefore, we studied the role of GATs in the regulation of hippocampal e[GABA] using in vivo microdialysis in freely moving rats. Here we show that GAT-1, which is predominantly presynaptically located, is the major GABA transporter under baseline, quiescent conditions. Furthermore, a significant contribution of GAT-3 in regulating e[GABA] was revealed by administration of the GAT-3 inhibitor SNAP-5114 during simultaneous blockade of GAT-1 by NNC-711. Thus, the GABA transporting activity of GAT-3 (the expression of which is confined to astrocytes) is apparent under conditions in which GAT-1 is blocked. However, sustained neuronal activation by K+-induced depolarization caused a profound spillover of GABA into the extrasynaptic space and this increase in e[GABA] was significantly potentiated by sole blockade of GAT-3 (i.e. even when uptake of GAT-1 is intact). Furthermore, experiments using tetrodotoxin to block action potentials revealed that GAT-3 regulates extrasynaptic GABA levels from action potential-independent sources when GAT-1 is blocked. Importantly, changes in e[GABA] resulting from both GAT-1 and GAT-3 inhibition directly precipitate changes in tonic conductances in dentate granule cells as measured by whole-cell patch-clamp recording. Thus, astrocytic GAT-3 contributes to the regulation of e[GABA] in the hippocampus in vivo and may play an important role in controlling the excitability of hippocampal cells when network activity is increased. PMID:23381899

  20. Expression of estrogen and progesterone receptors in glutamate and GABA neurons of the pubertal female monkey hypothalamus.

    PubMed

    Thind, K K; Goldsmith, P C

    1997-05-01

    We have previously reported direct glutamate (Glu) synapses upon GnRH-containing neurons in the primate hypothalamus, and extensive interactions between Glu and aminobutyric acid (GABA) neurons in areas associated with reproductive function. Both Glu and GABA are known to affect peripubertal GnRH neurohormone release, but their relative roles remain unclear. In a developmental survey, estrogen receptors (ER) and progesterone receptors (PR) were virtually undetectable after immunostaining the hypothalamus of prepubertal monkeys, but were clearly evident in neurons of adults. We hypothesized, therefore, that Glu and GABA neurons which develop ER or PR expression during puberty may participate in reactivation of the hypothalamic-pituitary-gonadal axis. To identify those neurons in midpubertal female cynomolgus monkeys, we performed immunofluorescence staining for ER or for PR in separate sets of hypothalamic sections, and then immunostained for Glu or for glutamate decarboxylase (GAD, to identify GABA neurons) using a contrasting fluorophore. ER and PR were localized in the cytoplasm and nuclei of Glu and GAD neurons in nine hypothalamic and related brain regions. Quantitation revealed intranuclear ER in an average of 80% of the Glu neurons in all regions analyzed, and an average of 84% of the GAD neurons in all regions except the supraoptic nucleus (28%). Intranuclear PR expression was more variable, occurring in an average of 93% of the Glu neurons in seven regions, but in only 41% in the medial preoptic area, and 0% in the arcuate-periventicular zone. In addition, while intranuclear PR was seen in 96% of the GAD neurons in the septum, it appeared in 67% of the GAD neurons in the paraventricular nucleus, 47% in the medial preoptic area, 40% in the periventricular zone, and was absent from neurons in the supraoptic nucleus and mammillary bodies. In summary, certain subpopulations of Glu and GABA neurons in principal hypothalamic regions of the female monkey express

  1. Evidence for a Revised Ion/Substrate Coupling Stoichiometry of GABA Transporters.

    PubMed

    Willford, Samantha L; Anderson, Cynthia M; Spencer, Shelly R; Eskandari, Sepehr

    2015-08-01

    Plasma membrane γ-aminobutyric acid (GABA) transporters (GATs) are electrogenic transport proteins that couple the cotranslocation of Na(+), Cl(-), and GABA across the plasma membrane of neurons and glia. A fundamental property of the transporter that determines its ability to concentrate GABA in cells and, hence, regulate synaptic and extra-synaptic GABA concentrations, is the ion/substrate coupling stoichiometry. Here, we scrutinized the currently accepted 2 Na(+):1 Cl(-):1 GABA stoichiometry because it is inconsistent with the measured net charge translocated per co-substrate (Na(+), Cl(-), and GABA). We expressed GAT1 and GAT3 in Xenopus laevis oocytes and utilized thermodynamic and uptake under voltage-clamp measurements to determine the stoichiometry of the GABA transporters. Voltage-clamped GAT1-expressing oocytes were internally loaded with GABA, and the reversal potential (V rev) of the transporter-mediated current was recorded at different external concentrations of Na(+), Cl(-), or GABA. The shifts in V rev for a tenfold change in the external Na(+), Cl(-), and GABA concentration were 84 ± 4, 30 ± 1, and 29 ± 1 mV, respectively. To determine the net charge translocated per Na(+), Cl(-), and GABA, we measured substrate fluxes under voltage clamp in cells expressing GAT1 or GAT3. Charge flux to substrate flux ratios were 0.7 ± 0.1 charge/Na(+), 2.0 ± 0.2 charges/Cl(-), and 2.1 ± 0.1 charges/GABA. Altogether, our results strongly suggest a 3 Na(+):1 Cl(-):1 GABA coupling stoichiometry for the GABA transporters. The revised stoichiometry has important implications for understanding the contribution of GATs to GABAergic signaling in health and disease.

  2. Effects of simulated microgravity on the expression of presynaptic proteins distorting the GABA/glutamate equilibrium--A proteomics approach.

    PubMed

    Wang, Yun; Iqbal, Javed; Liu, Yahui; Su, Rui; Lu, Song; Peng, Guang; Zhang, Yongqian; Qing, Hong; Deng, Yulin

    2015-11-01

    Microgravity may cause cognition-related changes in the animal nervous system due to the resulting uneven flow of fluids in the body. These changes may restrict the long-term stay of humans in space for various purposes. In this study, a rat tail suspension model (30°) was used to explore the effects of 21 days of prolonged simulated microgravity (SM) on the expression of proteins involved in cognitive functions in the rat hippocampus. SM decreased the content of γ-aminobutyric acid (GABA) and increased the content of glutamate (Glu) in the rat hippocampus. A comparative (18)O-labeled quantitative proteomics strategy was applied to detect the differential expression of synaptic proteins under SM. Fifty-three proteins were found to be differentially expressed under SM. Microgravity induces difficulty in the formation of the SNARE complex due to the down-regulation of vesicle-associated membrane protein 3(VAMP3) and syntaxin-1A. Synaptic vesicle recycling may also be affected due to the dysregulation of syntaxin-binding protein 5 (tomosyn), rab3A and its effector rim2. Both processes are disturbed, indicating that presynaptic proteins mediate a GABA/Glu imbalance under SM. These findings provide clues for understanding the mechanism of the GABA/Glu equilibrium in the hippocampus induced by microgravity in space and represent steps toward safe space travel.

  3. Neuroprotective effect of vitamin C against the ethanol and nicotine modulation of GABA(B) receptor and PKA-alpha expression in prenatal rat brain.

    PubMed

    Naseer, M I; Lee, H Y; Kim, M O

    2010-06-01

    Prenatal ethanol exposure has various deleterious effects on neuronal development and can induce various defects in developing brain, resulting in fetal alcohol syndrome (FAS). gamma-Aminobutyric acid (GABA(B)) receptor (R) is known to play an important role during the development of the central nervous system (CNS). Our study was designed to investigate the effect of ethanol (100 mM), nicotine (50 microM) (for 30 min and 1 h), vitamin C (vitC, 0.5 mM), ethanol plus vitC, and nicotine plus vitC on expression level of GABA(B1), GABA(B2)R, and protein kinase A-alpha (PKA) in prenatal rat cortical and hippocampal neurons at gestational days (GD) 17.5. The results showed that, upon ethanol and nicotine exposure, GABA(B1) and GABA(B2)R protein expression increased significantly in the cortex and hippocampus for a short (30 min) and long term (1 h), whereas only GABA(B2)R subunit was decreased upon nicotine exposure for a long term in the cortex. Furthermore, PKA expression in cortex and hippocampus increased with ethanol exposure during short term, whereas long-term exposure results increased in cortex and decreased in hippocampus. Moreover, the cotreatment of vitC with ethanol and nicotine showed significantly decreased expression of GABA(B1), GABA(B2)R, and PKA in cortex and hippocampus for a long-term exposure. Mitochondrial membrane potential, Fluoro-jade-B, and propidium iodide staining were used to elucidate possible neurodegeneration. Our results suggest the involvement of GABA(B)R and PKA in nicotine and ethanol-mediated neurodevelopmental defects and the potential use of vitC as a effective protective agent for FAS-related deficits.

  4. Selective expression of KCNS3 potassium channel α-subunit in parvalbumin-containing GABA neurons in the human prefrontal cortex.

    PubMed

    Georgiev, Danko; González-Burgos, Guillermo; Kikuchi, Mitsuru; Minabe, Yoshio; Lewis, David A; Hashimoto, Takanori

    2012-01-01

    The cognitive deficits of schizophrenia appear to be associated with altered cortical GABA neurotransmission in the subsets of inhibitory neurons that express either parvalbumin (PV) or somatostatin (SST). Identification of molecular mechanisms that operate selectively in these neurons is essential for developing targeted therapeutic strategies that do not influence other cell types. Consequently, we sought to identify, in the human cortex, gene products that are expressed selectively by PV and/or SST neurons, and that might contribute to their distinctive functional properties. Based on previously reported expression patterns in the cortex of mice and humans, we selected four genes: KCNS3, LHX6, KCNAB1, and PPP1R2, encoding K(+) channel Kv9.3 modulatory α-subunit, LIM homeobox protein 6, K(+) channel Kvβ1 subunit, and protein phosphatase 1 regulatory subunit 2, respectively, and examined their colocalization with PV or SST mRNAs in the human prefrontal cortex using dual-label in situ hybridization with (35)S- and digoxigenin-labeled antisense riboprobes. KCNS3 mRNA was detected in almost all PV neurons, but not in SST neurons, and PV mRNA was detected in >90% of KCNS3 mRNA-expressing neurons. LHX6 mRNA was detected in almost all PV and >90% of SST neurons, while among all LHX6 mRNA-expressing neurons 50% expressed PV mRNA and >44% expressed SST mRNA. KCNAB1 and PPP1R2 mRNAs were detected in much larger populations of cortical neurons than PV or SST neurons. These findings indicate that KCNS3 is a selective marker of PV neurons, whereas LHX6 is expressed by both PV and SST neurons. KCNS3 and LHX6 might be useful for characterizing cell-type specific molecular alterations of cortical GABA neurotransmission and for the development of novel treatments targeting PV and/or SST neurons in schizophrenia.

  5. Parvalbumin and neuropeptide Y expressing hippocampal GABA-ergic inhibitory interneuron numbers decline in a model of Gulf War illness

    PubMed Central

    Megahed, Tarick; Hattiangady, Bharathi; Shuai, Bing; Shetty, Ashok K.

    2015-01-01

    Cognitive dysfunction is amongst the most conspicuous symptoms in Gulf War illness (GWI). Combined exposure to the nerve gas antidote pyridostigmine bromide (PB), pesticides and stress during the Persian Gulf War-1 (PGW-1) are presumed to be among the major causes of GWI. Indeed, our recent studies in rat models have shown that exposure to GWI-related (GWIR) chemicals and mild stress for 4 weeks engenders cognitive impairments accompanied with several detrimental changes in the hippocampus. In this study, we tested whether reduced numbers of hippocampal gamma-amino butyric acid (GABA)-ergic interneurons are among the pathological changes induced by GWIR-chemicals and stress. Animals were exposed to low doses of GWIR-chemicals and mild stress for 4 weeks. Three months after this exposure, subpopulations of GABA-ergic interneurons expressing the calcium binding protein parvalbumin (PV), the neuropeptide Y (NPY) and somatostatin (SS) in the hippocampus were stereologically quantified. Animals exposed to GWIR-chemicals and stress for 4 weeks displayed reduced numbers of PV-expressing GABA-ergic interneurons in the dentate gyrus and NPY-expressing interneurons in the CA1 and CA3 subfields. However, no changes in SS+ interneuron population were observed in the hippocampus. Furthermore, GABA-ergic interneuron deficiency in these animals was associated with greatly diminished hippocampus neurogenesis. Because PV+ and NPY+ interneurons play roles in maintaining normal cognitive function and neurogenesis, and controlling the activity of excitatory neurons in the hippocampus, reduced numbers of these interneurons may be one of the major causes of cognitive dysfunction and reduced neurogenesis observed in GWI. Hence, strategies that improve inhibitory neurotransmission in the hippocampus may prove beneficial for reversing cognitive dysfunction in GWI. PMID:25620912

  6. Expression of GABAB receptors in magnocellular neurosecretory cells of male, virgin female and lactating rats.

    PubMed

    Richards, D S; Villalba, R M; Alvarez, F J; Stern, J E

    2005-07-01

    GABA is one of the key neurotransmitters that regulate the firing activity of neurones in the supraoptic (SON) and paraventricular (PVN) nuclei. In the present study, we used immunohistochemical techniques to study the distribution and subcellular localisation of metabotropic GABA(B) receptors in magnocellular neurones in the SON and PVN. Robust GABA(B) receptor immunoreactivity (GABA(B)R; both subunit 1 and subunit 2 of the heterodimer), was observed in the SON and PVN. At the light microcope level, GABA(B)R immonoreactivity displayed a clustered pattern localised both intracytoplasmically and at the plasma membrane. Densitometry analysis indicated that GABA(B)R immunoreactivity was significantly more intense in vasopressin cells than in oxytocin cells, both in male, virgin female and lactating rats, and was denser in males than in virgin females. Light and electron microscope studies indicated that cytoplasmic GABA(B)R was localised in various organelles, including the Golgi, early endosomes and lysosomes, suggesting the cycling of the receptor within the endocytic and trafficking pathways. Some smaller clusters at the level of the cell plasma membrane were apposed to glutamic acid decarboxylase 67 immunoreactive boutons, and appeared to be colocalised with gephyrin, a constituent protein of the postsynaptic density at inhibitory synapses. The presence of GABA(B)R immunoreactivity at synaptic and extrasynaptic sites was supported by electron microscopy. These results provide anatomical evidence for the expression of postsynaptic GABA(B) receptors in magnocellular neurosecretory cells. PMID:15946159

  7. The canonical Notch pathway effector RBP-J regulates neuronal plasticity and expression of GABA transporters in hippocampal networks.

    PubMed

    Liu, Shuxi; Wang, Yue; Worley, Paul F; Mattson, Mark P; Gaiano, Nicholas

    2015-05-01

    Activation of the Notch pathway in neurons is essential for learning and memory in various species from invertebrates to mammals. However, it remains unclear how Notch signaling regulates neuronal plasticity, and whether the transcriptional regulator and canonical pathway effector RBP-J plays a role. Here, we report that conditional disruption of RBP-J in the postnatal hippocampus leads to defects in long-term potentiation, long-term depression, and in learning and memory. Using gene expression profiling and chromatin immunoprecipitation, we identified two GABA transporters, GAT2 and BGT1, as putative Notch/RBP-J pathway targets, which may function downstream of RBP-J to limit the accumulation of GABA in the Schaffer collateral pathway. Our results reveal an essential role for canonical Notch/RBP-J signaling in hippocampal synaptic plasticity and suggest that role, at least in part, is mediated by the regulation of GABAergic signaling. PMID:25515406

  8. The canonical Notch pathway effector RBP-J regulates neuronal plasticity and expression of GABA transporters in hippocampal networks

    PubMed Central

    Liu, Shuxi; Wang, Yue; Worley, Paul F.; Mattson, Mark P.; Gaiano, Nicholas

    2014-01-01

    Activation of the Notch pathway in neurons is essential for learning and memory in various species from invertebrates to mammals. However, it remains unclear how Notch signaling regulates neuronal plasticity, and whether the transcriptional regulator and canonical pathway effector RBP-J plays a role. Here we report that conditional disruption of RBP-J in the postnatal hippocampus leads to defects in long-term potentiation (LTP), long-term depression (LTD), and in learning and memory. Using gene expression profiling and chromatin immunoprecipitation, we identified two GABA transporters, GAT2 and BGT1, as putative Notch/RBP-J pathway targets, which may function downstream of RBP-J to limit the accumulation of GABA in the Schaffer collateral pathway. Our results reveal an essential role for canonical Notch/RBP-J signaling in hippocampal synaptic plasticity and suggest that role, at least in part, is mediated by the regulation of GABAergic signaling. PMID:25515406

  9. The canonical Notch pathway effector RBP-J regulates neuronal plasticity and expression of GABA transporters in hippocampal networks.

    PubMed

    Liu, Shuxi; Wang, Yue; Worley, Paul F; Mattson, Mark P; Gaiano, Nicholas

    2015-05-01

    Activation of the Notch pathway in neurons is essential for learning and memory in various species from invertebrates to mammals. However, it remains unclear how Notch signaling regulates neuronal plasticity, and whether the transcriptional regulator and canonical pathway effector RBP-J plays a role. Here, we report that conditional disruption of RBP-J in the postnatal hippocampus leads to defects in long-term potentiation, long-term depression, and in learning and memory. Using gene expression profiling and chromatin immunoprecipitation, we identified two GABA transporters, GAT2 and BGT1, as putative Notch/RBP-J pathway targets, which may function downstream of RBP-J to limit the accumulation of GABA in the Schaffer collateral pathway. Our results reveal an essential role for canonical Notch/RBP-J signaling in hippocampal synaptic plasticity and suggest that role, at least in part, is mediated by the regulation of GABAergic signaling.

  10. A Fluorescence-Coupled Assay for Gamma Aminobutyric Acid (GABA) Reveals Metabolic Stress-Induced Modulation of GABA Content in Neuroendocrine Cancer

    PubMed Central

    Ippolito, Joseph E.; Piwnica-Worms, David

    2014-01-01

    Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA) have been implicated in the pathogenesis of high grade neuroendocrine (NE) neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1), was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, GAD1 was found to be expressed in castrate-resistant prostate cancer cell lines, but not androgen-responsive cell lines. Using a novel fluorescence-coupled enzymatic microplate assay for GABA mediated through reduction of resazurin in a prostate neuroendocrine carcinoma (PNEC) cell line, acid microenvironment-induced stress increased GABA levels while alkaline microenvironment-induced stress decreased GABA through modulation of GAD1 and glutamine synthetase (GLUL) activities. Moreover, glutamine but not glucose deprivation decreased GABA through modulation of GLUL. Consistent with evidence in prokaryotic and eukaryotic organisms that GABA synthesis mediated through GAD1 may play a crucial role in surviving stress, GABA may be an important mediator of stress survival in neoplasms. These findings identify GABA synthesis and metabolism as a potentially important pathway for regulating cancer cell stress response as well as a potential target for therapeutic strategies. PMID:24551133

  11. A fluorescence-coupled assay for gamma aminobutyric acid (GABA) reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer.

    PubMed

    Ippolito, Joseph E; Piwnica-Worms, David

    2014-01-01

    Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA) have been implicated in the pathogenesis of high grade neuroendocrine (NE) neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1), was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, GAD1 was found to be expressed in castrate-resistant prostate cancer cell lines, but not androgen-responsive cell lines. Using a novel fluorescence-coupled enzymatic microplate assay for GABA mediated through reduction of resazurin in a prostate neuroendocrine carcinoma (PNEC) cell line, acid microenvironment-induced stress increased GABA levels while alkaline microenvironment-induced stress decreased GABA through modulation of GAD1 and glutamine synthetase (GLUL) activities. Moreover, glutamine but not glucose deprivation decreased GABA through modulation of GLUL. Consistent with evidence in prokaryotic and eukaryotic organisms that GABA synthesis mediated through GAD1 may play a crucial role in surviving stress, GABA may be an important mediator of stress survival in neoplasms. These findings identify GABA synthesis and metabolism as a potentially important pathway for regulating cancer cell stress response as well as a potential target for therapeutic strategies.

  12. Quantitative changes of GABA-immunoreactive cells in the hindlimb representation of the rat somatosensory cortex after 14-day hindlimb unloading by tail suspension

    NASA Technical Reports Server (NTRS)

    D'Amelio, F.; Fox, R. A.; Wu, L. C.; Daunton, N. G.

    1996-01-01

    The present study was aimed at evaluating quantitatively gamma-aminobutyric acid (GABA) immunoreactivity in the hindlimb representation of the rat somatosensory cortex after 14 days of hindlimb unloading by tail suspension. A reduction in the number of GABA-immunoreactive cells with respect to the control animals was observed in layer Va and Vb. GABA-containing terminals were also reduced in the same layers, particularly those terminals surrounding the soma and apical dendrites of pyramidal cells in layer Vb. On the basis of previous morphological and behavioral studies of the neuromuscular system of hindlimb-suspended animals, it is suggested that the unloading due to hindlimb suspension alters afferent signaling and feedback information from intramuscular receptors to the cerebral cortex due to modifications in the reflex organization of hindlimb muscle groups. We propose that the reduction in immunoreactivity of local circuit GABAergic neurons and terminals is an expression of changes in their modulatory activity to compensate for the alterations in the afferent information.

  13. Interaction of GABA-mimetics with the taurine transporter (TauT, Slc6a6) in hyperosmotic treated Caco-2, LLC-PK1 and rat renal SKPT cells.

    PubMed

    Rasmussen, Rune Nørgaard; Lagunas, Candela; Plum, Jakob; Holm, René; Nielsen, Carsten Uhd

    2016-01-20

    The aim of the present study was to investigate if basic GABA-mimetics interact with the taurine transporter (TauT, Slc6a6), and to find a suitable cell based model that is robust towards extracellular changes in osmolality during uptake studies. Taurine uptake was measured in human Caco-2 cells, porcine LLC-PK1 cells, and rat SKPT cells using radiolabelled taurine. Hyperosmotic conditions were obtained by incubation with raffinose (final osmolality of 500mOsm) for 24h prior to the uptake experiments. Expression of the taurine transporter, TauT, was investigated at the mRNA level by real-time PCR. Uptake of the GABA-mimetics gaboxadol and vigabatrin was investigated in SKPT cells, and quantified by liquid scintillation or HPLC-MS/MS analysis, respectively. The uptake rate of [(3)H]-taurine was Na(+) and Cl(-) and concentration dependent with taurine with an apparent Vmax of 6.3±1.6pmolcm(-2)min(-1) and a Km of 24.9±15.0μM. β-alanine, nipecotic acid, gaboxadol, GABA, vigabatrin, δ-ALA and guvacine inhibited the taurine uptake rate in a concentration dependent manner. The order of affinity for TauT was β-alanine>GABA>nipecotic acid>guvacine>δ-ALA>vigabatrin>gaboxadol with IC50-values of 0.04, 1.07, 2.02, 4.19, 4.94, 31.4 and 39.9mM, respectively. In conclusion, GABA mimetics inhibited taurine uptake in hyperosmotic rat renal SKPT cells. SKPT cells, which seem to be a useful model for investigating taurine transport in the short-term presence of high concentrations of osmolytes. Furthermore, analogues of β-alanine appear to have higher affinities for TauT than GABA-analogues.

  14. Variations in maternal care alter GABA(A) receptor subunit expression in brain regions associated with fear.

    PubMed

    Caldji, Christian; Diorio, Josie; Meaney, Michael J

    2003-11-01

    Maternal care influences the development of stress reactivity in the offspring. These effects are accompanied by changes in corticotropin-releasing factor (CRF) expression in brain regions that regulate responses to stress. However, such effects appear secondary to those involving systems that normally serve to inhibit CRF expression and release. Thus, maternal care over the first week of life alters GABA(A) (gamma-aminobutyric acid)(A) receptor mRNA subunit expression. The adult offspring of mothers that exhibit increased levels of pup licking/grooming and arched back-nursing (high LG-ABN mothers) show increased alpha1 mRNA levels in the medial prefrontal cortex, the hippocampus as well as the basolateral and central regions, of the amygdala and increased gamma2 mRNA in the amygdala. Western blot analyses confirm these effects at the level of protein. In contrast, the offspring of low LG-ABN mothers showed increased levels of alpha3 and alpha4 subunit mRNAs. The results of an adoption study showed that the biological offspring of low LG-ABN mothers fostered shortly after birth to high LG-ABN dams showed the increased levels of both alpha1 and gamma2 mRNA expression in the amygdala in comparison to peers fostered to other low LG-ABN mothers (the reverse was true for the biological offspring of high LG-ABN mothers). These findings are consistent with earlier reports of the effects of maternal care on GABA(A)/benzodiazepine receptor binding and suggest that maternal care can permanently alter the subunit composition of the GABA(A) receptor complex in brain regions that regulate responses to stress.

  15. The effects of lowered extracellular sodium on gamma-aminobutyric acid (GABA)-induced currents of Muller (glial) cells of the skate retina.

    PubMed

    Qian, H; Malchow, R P; Ripps, H

    1993-04-01

    1. The effects of external sodium on GABA-induced chloride currents were examined with whole-cell voltage-clamp recordings obtained from enzymatically dissociated solitary Muller cells in culture. Our goal was to determine whether a sodium-dependent GABA uptake mechanism influences the GABAa-mediated responses of skate Muller cells. 2. At low concentrations of GABA (0.01 to 0.5 microM), removal of sodium from the external solution resulted in a marked increase in the ligand-gated currents mediated by activation of GABAa receptors. The enhancement by lowered sodium was greatest at hyperpolarizing potentials and decreased progressively as the cell was depolarized. 3. The reversal potential for the GABA-induced response was not significantly altered by the removal of sodium, suggesting that sodium ions did not directly contribute to the GABAa-mediated current. 4. Lowering external sodium had no effect on the currents induced by the GABAa-agonist muscimol, consistent with its much lower affinity for the GABA transport carrier. 5. Application of the GABA uptake blocker nipecotic acid also abolished the effects of lowered sodium. 6. These findings suggest that the effects of lowered external sodium resulted from a decrease in the uptake of GABA into the Muller cells, thus raising the effective concentration of GABA acting upon the GABAa receptors. PMID:8394215

  16. Expression of GABA transporters, GAT-1 and GAT-3, in the cerebral cortex and thalamus of the rat during postnatal development.

    PubMed

    Vitellaro-Zuccarello, L; Calvaresi, N; De Biasi, S

    2003-09-01

    The cellular and subcellular localization of two GABA transporters, GAT-1 and GAT-3, was investigated using immunocytochemical methods in the rat cerebral cortex and thalamus during postnatal development. The distribution of the transporters is compared with that of the neuronal marker GABA, and with that of vimentin and of glial fibrillary acidic protein, which identify immature and mature astrocytes, respectively. Our observations show that the two transporters are already expressed at birth in both brain areas with the same cellular localization as in adult rats, as GAT-1 is present in growth cones and terminals only in the cortex, whereas both transporters are expressed in astrocytes in the cortex and thalamus. The distribution of GAT-1 and GAT-3 undergoes postnatal changes reflecting in general the neurogenetic events of the neocortex and thalamus and, more specifically, the maturation of GABAergic innervation. The adult-like pattern of expression is achieved in the third postnatal week in the cortex and in the second postnatal week in the thalamus. The early expression of GAT-1 in GABAergic terminals confirms previous studies showing the existence of neuronal mechanisms of GABA uptake from the embryonic stages. As for the glial localization, the precocious existence of two astrocytic GABA transporters suggests that they operate through different functional mechanisms from birth, whereas their exclusively glial expression in the thalamus indicates that the astroglia plays a major role in the transport, recycling and metabolism of thalamic GABA.

  17. Anion transport and GABA signaling

    PubMed Central

    Hübner, Christian A.; Holthoff, Knut

    2013-01-01

    Whereas activation of GABAA receptors by GABA usually results in a hyperpolarizing influx of chloride into the neuron, the reversed chloride driving force in the immature nervous system results in a depolarizing efflux of chloride. This GABAergic depolarization is deemed to be important for the maturation of the neuronal network. The concept of a developmental GABA switch has mainly been derived from in vitro experiments and reliable in vivo evidence is still missing. As GABAA receptors are permeable for both chloride and bicarbonate, the net effect of GABA also critically depends on the distribution of bicarbonate. Whereas chloride can either mediate depolarizing or hyperpolarizing currents, bicarbonate invariably mediates a depolarizing current under physiological conditions. Intracellular bicarbonate is quickly replenished by cytosolic carbonic anhydrases. Intracellular bicarbonate levels also depend on different bicarbonate transporters expressed by neurons. The expression of these proteins is not only developmentally regulated but also differs between cell types and even subcellular regions. In this review we will summarize current knowledge about the role of some of these transporters for brain development and brain function. PMID:24187533

  18. Conserved site for neurosteroid modulation of GABA A receptors.

    PubMed

    Hosie, Alastair M; Clarke, Laura; da Silva, Helena; Smart, Trevor G

    2009-01-01

    This study addresses whether the potentiation site for neurosteroids on GABA(A) receptors is conserved amongst different GABA(A) receptor isoforms. The neurosteroid potentiation site was previously identified in the alpha1beta2gamma2S receptor by mutation of Q241 to methionine or leucine, which reduced the potentiation of GABA currents by the naturally occurring neurosteroids, allopregnanolone or tetrahydrodeoxycorticosterone (THDOC). By using heterologous expression of GABA(A) receptors in HEK cells, in combination with whole-cell patch clamp recording methods, a relatively consistent potentiation by allopregnanolone of GABA-activated currents was evident for receptors composed of one alpha subunit isoform (alpha2-5) assembled with beta3 and gamma2S subunits. Using mutant alphabetagamma receptors, the neurosteroid potentiation was universally dependent on the conserved glutamine residue in M1 of the respective alpha subunit. Studying wild-type and mutant receptors composed of alpha4beta3delta subunits revealed that the delta subunit is unlikely to contribute to the neurosteroid potentiation binding site and probably affects the efficacy of potentiation. Thus, in keeping with the ability of neurosteroids to potentiate GABA currents via a broad variety of GABA(A) receptor isoforms in neurons, the potentiation site is structurally highly conserved on this important neurotransmitter receptor family.

  19. Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics.

    PubMed

    Jin, Zhe; Bhandage, Amol K; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R; Birnir, Bryndis

    2014-01-01

    The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence. PMID:25278838

  20. GABA(B) receptors in neuroendocrine regulation.

    PubMed

    Lux-Lantos, Victoria A; Bianchi, María S; Catalano, Paolo N; Libertun, Carlos

    2008-09-01

    Gamma-amino butyric acid (GABA), in addition to being a metabolic intermediate and the main inhibitory neurotransmitter in the synaptic cleft, is postulated as a neurohormone, a paracrine signaling molecule, and a trophic factor. It acts through pre- and post-synaptic receptors, named GABA(A) and GABA(C) (ionotropic receptors) and GABA(B) (metabotropic receptor). Here we reviewed the participation of GABA(B) receptors in the regulation of the hypothalamic-pituitary-gonadal axis, using physiological, biochemical, and pharmacological approaches in rats, as well as in GABA(B1) knock-out mice, that lack functional GABA(B) receptors. Our general conclusion indicates that GABA(B )receptors participate in the regulation of pituitary hormone secretion acting both in the central nervous system and directly on the gland. PRL and gonadotropin axes are affected by GABA(B) receptor activation, as demonstrated in the rat and also in the GABA(B1) knock-out mouse. In addition, hypothalamic and pituitary GABA(B) receptor expression is modulated by steroid hormones. GABA participation in the brain control of pituitary secretion through GABA(B) receptors depends on physiological conditions, being age and sex critical factors.These results indicate that patients receiving GABA(B) agonists/antagonists should be monitored for possible endocrine side effects.

  1. Unique insecticide specificity of human homomeric rho 1 GABA(C) receptor.

    PubMed

    Ratra, Gurpreet S; Erkkila, Brian E; Weiss, David S; Casida, John E

    2002-03-24

    Several convulsants and major insecticides block the gamma-aminobutyric acid (GABA)-gated chloride channel in brain on binding to the GABA(A) receptor. The GABA(C) receptor, important in retina and present in brain, is also coupled to a chloride channel and is therefore a potential target for toxicant action examined here in radioligand binding and electrophysiological experiments. Human homomeric rho 1 GABA(C) receptor expressed in human embryonic kidney cells (HEK293) undergoes specific and saturable high-affinity binding of 4-n-[3H]propyl-4' -ethynylbicycloorthobenzoate ([3H]EBOB) using a cyano analog (CNBOB) to determine non-specific binding. This GABA(C) rho 1 receptor is very sensitive to CNBOB and lindane relative to alpha-endosulfan, tert-butylbicyclophosphorothionate, picrotoxinin and fipronil (IC(50) values of 23, 91, 800, 1080, 4000 and >10000 nM, respectively, in displacing [3H]EBOB). A similar potency sequence (except for picrotoxinin) is observed for inhibition of GABA-induced currents of rho 1 receptor expressed in Xenopus oocytes. The present study does not consider rho 2 homomeric and rho 1 rho 2 heteromeric GABA(C) receptors which are known to be more sensitive than rho 1 to picrotoxinin. The inhibitor sensitivity and specificity of this rho 1 GABA(C) receptor differ greatly from those of human homomeric beta 3 and native GABA(A) receptors.

  2. Profound desensitization by ambient GABA limits activation of δ-containing GABAA receptors during spillover.

    PubMed

    Bright, Damian P; Renzi, Massimiliano; Bartram, Julian; McGee, Thomas P; MacKenzie, Georgina; Hosie, Alastair M; Farrant, Mark; Brickley, Stephen G

    2011-01-12

    High-affinity extrasynaptic GABA(A) receptors (GABA(A)Rs) are a prominent feature of cerebellar granule neurons and thalamic relay neurons. In both cell types, the presence of synaptic glomeruli would be expected to promote activation of these GABA(A)Rs, contributing to phasic spillover-mediated currents and tonic inhibition. However, the precise role of different receptor subtypes in these two phenomena is unclear. To address this question, we made recordings from neurons in acute brain slices from mice, and from tsA201 cells expressing recombinant GABA(A)Rs. We found that δ subunit-containing GABA(A)Rs of both cerebellar granule neurons and thalamic relay neurons of the lateral geniculate nucleus contributed to tonic conductance caused by ambient GABA but not to spillover-mediated currents. In the presence of a low "ambient" GABA concentration, recombinant "extrasynaptic" δ subunit-containing GABA(A)Rs exhibited profound desensitization, rendering them insensitive to brief synaptic- or spillover-like GABA transients. Together, our results demonstrate that phasic spillover and tonic inhibition reflect the activation of distinct receptor populations.

  3. Loss of UBE3A from TH-expressing neurons suppresses GABA co-release and enhances VTA-NAc optical self-stimulation.

    PubMed

    Berrios, Janet; Stamatakis, Alice M; Kantak, Pranish A; McElligott, Zoe A; Judson, Matthew C; Aita, Megumi; Rougie, Marie; Stuber, Garret D; Philpot, Benjamin D

    2016-01-01

    Motivated reward-seeking behaviours are governed by dopaminergic ventral tegmental area projections to the nucleus accumbens. In addition to dopamine, these mesoaccumbal terminals co-release other neurotransmitters including glutamate and GABA, whose roles in regulating motivated behaviours are currently being investigated. Here we demonstrate that loss of the E3-ubiquitin ligase, UBE3A, from tyrosine hydroxylase-expressing neurons impairs mesoaccumbal, non-canonical GABA co-release and enhances reward-seeking behaviour measured by optical self-stimulation. PMID:26869263

  4. Loss of UBE3A from TH-expressing neurons suppresses GABA co-release and enhances VTA-NAc optical self-stimulation.

    PubMed

    Berrios, Janet; Stamatakis, Alice M; Kantak, Pranish A; McElligott, Zoe A; Judson, Matthew C; Aita, Megumi; Rougie, Marie; Stuber, Garret D; Philpot, Benjamin D

    2016-02-12

    Motivated reward-seeking behaviours are governed by dopaminergic ventral tegmental area projections to the nucleus accumbens. In addition to dopamine, these mesoaccumbal terminals co-release other neurotransmitters including glutamate and GABA, whose roles in regulating motivated behaviours are currently being investigated. Here we demonstrate that loss of the E3-ubiquitin ligase, UBE3A, from tyrosine hydroxylase-expressing neurons impairs mesoaccumbal, non-canonical GABA co-release and enhances reward-seeking behaviour measured by optical self-stimulation.

  5. Isolation, characterization, and tissue-specific expression of GABA A receptor α1 subunit gene of Carassius auratus gibelio after avermectin treatment.

    PubMed

    Zhao, Yini; Sun, Qi; Hu, Kun; Ruan, Jiming; Yang, Xianle

    2016-02-01

    Carassius auratus gibelio has been widely cultivated in fish farms in China, with avermectin (AVM) being used to prevent parasite infection. Recently, AVM was found to pass through the Carassius auratus gibelio blood-brain barrier (BBB). Although AVM acts mainly through a GABA receptor and specifically the α1 subunit gene, the most common isoform of the GABA A receptor, which is widely expressed in brain neurons and has been studied in other fish, Carassius auratus gibelio GABA A receptor α1 subunit gene cloning, and whether AVM passes through the BBB to induce Carassius auratus gibelio GABA A receptor α1 subunit gene expression have not been studied. The aim of this study was to clone, sequence, and phylogenetically analyze the GABA A receptor α1 subunit gene and to investigate the correlation of its expression with neurotoxicity in brain, liver, and kidney after AVM treatment by quantitative real-time reverse transcription polymerase chain reaction. The α1 subunit gene was 1550 bp in length with an open reading frame of 1380 bp encoding a predicted protein with 459 amino acid residues. The gene contained 128 bp of 5' terminal untranslated region (URT) and 72 bp of 3' terminal UTR. The α1 subunit structural features conformed to the Cys-loop ligand-gated ion channels family, which includes a signal peptide, an extracellular domain at the N-terminal, and four transmembrane domains. The established phylogenetic tree indicated that the α1 subunits of Carassius auratus gibelio and Danio rerio were the most closely related to each other. The α1 subunit was found to be highly expressed in brain and ovary, and the α1 mRNA transcription level increased significantly in brain. Moreover, the higher the concentration of AVM was, the higher the GABA A receptor expression was, indicating that AVM can induce significant neurotoxicity to Carassius auratus gibelio. Therefore, the α1 subunit mRNA expression was positively correlated with the neurotoxicity of AVM in

  6. Release of GABA from sensory neurons transduced with a GAD67-expressing vector occurs by non-vesicular mechanisms.

    PubMed

    Liu, Jun; Tai, Changfeng; de Groat, William C; Peng, Xiang-min; Mata, Marina; Fink, David J

    2006-02-16

    We have demonstrated that dorsal root ganglion neurons transduced with a recombinant replication-defective herpes simplex virus vector coding for glutamic acid decarboxylase (QHGAD67) release GABA to produce an analgesic effect in rodent models of pain. In this study, we examined the mechanism of transgene-mediated GABA release from dorsal root ganglion neurons in vitro and in vivo. Release of GABA from dorsal root ganglion neurons transduced with QHGAD67 was not increased by membrane depolarization induced by 60 mM extracellular K+ nor reduced by the removal of Ca2+ from the medium. Release of GABA from transduced dorsal root ganglion neurons was, however, blocked in a dose-dependent manner by NO-711, a selective inhibitor of the GABA transporter-1. The amount of GABA released from a spinal cord slice preparation, prepared from animals transduced by subcutaneous inoculation of QHGAD67 in the hind paws, was substantially increased compared to animals transduced with control vector Q0ZHG or normal animals, but the amount of GABA released was not changed by stimulation of the dorsal roots at either low (0.1 mA, 0.5-ms duration) or high (10 mA, 0.5-ms duration) intensity. We conclude that QHGAD67-mediated GABA release from dorsal root ganglion neurons is non-vesicular, independent of electrical depolarization, and that this efflux is mediated through reversal of the GABA transporter.

  7. Phasic, Nonsynaptic GABA-A Receptor-Mediated Inhibition Entrains Thalamocortical Oscillations

    PubMed Central

    Rovó, Zita; Mátyás, Ferenc; Barthó, Péter; Slézia, Andrea; Lecci, Sandro; Pellegrini, Chiara; Astori, Simone; Dávid, Csaba; Hangya, Balázs

    2014-01-01

    GABA-A receptors (GABA-ARs) are typically expressed at synaptic or nonsynaptic sites mediating phasic and tonic inhibition, respectively. These two forms of inhibition conjointly control various network oscillations. To disentangle their roles in thalamocortical rhythms, we focally deleted synaptic, γ2 subunit-containing GABA-ARs in the thalamus using viral intervention in mice. After successful removal of γ2 subunit clusters, spontaneous and evoked GABAergic synaptic currents disappeared in thalamocortical cells when the presynaptic, reticular thalamic (nRT) neurons fired in tonic mode. However, when nRT cells fired in burst mode, slow phasic GABA-AR-mediated events persisted, indicating a dynamic, burst-specific recruitment of nonsynaptic GABA-ARs. In vivo, removal of synaptic GABA-ARs reduced the firing of individual thalamocortical cells but did not abolish slow oscillations or sleep spindles. We conclude that nonsynaptic GABA-ARs are recruited in a phasic manner specifically during burst firing of nRT cells and provide sufficient GABA-AR activation to control major thalamocortical oscillations. PMID:24849349

  8. Seizure-induced alterations in fast-spiking basket cell GABA currents modulate frequency and coherence of gamma oscillation in network simulations

    SciTech Connect

    Proddutur, Archana; Yu, Jiandong; Elgammal, Fatima S.; Santhakumar, Vijayalakshmi

    2013-12-15

    Gamma frequency oscillations have been proposed to contribute to memory formation and retrieval. Fast-spiking basket cells (FS-BCs) are known to underlie development of gamma oscillations. Fast, high amplitude GABA synapses and gap junctions have been suggested to contribute to gamma oscillations in FS-BC networks. Recently, we identified that, apart from GABAergic synapses, FS-BCs in the hippocampal dentate gyrus have GABAergic currents mediated by extrasynaptic receptors. Our experimental studies demonstrated two specific changes in FS-BC GABA currents following experimental seizures [Yu et al., J. Neurophysiol. 109, 1746 (2013)]: increase in the magnitude of extrasynaptic (tonic) GABA currents and a depolarizing shift in GABA reversal potential (E{sub GABA}). Here, we use homogeneous networks of a biophysically based model of FS-BCs to examine how the presence of extrasynaptic GABA conductance (g{sub GABA-extra}) and experimentally identified, seizure-induced changes in g{sub GABA-extra} and E{sub GABA} influence network activity. Networks of FS-BCs interconnected by fast GABAergic synapses developed synchronous firing in the dentate gamma frequency range (40–100 Hz). Systematic investigation revealed that the biologically realistic range of 30 to 40 connections between FS-BCs resulted in greater coherence in the gamma frequency range when networks were activated by Poisson-distributed dendritic synaptic inputs rather than by homogeneous somatic current injections, which were balanced for FS-BC firing frequency in unconnected networks. Distance-dependent conduction delay enhanced coherence in networks with 30–40 FS-BC interconnections while inclusion of gap junctional conductance had a modest effect on coherence. In networks activated by somatic current injections resulting in heterogeneous FS-BC firing, increasing g{sub GABA-extra} reduced the frequency and coherence of FS-BC firing when E{sub GABA} was shunting (−74 mV), but failed to alter average

  9. Rapid substrate-induced charge movements of the GABA transporter GAT1.

    PubMed

    Bicho, Ana; Grewer, Christof

    2005-07-01

    The GABA transporter GAT1 removes the neurotransmitter GABA from the synaptic cleft by coupling of GABA uptake to the co-transport of two sodium ions and one chloride ion. The aim of this work was to investigate the individual reaction steps of GAT1 after a GABA concentration jump. GAT1 was transiently expressed in HEK293 cells and its pre-steady-state kinetics were studied by combining the patch-clamp technique with the laser-pulse photolysis of caged GABA, which allowed us to generate GABA concentration jumps within <100 micros. Recordings of transport currents generated by GAT1, both in forward and exchange transport modes, showed multiple charge movements that can be separated along the time axis. The individual reactions associated with these charge movements differ from the well-characterized electrogenic "sodium-occlusion" reaction by GAT1. One of the observed electrogenic reactions is shown to be associated with the GABA-translocating half-cycle of the transporter, in contradiction to previous studies that showed no charge movements associated with these reactions. Interestingly, reactions of the GABA-bound transporter were not affected by the absence of extracellular chloride, suggesting that Cl- may not be co-translocated with GABA. Based on the results, a new alternating access sequential-binding model is proposed for GAT1's transport cycle that describes the results presented here and those by others. PMID:15849242

  10. Oxytocin depolarizes fast-spiking hilar interneurons and induces GABA release onto mossy cells of the rat dentate gyrus.

    PubMed

    Harden, Scott W; Frazier, Charles J

    2016-09-01

    Delivery of exogenous oxytocin (OXT) to central oxytocin receptors (OXT-Rs) is currently being investigated as a potential treatment for conditions such as post-traumatic stress disorder (PTSD), depression, social anxiety, and autism spectrum disorder (ASD). Despite significant research implicating central OXT signaling in modulation of mood, affect, social behavior, and stress response, relatively little is known about the cellular and synaptic mechanisms underlying these complex actions, particularly in brain regions which express the OXT-R but lie outside of the hypothalamus (where OXT-synthesizing neurons reside). We report that bath application of low concentrations of the selective OXT-R agonist Thr4,Gly7-OXT (TGOT) reliably and robustly drives GABA release in the dentate gyrus in an action potential dependent manner. Additional experiments led to identification of a small subset of small hilar interneurons that are directly depolarized by acute application of TGOT. From a physiological perspective, TGOT-responsive hilar interneurons have high input resistance, rapid repolarization velocity during an action potential, and a robust afterhyperpolarization. Further, they fire irregularly (or stutter) in response to moderate depolarization, and fire quickly with minimal spike frequency accommodation in response to large current injections. From an anatomical perspective, TGOT responsive hilar interneurons have dense axonal arborizations in the hilus that were found in close proximity with mossy cell somata and/or proximal dendrites, and also invade the granule cell layer. Further, they have primary dendrites that always extend into the granule cell layer, and sometimes have clear arborizations in the molecular layer. Overall, these data reveal a novel site of action for OXT in an important limbic circuit, and represent a significant step towards better understanding how endogenous OXT may modulate flow of information in hippocampal networks. © 2016 Wiley

  11. Embryonic GABA(B) receptor blockade alters cell migration, adult hypothalamic structure, and anxiety- and depression-like behaviors sex specifically in mice.

    PubMed

    Stratton, Matthew S; Staros, Michelle; Budefeld, Tomaz; Searcy, Brian T; Nash, Connor; Eitel, Chad; Carbone, David; Handa, Robert J; Majdic, Gregor; Tobet, Stuart A

    2014-01-01

    Neurons of the paraventricular nucleus of the hypothalamus (PVN) regulate the hypothalamic- pituitary-adrenal (HPA) axis and the autonomic nervous system. Females lacking functional GABA(B) receptors because of a genetic disruption of the R1 subunit have altered cellular characteristics in and around the PVN at birth. The genetic disruption precluded appropriate assessments of physiology or behavior in adulthood. The current study was conducted to test the long term impact of a temporally restricting pharmacological blockade of the GABA(B) receptor to a 7-day critical period (E11-E17) during embryonic development. Experiments tested the role of GABA(B) receptor signaling in fetal development of the PVN and later adult capacities for adult stress related behaviors and physiology. In organotypic slices containing fetal PVN, there was a female specific, 52% increase in cell movement speeds with GABA(B) receptor antagonist treatment that was consistent with a sex-dependent lateral displacement of cells in vivo following 7 days of fetal exposure to GABA(B) receptor antagonist. Anxiety-like and depression-like behaviors, open-field activity, and HPA mediated responses to restraint stress were measured in adult offspring of mothers treated with GABA(B) receptor antagonist. Embryonic exposure to GABA(B) receptor antagonist resulted in reduced HPA axis activation following restraint stress and reduced depression-like behaviors. There was also increased anxiety-like behavior selectively in females and hyperactivity in males. A sex dependent response to disruptions of GABA(B) receptor signaling was identified for PVN formation and key aspects of physiology and behavior. These changes correspond to sex specific prevalence in similar human disorders, namely anxiety disorders and hyperactivity.

  12. GABA(A) receptors implicated in REM sleep control express a benzodiazepine binding site.

    PubMed

    Nguyen, Tin Quang; Liang, Chang-Lin; Marks, Gerald A

    2013-08-21

    It has been reported that non-subtype-selective GABAA receptor antagonists injected into the nucleus pontis oralis (PnO) of rats induced long-lasting increases in REM sleep. Characteristics of these REM sleep increases were identical to those resulting from injection of muscarinic cholinergic agonists. Both actions were blocked by the muscarinic antagonist, atropine. Microdialysis of GABAA receptor antagonists into the PnO resulted in increased acetylcholine levels. These findings were consistent with GABAA receptor antagonists disinhibiting acetylcholine release in the PnO to result in an acetylcholine-mediated REM sleep induction. Direct evidence has been lacking for localization in the PnO of the specific GABAA receptor-subtypes mediating the REM sleep effects. Here, we demonstrated a dose-related, long-lasting increase in REM sleep following injection (60 nl) in the PnO of the inverse benzodiazepine agonist, methyl-6,7-dimethoxy-4-ethyl-β-carboline (DMCM, 10(-2)M). REM sleep increases were greater and more consistently produced than with the non-selective antagonist gabazine, and both were blocked by atropine. Fluorescence immunohistochemistry and laser scanning confocal microscopy, colocalized in PnO vesicular acetylcholine transporter, a presynaptic marker of cholinergic boutons, with the γ2 subunit of the GABAA receptor. These data provide support for the direct action of GABA on mechanisms of acetylcholine release in the PnO. The presence of the γ2 subunit at this locus and the REM sleep induction by DMCM are consistent with binding of benzodiazepines by a GABAA receptor-subtype in control of REM sleep.

  13. Gad1 mRNA as a reliable indicator of altered GABA release from orexigenic neurons in the hypothalamus

    PubMed Central

    Dicken, Matthew S.; Hughes, Alexander R.; Hentges, Shane T.

    2016-01-01

    The strength of γ-aminobutyric acid (GABA)-mediated inhibitory synaptic input is a principle determinant of neuronal activity. However, because of differences in the number of GABA afferent inputs and the sites of synapses, it is difficult to directly assay for altered GABA transmission between specific cells. The present study tested the hypothesis that the level of mRNA for the GABA synthetic enzyme glutamate decarboxylase (GAD) can provide a reliable proxy for GABA release. This was tested in a mouse hypothalamic circuit important in the regulation of energy balance. Fluorescent in situ hybridization results show that the expression of Gad1 mRNA (encoding the GAD67 enzyme) was increased in hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons after an overnight fast, consistent with the ability of GABA from these neurons to stimulate food intake. Optogenetic studies confirmed that the observed increase in Gad1 mRNA correlated with an increase in the probability of GABA release from NPY/AgRP neurons onto downstream proopiomelanocortin neurons. Likewise, there was an increase in the readily releasable pool of GABA in NPY/AgRP neurons. Selective inhibition of GAD activity in NPY/AgRP neurons decreased GABA release, indicating that GAD67 activity, which is largely dictated by expression level, is a key determinant of GABA release. Altogether, it appears that Gad expression may be a reliable proxy of altered GABAergic transmission. Examining changes in Gad mRNA as a proxy for GABA release may be particularly helpful when the downstream targets are not known or when limited tools exist for detecting GABA release at a particular synapse. PMID:26370162

  14. GABA release from mouse axonal growth cones

    PubMed Central

    Gao, Xiao-Bing; van den Pol, Anthony N

    2000-01-01

    Using developing hypothalamic neurons from transgenic mice that express high levels of green fluorescent protein in growing axons, and an outside-out patch from mature neuronal membranes that contain neurotransmitter receptors as a sensitive detector, we found that GABA is released by a vesicular mechanism from the growth cones of developing axons prior to synapse formation. A low level of GABA release occurs spontaneously from the growth cone, and this is substantially increased by evoked action potentials. Neurotransmitters such as acetylcholine can enhance protein kinase C (PKC) activity even prior to synapse formation; PKC activation caused a substantial increase in spontaneous GABA release from the growth cone, probably acting at the axon terminal. These data indicate that GABA is secreted from axons during a stage of neuronal development when GABA is excitatory, and that neuromodulators could alter GABA release from the growing axon, potentially enabling other developing neurons of different transmitter phenotype to modulate the early actions of GABA. PMID:10718743

  15. GABA maintains the proliferation of progenitors in the developing chick ciliary marginal zone and non-pigmented ciliary epithelium.

    PubMed

    Ring, Henrik; Mendu, Suresh Kumar; Shirazi-Fard, Shahrzad; Birnir, Bryndis; Hallböök, Finn

    2012-01-01

    GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABA(A) receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABA(A) receptor system. To quantify the effects on proliferation by GABA(A) receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABA(A) receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABA(A) receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl-transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABA(A) receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABA(A) receptors. This supported the depolarising role for the GABA(A) receptors. Inhibition of L-type voltage-gated Ca(2+) channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABA(A) receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27(KIP1), along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27(KIP1) after inhibition of either the GABA(A) receptors or the L-type VGCCs suggests a link between the GABA(A) receptors, membrane potential, and intracellular Ca

  16. Enhancement of GABA release through endogenous activation of axonal GABA(A) receptors in juvenile cerebellum.

    PubMed

    Trigo, Federico F; Chat, Mireille; Marty, Alain

    2007-11-14

    Recent evidence indicates the presence of presynaptic GABA(A) receptors (GABA(A)Rs) in the axon domain of several classes of central neurons, including cerebellar basket and stellate cells. Here, we investigate the possibility that these receptors could be activated in the absence of electrical or chemical stimulation. We find that low concentrations of GABA increase the frequency of miniature GABAergic synaptic currents. Submaximal concentrations of a GABA(A)R blocker, gabazine, decrease both the miniature current frequency and the probability of evoked GABA release. Zolpidem, an agonist of the benzodiazepine binding site, and NO-711 (1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride), a blocker of GABA uptake, both increase the frequency of miniature currents. These effects occur up to postnatal day 14, but not later. Immunohistochemistry indicates the presence of alpha1-containing GABA(A)Rs in interneuron presynaptic terminals with a similar age dependence. We conclude that, under resting conditions, axonal GABA(A)Rs are significantly activated, that this activation results in enhanced GABA release, and that it can be augmented by increasing the affinity of GABA(A)Rs or reducing GABA uptake. Our findings suggest the existence of a positive-feedback mechanism involving presynaptic GABA(A)Rs that maintains a high release rate and a high local GABA concentration in the immature cerebellar network.

  17. Enhanced neurosteroid potentiation of ternary GABA(A) receptors containing the delta subunit.

    PubMed

    Wohlfarth, Kai M; Bianchi, Matt T; Macdonald, Robert L

    2002-03-01

    Attenuated behavioral sensitivity to neurosteroids has been reported for mice deficient in the GABA(A) receptor delta subunit. We therefore investigated potential subunit-specific neurosteroid pharmacology of the following GABA(A) receptor isoforms in a transient expression system: alpha1beta3gamma2L, alpha1beta3delta, alpha6beta3gamma2L, and alpha6beta3delta. Potentiation of submaximal GABA(A) receptor currents by the neurosteroid tetrahydrodeoxycorticosterone (THDOC) was greatest for the alpha1beta3delta isoform. Whole-cell GABA concentration--response curves performed with and without low concentrations (30 nm) of THDOC revealed enhanced peak GABA(A) receptor currents for isoforms tested without affecting the GABA EC50. Alpha1beta3delta currents were enhanced the most (>150%), whereas the other isoform currents were enhanced 15-50%. At a higher concentration (1 microm), THDOC decreased peak alpha1beta3gamma2L receptor current amplitude evoked by GABA (1 mm) concentration jumps and prolonged deactivation but had little effect on the rate or extent of apparent desensitization. Thus the polarity of THDOC modulation depended on GABA concentration for alpha1beta3gamma2L GABA(A) receptors. However, the same protocol applied to alpha1beta3delta receptors resulted in peak current enhancement by THDOC of >800% and prolonged deactivation. Interestingly, THDOC induced pronounced desensitization in the minimally desensitizing alpha1beta3delta receptors. Single channel recordings obtained from alpha1beta3delta receptors indicated that THDOC increased the channel opening duration, including the introduction of an additional longer duration open state. Our results suggest that the GABA(A) receptor delta subunit confers increased sensitivity to neurosteroid modulation and that the intrinsic gating and desensitization kinetics of alpha1beta3delta GABA(A) receptors are altered by THDOC.

  18. Dark-rearing-induced reduction of GABA and GAD and prevention of the effect by BDNF in the mouse retina.

    PubMed

    Lee, Eun-Jin; Gibo, Tricia L; Grzywacz, Norberto M

    2006-10-01

    Gamma-aminobutyric acid (GABA) is an important retinal neurotransmitter. We studied the expression of GABA, glutamate decarboxylase 65 (GAD65) and GAD67 by immunocytochemistry and Western blot, in the retinas of control and dark-reared C57BL/6J black mice. This study asked three questions. First, is visual input necessary for the normal expression of GABA, GAD65 and GAD67? Second, can the retina recover from the effects of dark-rearing if returned to a normal light-dark cycle? Third, does BDNF prevent the influence of dark-rearing on the expression of GABA and GAD? At postnatal day 10 (P10), before eye opening, GABA immunoreactivity was present in the ganglion cell layer (GCL), in the innermost rows of the inner nuclear layer (INL) and throughout the inner plexiform layer (IPL) of control and dark-reared retinas. In P30 control retinas, GABA immunoreactivity showed similar patterns to those at P10. However, in P30 dark-reared retinas, the density of GABA-immunoreactive cells was lower in both the INL and GCL than in control retinas. In addition, visual deprivation retarded GABA immunoreactivity in the IPL. Western blot analysis showed corresponding differences in the levels of GAD65 but not of GAD67 expression between control and dark-rearing conditions. In our study, dark-rearing effects were reversed when the mice were put in normal cyclic light-dark conditions for 2 weeks. Moreover, dark-reared retinas treated with BDNF showed normal expression of both GABA and GAD65. Our data indicate that normal expression of GABA and GAD65 is dependent on visual input. Furthermore, the data suggest that BDNF controls this dependence.

  19. Biochemical and electrophysiological characteristics of mammalian GABA receptors.

    PubMed

    Enna, S J; Gallagher, J P

    1983-01-01

    The concept that GABA is a neurotransmitter in the mammalian CNS is supported by both electrophysiological and biochemical data. Whereas the electrophysiological studies are essential for demonstrating a specific functional response to GABA, the biochemical approach is useful for characterizing the molecular properties of this site. As a result of these studies the concept of the GABA receptor has progressed from a simple model of a single recognition site associated with a chloride channel to a more complex structure having a variety of interacting components. Thus, both electrophysiological and biochemical data support the existence of at least two pharmacologically distinct types of GABA receptors, based on the sensitivity to bicuculline. Also, anatomically, there appear to be two different types of receptors, those located postsynaptically on the soma or dendrites of a neighboring cell and those found presynaptically on GABAergic and other neurotransmitter terminals. From biochemical studies it appears that the GABA receptor may be composed of at least three distinct interacting components. One of these, the recognition site, may exist in two conformations, with one preferring agonists and the other having a higher affinity for antagonists. Ion channels may be considered a second component, with some of these regulating the passage of chloride ion, whereas others may be associated with calcium transport. The third major element of GABA receptors appears to be a benzodiazepine recognition site, although only a certain population of GABA receptors may be endowed with this property. In addition to these, the GABA receptor complex appears to contain substances that modulate the recognition site by influencing the availability of higher affinity binding proteins. It would appear therefore that changes affecting any one of these constituents can influence the characteristics of the others. While increasing the complexity of the system, this arrangement makes for a

  20. Effect of GABA, a bacterial metabolite, on Pseudomonas fluorescens surface properties and cytotoxicity.

    PubMed

    Dagorn, Audrey; Chapalain, Annelise; Mijouin, Lily; Hillion, Mélanie; Duclairoir-Poc, Cécile; Chevalier, Sylvie; Taupin, Laure; Orange, Nicole; Feuilloley, Marc G J

    2013-01-01

    Different bacterial species and, particularly Pseudomonas fluorescens, can produce gamma-aminobutyric acid (GABA) and express GABA-binding proteins. In this study, we investigated the effect of GABA on the virulence and biofilm formation activity of different strains of P. fluorescens. Exposure of a psychotropic strain of P. fluorescens (MF37) to GABA (10-5 M) increased its necrotic-like activity on eukaryotic (glial) cells, but reduced its apoptotic effect. Conversely, muscimol and bicuculline, the selective agonist and antagonist of eukaryote GABAA receptors, respectively, were ineffective. P. fluorescens MF37 did not produce biosurfactants, and its caseinase, esterase, amylase, hemolytic activity or pyoverdine productions were unchanged. In contrast, the effect of GABA was associated to rearrangements of the lipopolysaccharide (LPS) structure, particularly in the lipid A region. The surface hydrophobicity of MF37 was marginally modified, and GABA reduced its biofilm formation activity on PVC, but not on glass, although the initial adhesion was increased. Five other P. fluorescens strains were studied, and only one, MFP05, a strain isolated from human skin, showed structural differences of biofilm maturation after exposure to GABA. These results reveal that GABA can regulate the LPS structure and cytotoxicity of P. fluorescens, but that this property is specific to some strains. PMID:23743829

  1. Impaired surface αβγ GABA(A) receptor expression in familial epilepsy due to a GABRG2 frameshift mutation.

    PubMed

    Tian, Mengnan; Mei, Davide; Freri, Elena; Hernandez, Ciria C; Granata, Tiziana; Shen, Wangzhen; Macdonald, Robert L; Guerrini, Renzo

    2013-02-01

    The purpose of the study was to explore the pathogenic mechanisms underlying generalized epilepsy and febrile seizures plus (GEFS+) in a family with a novel γ2 subunit gene (GABRG2) frameshift mutation. Four affected and one unaffected individuals carried a c.1329delC GABRG2 mutation resulting in a subunit [γ2S(S443delC)] with a modified and elongated carboxy-terminus that is different from that of the wildtype γ2S subunit. We expressed the wildtype γ2S subunit and the predicted mutant γ2S(S443delC) subunit cDNAs in HEK293T cells and performed immunoblotting, flow cytometry and electrophysiology studies. The mutant subunit was translated as a stable protein that was larger than the wildtype γ2S subunit and was retained in the ER and not expressed on the cell surface membrane, suggesting GABRG2 haploinsufficiency. Peak GABA-evoked currents recorded from cells cotransfected with wildtype α1 and β2 subunits and mutant γ2S subunits were significantly decreased and were comparable to α1β2 receptor currents. S443delC is the first GABR epilepsy mutation predicted to abolish the natural stop codon and produce a stop codon in the 3' UTR that leads to translation of an extended peptide. The GEFS+ phenotype observed in this family is likely caused by γ2S subunit loss-of-function and possibly to dominant-negative suppression of α1β2γ2 receptors. Many GABRG2 truncation mutations result in GEFS+, but the spectrum of phenotypic severity is wider, ranging from asymptomatic individuals to the Dravet syndrome. Mechanisms influencing the severity of the phenotype are therefore complex and difficult to correlate with its demonstrable functional effects.

  2. Protein kinase C regulates tonic GABA(A) receptor-mediated inhibition in the hippocampus and thalamus.

    PubMed

    Bright, Damian P; Smart, Trevor G

    2013-11-01

    Tonic inhibition mediated by extrasynaptic GABA(A) receptors (GABA(A) Rs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABA(A) Rs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABA(A) R-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABA(A) R activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABA(A) Rs, which represent a key extrasynaptic GABA(A) R isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABA(A) Rs. The inhibitory effects of PKC activation on α4β2δ GABA(A) R activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABA(A) R-mediated inhibition.

  3. Role for pro-inflammatory cytokines in regulating expression of GABA transporter type 1 and 3 in specific brain regions of kainic acid-induced status epilepticus.

    PubMed

    Su, Jing; Yin, Jian; Qin, Wei; Sha, Suxu; Xu, Jun; Jiang, Changbin

    2015-03-01

    In general, pro-inflammatory cytokines (PICs) contribute to regulation of epilepsy-associated pathophysiological processes in the central nerve system. In this report, we examined the specific activation of PICs, namely IL-1β, IL-6 and TNF-α in rat brain after kainic acid (KA)-induced status epilepticus (SE). Also, we examined the role played by PICs in regulating expression of GABA transporter type 1 and 3 (GAT-1 and GAT-3, respectively), which are the two important subtypes of GATs responsible for the regulation of extracellular GABA levels in the brain. Our results show that IL-1β, IL-6 and TNF-α were significantly increased in the parietal cortex, hippocampus and amygdala of KA-rats as compared with sham control animals (P < 0.05, KA rats vs. control rats). KA-induced SE also significantly increased (P < 0.05 vs. controls) the protein expression of GAT-1 and GAT-3 in those brain regions. In addition, central administration of antagonists to IL-1β and TNF-α receptors significantly attenuated amplified GAT-1 and GAT-3 (P < 0.05 vs. vehicle control for each antagonist group). However, antagonist to IL-6 receptor failed to attenuate enhancement in expression of GAT-1 and GAT-3 induced by KA-induced SE. Overall, our data demonstrate that PIC pathways are activated in the specific brain regions during SE which thereby selectively leads to upregulation of GABA transporters. As a result, it is likely that de-inhibition of GABA system is increased in the brain. This support a role for PICs in engagement of the adaptive mechanisms associated with epileptic activity, and has pharmacological implications to target specific PICs for neuronal dysfunction and vulnerability related to epilepsy. PMID:25708016

  4. Development of imidazole alkanoic acids as mGAT3 selective GABA uptake inhibitors.

    PubMed

    Hack, Silke; Wörlein, Babette; Höfner, Georg; Pabel, Jörg; Wanner, Klaus T

    2011-05-01

    A new series of potential GABA uptake inhibitors starting from of 1H-imidazol-4-ylacetic acid with the carboxylic acid side chain originating from different positions and varying in length have been synthesized and tested for the inhibitory potency at the four GABA uptake transporters mGAT1-4 stably expressed in HEK cells. Further two bicyclic compounds with a rigidified carboxylic acid side chain were included in this study. The results of the biological tests indicated that most ω-imidazole alkanoic and alkenoic acid derivatives exhibit the highest potencies as GABA uptake inhibitors at mGAT3.

  5. KCC2 expression supersedes NKCC1 in mature fiber cells in mouse and rabbit lenses

    PubMed Central

    Kasinathan, Chinnaswamy

    2015-01-01

    Purpose Na-K-Cl cotransporter 1 (NKCC1) and K-Cl cotransporter 2 (KCC2) have fundamental roles in neuron differentiation that are integrated with gamma-aminobutyric acid (GABA) and glutamate receptors, GABA synthesized by GAD25/65/67 encoded by GAD1/GAD2 genes, and GABA transporters (GATs). Cells in the eye lens express at least 13 GABA receptor subunits, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl D-aspartate (NMDA) glutamate receptors, GAD1/GAD2, GAT1–4 and vGAT, and NKCC1. NKCC1:KCC2 ratios determine the switch in GABA actions from trophic/growth promoting early in development to their classic inhibitory roles in adult neurons. Lens epithelial cells cover the anterior surface and differentiate to elongated fiber cells in the lens interior with comparable morphology and sub-cellular structures as neurons. NKCC1 is expressed before KCC2 in neuron development and increases cell chloride, which stimulates differentiation and process formation. Subsequently, KCC2 increases and extrudes cell chloride linked with maturation. KCC2 has an additional structural moonlighting role interacting with F-actin scaffolding in dendritic spine morphogenesis. We examined KCC2 versus NKCC1 spatial expression in relation to fiber cell developmental status within the lens. Methods Immunofluorescence and immunoblots were used to detect expression in mouse and rabbit lenses. Results NKCC1 was restricted to peripheral elongating lens fiber cells in young adult mouse and rabbit lenses. Lens KCC2 expression included the major KCC2b neuronal isoform and was detected in interior fiber cells with decreased NKCC1 expression and localized at the membranes. Lens expression of RE-1 silencing transcription factor (REST) regulated KCC2 is consistent with GAD1 and GAD2, several GABA and glutamate receptor subunits, miR-124, and other REST-regulated genes expressed in lenses. Conclusions NKCC1 in peripheral elongating fiber cells is superseded by KCC2 expression in

  6. Inhibition of protein kinase C decreases sensitivity of GABA receptor subtype to fipronil insecticide in insect neurosecretory cells.

    PubMed

    Murillo, Laurence; Hamon, Alain; Es-Salah-Lamoureux, Zeineb; Itier, Valérie; Quinchard, Sophie; Lapied, Bruno

    2011-12-01

    Phosphorylation by serine/threonine kinases has been described as a new mechanism for regulating the effects of insecticides on insect neuronal receptors and channels. Although insect GABA receptors are commercially important targets for insecticides (e.g. fipronil), their modulation by kinases is poorly understood and the influence of phosphorylation on insecticide sensitivity is unknown. Using the whole-cell patch-clamp technique, we investigated the modulatory effect of PKC and CaMKinase II on GABA receptor subtypes (GABAR1 and GABAR2) in DUM neurons isolated from the terminal abdominal ganglion (TAG) of Periplaneta americana. Chloride currents through GABAR2 were selectively abolished by PMA and PDBu (the PKC activators) and potentiated by Gö6983, an inhibitor of PKC. Furthermore, using KN-62, a specific CaMKinase II inhibitor, we demonstrated that CaMKinase II activation was also involved in the regulation of GABAR2 function. In addition, using CdCl(2) (the calcium channel blocker) and LOE-908, a blocker of TRPγ, we revealed that calcium influx through TRPγ played an important role in kinase activations. Comparative studies performed with CACA, a selective agonist of GABAR1 in DUM neurons confirmed the involvement of these kinases in the specific regulation of GABAR2. Furthermore, our study reported that GABAR1 was less sensitive than GABAR2 to fipronil. This was demonstrated by the biphasic concentration-response curve and the current-voltage relationship established with both GABA and CACA. Finally, we demonstrated that GABAR2 was 10-fold less sensitive to fipronil following inhibition of PKC, whereas inhibition of CaMKinase II did not alter the effect of fipronil. PMID:21684305

  7. Chronic Intermittent Ethanol Regulates Hippocampal GABA(A) Receptor Delta Subunit Gene Expression

    PubMed Central

    Follesa, Paolo; Floris, Gabriele; Asuni, Gino P.; Ibba, Antonio; Tocco, Maria G.; Zicca, Luca; Mercante, Beniamina; Deriu, Franca; Gorini, Giorgio

    2015-01-01

    Chronic ethanol consumption causes structural and functional reorganization in the hippocampus and induces alterations in the gene expression of gamma-aminobutyric acid type A receptors (GABAARs). Distinct forced intermittent exposure models have been used previously to investigate changes in GABAAR expression, with contrasting results. Here, we used repeated cycles of a Chronic Intermittent Ethanol paradigm to examine the relationship between voluntary, dependence-associated ethanol consumption, and GABAAR gene expression in mouse hippocampus. Adult male C57BL/6J mice were exposed to four 16-h ethanol vapor (or air) cycles in inhalation chambers alternated with limited-access two-bottle choice between ethanol (15%) and water consumption. The mice exposed to ethanol vapor showed significant increases in ethanol consumption compared to their air-matched controls. GABAAR alpha4 and delta subunit gene expression were measured by qRT-PCR at different stages. There were significant changes in GABAAR delta subunit transcript levels at different time points in ethanol-vapor exposed mice, while the alpha4 subunit levels remained unchanged. Correlated concurrent blood ethanol concentrations suggested that GABAAR delta subunit mRNA levels fluctuate depending on ethanol intoxication, dependence, and withdrawal state. Using a vapor-based Chronic Intermittent Ethanol procedure with combined two-bottle choice consumption, we corroborated previous evidences showing that discontinuous ethanol exposure affects GABAAR delta subunit expression but we did not observe changes in alpha4 subunit. These findings indicate that hippocampal GABAAR delta subunit expression changes transiently over the course of a Chronic Intermittent Ethanol paradigm associated with voluntary intake, in response to ethanol-mediated disturbance of GABAergic neurotransmission. PMID:26617492

  8. Effects of Salt Loading on the Regulation of Rat Hypothalamic Magnocellular Neurosecretory Cells by Ionotropic GABA and Glycine Receptors.

    PubMed

    Choe, K Y; Trudel, E; Bourque, C W

    2016-04-01

    Synaptic and extrasynaptic transmission mediated by ionotropic GABA and glycine receptors plays a critical role in shaping the action potential firing (spiking) activity of hypothalamic magnocellular neurosecretory cells and therefore determines the rate at which vasopressin and oxytocin are released from the neurohypophysis. The inhibitory effect of these transmitters relies on the maintenance of a low concentration of intracellular chloride ions such that, when activated by GABA or glycine, a hyperpolarisation of the neuronal membrane potential results. In this review, we highlight the various ways by which the two types of inhibitory receptors contribute to homeostasis by fine-tuning the spiking rate of vasopressin-releasing magnocellular neurosecretory cells in a manner dependent on the hydration state of the animal. In addition, we review the currently available evidence on how the strength of these inhibitory pathways can be regulated during chronic hypernatraemia via a form of activity-dependent depolarisation of the chloride reversal potential, leading to an abolition of these inhibitory pathways potentially causing sodium-dependent elevations in blood pressure. PMID:26833894

  9. The sushi domains of secreted GABA(B1) isoforms selectively impair GABA(B) heteroreceptor function.

    PubMed

    Tiao, Jim Y; Bradaia, Amyaouch; Biermann, Barbara; Kaupmann, Klemens; Metz, Michaela; Haller, Corinne; Rolink, Antonius G; Pless, Elin; Barlow, Paul N; Gassmann, Martin; Bettler, Bernhard

    2008-11-01

    GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. GABA(B) receptors are promising drug targets for a wide spectrum of psychiatric and neurological disorders. Receptor subtypes exhibit no pharmacological differences and are based on the subunit isoforms GABA(B1a) and GABA(B1b). GABA(B1a) differs from GABA(B1b) in its ectodomain by the presence of a pair of conserved protein binding motifs, the sushi domains (SDs). Previous work showed that selectively GABA(B1a) contributes to heteroreceptors at glutamatergic terminals, whereas both GABA(B1a) and GABA(B1b) contribute to autoreceptors at GABAergic terminals or to postsynaptic receptors. Here, we describe GABA(B1j), a secreted GABA(B1) isoform comprising the two SDs. We show that the two SDs, when expressed as a soluble protein, bind to neuronal membranes with low nanomolar affinity. Soluble SD protein, when added at nanomolar concentrations to dissociated hippocampal neurons or to acute hippocampal slices, impairs the inhibitory effect of GABA(B) heteroreceptors on evoked and spontaneous glutamate release. In contrast, soluble SD protein neither impairs the activity of GABA(B) autoreceptors nor impairs the activity of postsynaptic GABA(B) receptors. We propose that soluble SD protein scavenges an extracellular binding partner that retains GABA(B1a)-containing heteroreceptors in proximity of the presynaptic release machinery. Soluble GABA(B1) isoforms like GABA(B1j) may therefore act as dominant-negative inhibitors of heteroreceptors and control the level of GABA(B)-mediated inhibition at glutamatergic terminals. Of importance for drug discovery, our data also demonstrate that it is possible to selectively impair GABA(B) heteroreceptors by targeting their SDs.

  10. Effects of GABA receptor antagonists on thresholds of P23H rat retinal ganglion cells to electrical stimulation of the retina

    NASA Astrophysics Data System (ADS)

    Jensen, Ralph J.; Rizzo, Joseph F., III

    2011-06-01

    An electronic retinal prosthesis may provide useful vision for patients suffering from retinitis pigmentosa (RP). In animal models of RP, the amount of current needed to activate retinal ganglion cells (RGCs) is higher than in normal, healthy retinas. In this study, we sought to reduce the stimulation thresholds of RGCs in a degenerate rat model (P23H-line 1) by blocking GABA receptor mediated inhibition in the retina. We examined the effects of TPMPA, a GABAC receptor antagonist, and SR95531, a GABAA receptor antagonist, on the electrically evoked responses of RGCs to biphasic current pulses delivered to the subretinal surface through a 400 µm diameter electrode. Both TPMPA and SR95531 reduced the stimulation thresholds of ON-center RGCs on average by 15% and 20% respectively. Co-application of the two GABA receptor antagonists had the greatest effect, on average reducing stimulation thresholds by 32%. In addition, co-application of the two GABA receptor antagonists increased the magnitude of the electrically evoked responses on average three-fold. Neither TPMPA nor SR95531, applied alone or in combination, had consistent effects on the stimulation thresholds of OFF-center RGCs. We suggest that the effects of the GABA receptor antagonists on ON-center RGCs may be attributable to blockage of GABA receptors on the axon terminals of ON bipolar cells.

  11. GABA excitation in mouse hilar neuropeptide Y neurons

    PubMed Central

    Fu, Li-Ying; van den Pol, Anthony N

    2007-01-01

    Neuropeptide Y-containing interneurons in the dentate hilar area play an important role in inhibiting the activity of hippocampal circuitry. Hilar cells are often among the first lost in hippocampal epilepsy. As many types of neurons are found in the hilus, we used a new transgenic mouse expressing green fluorescent protein (GFP) in a subset of neurons that colocalized neuropeptide Y (NPY), somatostatin (SST), and GABA for whole-cell, perforated, and cell-attached recording in 240 neurons. As these neurons have not previously been identifiable in live slices, they have not been the focus of physiological analysis. Hilar NPY neurons showed modest spike frequency adaptation, a large 15.6 ± 1.0 mV afterhyperpolarization, a mean input resistance of 335 ± 26 mΩ, and were capable of fast-firing. Muscimol-mediated excitatory actions were found in a nominally Ca2+-free/high-Mg2+ bath solution using cell-attached recording. GABAA receptor antagonists inhibited half the recorded neurons and blocked burst firing. Gramicidin perforated-patch recording revealed a GABA reversal potential positive to both the resting membrane potential and spike threshold. Together, these data suggest GABA is excitatory to many NPY cells. NPY and SST consistently hyperpolarized and reduced spike frequency in these neurons. No hyperpolarization of NPY on membrane potential was detected in the presence of tetrodotoxin, AP5, CNQX and bicuculline, supporting an indirect effect. Under similar conditions, SST hyperpolarized the cells, suggesting a direct postsynaptic action. Depolarizing actions of GABA and GABA-dependent burst-firing may synchronize a rapid release of GABA, NPY, and SST, leading to pre- and postsynaptic inhibition of excitatory hippocampal circuits. PMID:17204505

  12. Unpredictable neonatal stress enhances adult anxiety and alters amygdala gene expression related to serotonin and GABA

    PubMed Central

    Sarro, Emma C; Sullivan, Regina M; Barr, Gordon

    2014-01-01

    Anxiety-related disorders are among the most common psychiatric illnesses, thought to have both genetic and environmental causes. Early-life trauma, such as abuse from a caregiver, can be predictable or unpredictable, each resulting in increased prevalence and severity of a unique set of disorders. In this study, we examined the influence of early unpredictable trauma on both the behavioral expression of adult anxiety and gene expression within the amygdala. Neonatal rats were exposed to unpaired odor-shock conditioning for 5 days, which produces deficits in adult behavior and amygdala dysfunction. In adulthood, we used the Light/Dark box test to measure anxiety-related behaviors, measuring the latency to enter the lit area and quantified urination and defecation. The amygdala was then dissected and a microarray analysis was performed to examine changes in gene expression. Animals that had received early unpredictable trauma displayed significantly longer latencies to enter the lit area and more defecation and urination. The microarray analysis revealed over-represented genes related to learning and memory, synaptic transmission and trans-membrane transport. Gene ontology and pathway analysis identified highly represented disease states related to anxiety phenotypes, including social anxiety, obsessive-compulsive disorders, PTSD and bipolar disorder. Addiction related genes were also overrepresented in this analysis. Unpredictable shock during early development increased anxiety-like behaviors in adulthood with concomitant changes in genes related to neurotransmission, resulting in gene expression patterns similar to anxiety-related psychiatric disorders. PMID:24240029

  13. Suppression of γ-aminobutyric acid (GABA) transaminases induces prominent GABA accumulation, dwarfism and infertility in the tomato (Solanum lycopersicum L.).

    PubMed

    Koike, Satoshi; Matsukura, Chiaki; Takayama, Mariko; Asamizu, Erika; Ezura, Hiroshi

    2013-05-01

    Tomatoes accumulate γ-aminobutyric acid (GABA) at high levels in the immature fruits. GABA is rapidly converted to succinate during fruit ripening through the activities of GABA transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH). Although three genes encoding GABA-T and both pyruvate- and α-ketoglutarate-dependent GABA-T activities have been detected in tomato fruits, the mechanism underlying the GABA-T-mediated conversion of GABA has not been fully understood. In this work, we conducted loss-of-function analyses utilizing RNA interference (RNAi) transgenic plants with suppressed pyruvate- and glyoxylate-dependent GABA-T gene expression to clarify which GABA-T isoforms are essential for its function. The RNAi plants with suppressed SlGABA-T gene expression, particularly SlGABA-T1, showed severe dwarfism and infertility. SlGABA-T1 expression was inversely associated with GABA levels in the fruit at the red ripe stage. The GABA contents in 35S::SlGABA-T1(RNAi) lines were 1.3-2.0 times and 6.8-9.2 times higher in mature green and red ripe fruits, respectively, than the contents in wild-type fruits. In addition, SlGABA-T1 expression was strongly suppressed in the GABA-accumulating lines. These results indicate that pyruvate- and glyoxylate-dependent GABA-T is the essential isoform for GABA metabolism in tomato plants and that GABA-T1 primarily contributes to GABA reduction in the ripening fruits.

  14. Inhibitory interaction between P2X4 and GABA(C) rho1 receptors.

    PubMed

    Xia, Rong; Mei, Zhu-Zhong; Milligan, Carol; Jiang, Lin-Hua

    2008-10-10

    Reciprocal functional inhibition between P2X and GABA(A/C) receptors represents a novel mechanism fine-tuning neuronal excitability. However, the participating receptors and underlying mechanisms are not fully understood. P2X(4) receptor is widely found in neurons that express GABA(C) rho1 receptor. Thus, we co-expressed P2X(4) and rho1 receptors in HEK293 cells and, using patch-clamp recording, examined whether they have mutual functional inhibition. Currents evoked by simultaneous application of ATP and GABA (I(ATP+GABA)) were significantly smaller compared to the addition of I(ATP) and I(GABA). Furthermore, I(ATP) were strongly suppressed during rho1 receptor activation. Similarly, I(GABA) were greatly attenuated during P2X(4) receptor activation. Such mutual inhibition was absent in cells only expressing P2X(4) or rho1 receptor. Taken together, these functional data support negative cross-talk between P2X(4) and rho1 receptors.

  15. GABA localization in the nematode Ascaris

    SciTech Connect

    Guastella, J.

    1988-01-01

    A histochemical approach was used to examine the distribution of GABA-associated neurons in the nematode Ascaris, an organism whose small number of morphologically simple neurons make it an excellent preparation for analyzing neuronal phenotypes. Two GABAergic markers were examined: GABA-like immunoreactivity (GLIR), a marker for endogenous stores of GABA; and ({sup 3}H)-GABA uptake, a marker for GABA uptake sites. Strong GLIR was present in the cell bodies, neurites and commissures of dorsal and ventral inhibitory motorneurons present in this region. Strong GLIR was also present in the cell bodies and processes of the four RME neurons in the nerve ring and in several other ganglionic neurons. Staining was absent in excitatory motorneurons, in ventral cord interneurons and in muscle cells and hypodermis. GABA uptake sites were found in single neural processes in both the ventral and dorsal nerve cords. ({sup 3}H)-GABA labeling was also observed in the other two RME cells and several other cephalic neurons. Four putative cholinergic excitatory motorneurons in the retrovesicular ganglion (RVG) were heavily labeled. Ventral and dorsal nerve cord inhibitory motorneurons did not take up ({sup 3}H)-GABA. Labeling of the ventral cord excitatory motorneuron somata and cell bodies was at or slightly above background. Heavy labeling of muscle cells was also observed.

  16. Gating allosterism at a single class of etomidate sites on alpha1beta2gamma2L GABA A receptors accounts for both direct activation and agonist modulation.

    PubMed

    Rüsch, Dirk; Zhong, Huijun; Forman, Stuart A

    2004-05-14

    At clinical concentrations, the potent intravenous general anesthetic etomidate enhances gamma-aminobutyric acid, type A (GABA(A)) receptor activity elicited with low gamma-aminobutyric acid (GABA) concentrations, whereas much higher etomidate concentrations activate receptors in the absence of GABA. Therefore, GABA(A) receptors may possess two types of etomidate sites: high affinity GABA-modulating sites and low affinity channel-activating sites. However, GABA modulation and direct activation share stereoselectivity for the (R)(+)-etomidate isomer and display parallel dependence on GABA(A) beta subunit isoforms, suggesting that these two actions may be mediated by a single class of etomidate site(s) that exert one or more molecular effects. In this study, we assessed GABA modulation by etomidate using leftward shifts of electrophysiological GABA concentration responses in cells expressing human alpha1beta2gamma2L receptors. Etomidate at up to 100 microm reduced GABA EC(50) values by over 100-fold but without apparent saturation, indicating the absence of high affinity etomidate sites. In experiments using a partial agonist, P4S, etomidate both reduced EC(50) and increased maximal efficacy, demonstrating that etomidate shifts the GABA(A) receptor gating equilibrium toward open states. Results were quantitatively analyzed using equilibrium receptor gating models, wherein a postulated class of equivalent etomidate sites both directly activates receptors and enhances agonist gating. A Monod-Wyman-Changeux co-agonist mechanism with two equivalent etomidate sites that allosterically enhance GABA(A) receptor gating independently of agonist binding most simply accounts for direct activation and agonist modulation. This model also correctly predicts the actions of etomidate on GABA(A) receptors containing a point mutation that increases constitutive gating activity.

  17. Further evidence for involvement of the dorsal hippocampus serotonergic and γ-aminobutyric acid (GABA)ergic pathways in the expression of contextual fear conditioning in rats.

    PubMed

    Almada, Rafael C; Albrechet-Souza, Lucas; Brandão, Marcus L

    2013-12-01

    Intra-dorsal hippocampus (DH) injections of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), a serotonin-1A (5-hydroxytryptamine (5-HT)-1A) receptor agonist, were previously shown to inhibit the expression of contextual fear when administered six hours after conditioning. However, further understanding of the consolidation and expression of aversive memories requires investigations of these and other mechanisms at distinct time points and the regions of the brain to which they are transferred. Thus, the purpose of the present study was to investigate the role of DH serotonergic and γ-aminobutyric acid (GABA)ergic mechanisms in the expression of contextual fear 24 h after conditioning, reflected by fear-potentiated startle (FPS) and freezing behavior. The recruitment of the amygdala and medial prefrontal cortex (mPFC) in these processes was also evaluated by measuring Fos protein immunoreactivity. Although intra-DH injections of 8-OH-DPAT did not produce behavioral changes, muscimol reduced both FPS and the freezing response. Fos protein immunoreactivity revealed that contextual fear promoted wide activation of the mPFC, which was significantly reduced after intra-DH infusions of muscimol. The present findings, together with previous data, indicate that in contrast to 5-HT, which appears to play a role during the early phases of contextual aversive memory consolidation, longer-lasting GABA-mediated mechanisms are recruited during the expression of contextual fear memories.

  18. Microtransplantation of cellular membranes from squid stellate ganglion reveals ionotropic GABA receptors.

    PubMed

    Conti, Luca; Limon, Agenor; Palma, Eleonora; Miledi, Ricardo

    2013-02-01

    The squid has been the most studied cephalopod, and it has served as a very useful model for investigating the events associated with nerve impulse generation and synaptic transmission. While the physiology of squid giant axons has been extensively studied, very little is known about the distribution and function of the neurotransmitters and receptors that mediate inhibitory transmission at the synapses. In this study we investigated whether γ-aminobutyric acid (GABA) activates neurotransmitter receptors in stellate ganglia membranes. To overcome the low abundance of GABA-like mRNAs in invertebrates and the low expression of GABA in cephalopods, we used a two-electrode voltage clamp technique to determine if Xenopus laevis oocytes injected with cell membranes from squid stellate ganglia responded to GABA. Using this method, membrane patches containing proteins and ion channels from the squid's stellate ganglion were incorporated into the surface of oocytes. We demonstrated that GABA activates membrane receptors in cellular membranes isolated from squid stellate ganglia. Using the same approach, we were able to record native glutamate-evoked currents. The squid's GABA receptors showed an EC(50) of 98 μmol l(-1) to GABA and were inhibited by zinc (IC(50) = 356 μmol l(-1)). Interestingly, GABA receptors from the squid were only partially blocked by bicuculline. These results indicate that the microtransplantation of native cell membranes is useful to identify and characterize scarce membrane proteins. Moreover, our data also support the role of GABA as an ionotropic neurotransmitter in cephalopods, acting through chloride-permeable membrane receptors.

  19. Seizure-induced alterations in fast-spiking basket cell GABA currents modulate frequency and coherence of gamma oscillation in network simulations

    NASA Astrophysics Data System (ADS)

    Proddutur, Archana; Yu, Jiandong; Elgammal, Fatima S.; Santhakumar, Vijayalakshmi

    2013-12-01

    Gamma frequency oscillations have been proposed to contribute to memory formation and retrieval. Fast-spiking basket cells (FS-BCs) are known to underlie development of gamma oscillations. Fast, high amplitude GABA synapses and gap junctions have been suggested to contribute to gamma oscillations in FS-BC networks. Recently, we identified that, apart from GABAergic synapses, FS-BCs in the hippocampal dentate gyrus have GABAergic currents mediated by extrasynaptic receptors. Our experimental studies demonstrated two specific changes in FS-BC GABA currents following experimental seizures [Yu et al., J. Neurophysiol. 109, 1746 (2013)]: increase in the magnitude of extrasynaptic (tonic) GABA currents and a depolarizing shift in GABA reversal potential (EGABA). Here, we use homogeneous networks of a biophysically based model of FS-BCs to examine how the presence of extrasynaptic GABA conductance (gGABA-extra) and experimentally identified, seizure-induced changes in gGABA-extra and EGABA influence network activity. Networks of FS-BCs interconnected by fast GABAergic synapses developed synchronous firing in the dentate gamma frequency range (40-100 Hz). Systematic investigation revealed that the biologically realistic range of 30 to 40 connections between FS-BCs resulted in greater coherence in the gamma frequency range when networks were activated by Poisson-distributed dendritic synaptic inputs rather than by homogeneous somatic current injections, which were balanced for FS-BC firing frequency in unconnected networks. Distance-dependent conduction delay enhanced coherence in networks with 30-40 FS-BC interconnections while inclusion of gap junctional conductance had a modest effect on coherence. In networks activated by somatic current injections resulting in heterogeneous FS-BC firing, increasing gGABA-extra reduced the frequency and coherence of FS-BC firing when EGABA was shunting (-74 mV), but failed to alter average FS-BC frequency when EGABA was depolarizing

  20. Ion binding and permeation at the GABA transporter GAT1.

    PubMed

    Mager, S; Kleinberger-Doron, N; Keshet, G I; Davidson, N; Kanner, B I; Lester, H A

    1996-09-01

    This study addresses the binding of ions and the permeation of substrates during function of the GABA transporter GAT1. GAT1 was expressed in Xenopus oocytes and studied electrophysiologically as well as with [3H]GABA flux; GAT1 was also expressed in mammalian cells and studied with [3H]GABA and [3H]tiagabine binding. Voltage jumps, Na+ and Cl- concentration jumps, and exposure to high-affinity blockers (NO-05-711 and SKF-100330A) all produce capacitive charge movements. Occlusive interactions among these three types of perturbations show that they all measure the same population of charges. The concentration dependences of the charge movements reveal (1) that two Na+ ions interact with the transporter even in the absence of GABA, and (2) that Cl- facilitates the binding of Na+. Comparison between the charge movements and the transport-associated current shows that this initial Na(+)-transporter interaction limits the overall transport rate when [GABA] is saturating. However, two classes of manipulation--treatment with high-affinity uptake blockers and the W68L mutation-"lock" Na+ onto the transporter by slowing or preventing the subsequent events that release the substrates to the intracellular medium. The Na+ substitutes Li+ and Cs+ do not support charge movements, but they can permeate the transporter in an uncoupled manner. Our results (1) support the hypothesis that efficient removal of synaptic transmitter by the GABA transporter GAT1 depends on the previous binding of Na+ and Cl-, and (2) indicate the important role of the conserved putative transmembrane domain 1 in interactions with the permeant substrates. PMID:8757253

  1. Proton sensitivity of the GABA(A) receptor is associated with the receptor subunit composition.

    PubMed Central

    Krishek, B J; Amato, A; Connolly, C N; Moss, S J; Smart, T G

    1996-01-01

    1. Modulation of GABA(A) receptors by external H(+) was examined in cultured rat sympathetic neurones, and in Xenopus laevis oocytes and human embryonic kidney (HEK) cells expressing recombinant GABA(A) receptors composed of combinations of alpha 1, beta 1, beta 2, gamma 2S and delta subunits. 2. Changing the external pH from 7.4 reduced GABA-activated currents in sympathetic neurones. pH titration of the GABA-induced current was fitted with a pH model which predicted that H(+) interact with two sites (PK(a) values of 6.4 and 7.2). 3. For alpha 1 beta 1 GABA(A) receptors, low external pH (< 7.4) enhanced responses to GABA. pH titration predicted the existence of two sites with PK(a) values of 6.6 and 7.5. The GABA concentration-response curve was shifted to the left by low pH and non-competitively inhibited at high pH (> 7.4). 4. alpha 1 beta 1 gamma 2S receptor constructs were not affected by external pH, whereas exchanging the beta 1 subunit for beta 2 conferred a sensitivity to pH, with predicted PK(a) values of 5.16 and 9.44. 5. Low pH enhanced the responses to GABA on alpha 1 beta 1 delta subunits, whilst high pH caused an inhibition (PK(a) values of 6.6 and 9.9). The GABA concentration-response curves were enhanced (pH 5.4) or reduced (pH 9.4) with no changes in the GABA EC(50). 6. Immunoprecipitation with subunit and epitope-specific antisera to alpha 1, beta 1 and delta subunits demonstrated that these subunits could co-assemble in cell membranes. 7. Expression of alpha 1 beta 1 gamma 2S delta constructs resulted in a 'bell-shaped' pH titration relationship. Increasing or decreasing external pH inhibited the responses to GABA. 8. The pH sensitivity of recombinant GABA(A) receptors expressed in HEK cells was generally in accordance with data accrued from Xenopus oocytes. However, rapid application of GABA to alpha 1 beta 1 constructs at high pH (> 7.4) caused an increased peak and reduced steady-state current, with a correspondingly increased rate of

  2. Immunoreactivity for GABA, GAD65, GAD67 and Bestrophin-1 in the meninges and the choroid plexus: implications for non-neuronal sources for GABA in the developing mouse brain.

    PubMed

    Tochitani, Shiro; Kondo, Shigeaki

    2013-01-01

    Neural progenitors in the developing neocortex, neuroepithelial cells and radial glial cells, have a bipolar shape with a basal process contacting the basal membrane of the meninge and an apical plasma membrane facing the lateral ventricle, which the cerebrospinal fluid is filled with. Recent studies revealed that the meninges and the cerebrospinal fluid have certain roles to regulate brain development. γ-aminobutyric acid (GABA) is a neurotransmitter which appears first during development and works as a diffusible factor to regulate the properties of neural progenitors. In this study, we examined whether GABA can be released from the meninges and the choroid plexus in the developing mouse brain. Immunohistochemical analyses showed that glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67), both of which are GABA-synthesizing enzymes, are expressed in the meninges. The epithelial cells in the choroid plexus express GAD65. GABA immunoreactivity could be observed beneath the basal membrane of the meninge and in the epithelial cells of the choroid plexus. Expression analyses on Bestrophin-1, which is known as a GABA-permeable channel in differentiated glial cells, suggested that the cells in the meninges and the epithelial cells in the choroid plexus have the channels able to permeate non-synaptic GABA into the extracellular space. Further studies showed that GAD65/67-expressing meningeal cells appear in a manner with rostral to caudal and lateral to dorsal gradient to cover the entire neocortex by E14.5 during development, while the cells in the choroid plexus in the lateral ventricle start to express GAD65 on E11-E12, the time when the choroid plexus starts to develop in the developing brain. These results totally suggest that the meninges and the choroid plexus can work as non-neuronal sources for ambient GABA which can modulate the properties of neural progenitors during neocortical development.

  3. The transporter GAT1 plays an important role in GABA-mediated carbon-nitrogen interactions in Arabidopsis

    PubMed Central

    Batushansky, Albert; Kirma, Menny; Grillich, Nicole; Pham, Phuong A.; Rentsch, Doris; Galili, Gad; Fernie, Alisdair R.; Fait, Aaron

    2015-01-01

    Glutamate derived γ-aminobutyric acid (GABA) is synthetized in the cytosol prior to delivery to the mitochondria where it is catabolized via the TCA cycle. GABA accumulates under various environmental conditions, but an increasing number of studies show its involvement at the crossroad between C and N metabolism. To assess the role of GABA in modulating cellular metabolism, we exposed seedlings of A. thaliana GABA transporter gat1 mutant to full nutrition medium and media deficient in C and N combined with feeding of different concentrations (0.5 and 1 mM) of exogenous GABA. GC-MS based metabolite profiling showed an expected effect of medium composition on the seedlings metabolism of mutant and wild type alike. That being said, a significant interaction between GAT1 deficiency and medium composition was determined with respect to magnitude of change in relative amino acid levels. The effect of exogenous GABA treatment on metabolism was contingent on both the medium and the genotype, leading for instance to a drop in asparagine under full nutrition and low C conditions and glucose under all tested media, but not to changes in GABA content. We additionally assessed the effect of GAT1 deficiency on the expression of glutamate metabolism related genes and genes involved in abiotic stress responses. These results suggest a role for GAT1 in GABA-mediated metabolic alterations in the context of the C-N equilibrium of plant cells. PMID:26483804

  4. Gamma-amino butyric acid (GABA) release in the ciliated protozoon Paramecium occurs by neuronal-like exocytosis.

    PubMed

    Ramoino, P; Milanese, M; Candiani, S; Diaspro, A; Fato, M; Usai, C; Bonanno, G

    2010-04-01

    Paramecium primaurelia expresses a significant amount of gamma-amino butyric acid (GABA). Paramecia possess both glutamate decarboxylase (GAD)-like and vesicular GABA transporter (vGAT)-like proteins, indicating the ability to synthesize GABA from glutamate and to transport GABA into vesicles. Using antibodies raised against mammalian GAD and vGAT, bands with an apparent molecular weight of about 67 kDa and 57 kDa were detected. The presence of these bands indicated a similarity between the proteins in Paramecium and in mammals. VAMP, syntaxin and SNAP, putative proteins of the release machinery that form the so-called SNARE complex, are present in Paramecium. Most VAMP, syntaxin and SNAP fluorescence is localized in spots that vary in size and density and are primarily distributed near the plasma membrane. Antibodies raised against mammal VAMP-3, sintaxin-1 or SNAP-25 revealed protein immunoblot bands having molecular weights consistent with those observed in mammals. Moreover, P. primaurelia spontaneously releases GABA into the environment, and this neurotransmitter release significantly increases after membrane depolarization. The depolarization-induced GABA release was strongly reduced not only in the absence of extracellular Ca(2+) but also by pre-incubation with bafilomycin A1 or with botulinum toxin C1 serotype. It can be concluded that GABA occurs in Paramecium, where it is probably stored in vesicles capable of fusion with the cell membrane; accordingly, GABA can be released from Paramecium by stimulus-induced, neuronal-like exocytotic mechanisms.

  5. GABA[subscript A] Receptor Downregulation in Brains of Subjects with Autism

    ERIC Educational Resources Information Center

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Thuras, Paul D.

    2009-01-01

    Gamma-aminobutyric acid A (GABA[subscript A]) receptors are ligand-gated ion channels responsible for mediation of fast inhibitory action of GABA in the brain. Preliminary reports have demonstrated altered expression of GABA receptors in the brains of subjects with autism suggesting GABA/glutamate system dysregulation. We investigated the…

  6. Novel dose-dependent alterations in excitatory GABA during embryonic development associated with lead (Pb) neurotoxicity

    PubMed Central

    Wirbisky, Sara E.; Weber, Gregory J.; Lee, Jang-Won; Cannon, Jason R.; Freeman, Jennifer L.

    2014-01-01

    Lead (Pb) is a heavy metal that is toxic to numerous physiological processes. Its use in industrial applications is widespread and results in an increased risk of human environmental exposure. The central nervous system (CNS) is most sensitive to Pb exposure during early development due to rapid cell proliferation and migration, axonal growth, and synaptogenesis. One of the key components of CNS development is the Gamma-aminobutyric acid (GABA)ergic system. GABA is the primary inhibitory neurotransmitter in the adult brain. However, during development GABA acts as an excitatory neurotrophic factor which contributes to these cellular processes. Multiple studies report effects of Pb on GABA in the mature brain; however, little is known regarding the adverse effects of Pb exposure on the GABAergic system during embryonic development. To characterize the effects of Pb on the GABAergic system during development, zebrafish embryos were exposed to 10, 50, or 100 ppb Pb or a control treatment. Tissue up-take, gross morphological alterations, gene expression, and neurotransmitter levels were analyzed. Analysis revealed that alterations in gene expression throughout the GABAergic system and GABA levels were dose and developmental time point specific. These data provide a framework for further analysis of the effects of Pb on the GABAergic system during the excitatory phase and as GABA transitions to an inhibitory neurotransmitter during development. PMID:24875535

  7. Activation of GABA(B) receptors inhibits protein kinase B/glycogen synthase kinase 3 signaling.

    PubMed

    Lu, Frances Fangjia; Su, Ping; Liu, Fang; Daskalakis, Zafiris J

    2012-11-28

    Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt)/glycogen synthase kinase (GSK)-3 signaling. Here we report that activation of GABA(B) receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABA(B) receptors enhances the phosphorylation of Akt (Thr-308) and enhances the phosphorylation of GSK-3α (Ser-21)/β (Ser-9) in both HEK-293T cells expressing GABA(B) receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABA(B) receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABA(B) receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  8. Differential expression of gamma-aminobutyric acid type B receptor subunit mRNAs in the developing nervous system and receptor coupling to adenylyl cyclase in embryonic neurons.

    PubMed

    Martin, Stella C; Steiger, Janine L; Gravielle, María Clara; Lyons, Helen R; Russek, Shelley J; Farb, David H

    2004-05-17

    gamma-Aminobutyric acid type B receptors (GABA(B)Rs) mediate both slow inhibitory synaptic activity in the adult nervous system and motility signals for migrating embryonic cortical cells. Previous papers have described the expression of GABA(B)Rs in the adult brain, but the expression and functional significance of these gene products in the embryo are largely unknown. Here we examine GABA(B)R expression from rat embryonic day 10 (E10) to E18 compared with adult and ask whether embryonic cortical neurons contain functional GABA(B)R. GABA(B)R1 transcript levels greatly exceed GABA(B)R2 levels in the developing neural tube at E11, and olfactory bulb and striatum at E17 but equalize in most regions of adult nervous tissue, except for the glomerular and granule cell layers of the main olfactory bulb and the striatum. Consistent with expression differences, the binding affinity of GABA for GABA(B)Rs is significantly lower in adult striatum compared with cerebellum. Multiple lines of evidence from in situ hybridization, RNase protection, and real-time PCR demonstrate that GABA(B)R1a, GABA(B)R1b, GABA(B)R1h (a subunit subtype, lacking a sushi domain, that we have identified in embryonic rat brain), GABA(B)R2, and GABA(B)L transcript levels are not coordinately regulated. Despite the functional requirement for a heterodimer of GABA(B)R subunits, the expression of each subunit mRNA is under independent control during embryonic development, and, by E18, GABA(B)Rs are negatively coupled to adenylyl cyclase in neocortical neurons. The presence of embryonic GABA(B)R transcripts and protein and functional receptor coupling indicates potentially important roles for GABA(B)Rs in modulation of synaptic transmission in the developing embryonic nervous system.

  9. Neural Stem Cell or Human Induced Pluripotent Stem Cell-Derived GABA-ergic Progenitor Cell Grafting in an Animal Model of Chronic Temporal Lobe Epilepsy.

    PubMed

    Upadhya, Dinesh; Hattiangady, Bharathi; Shetty, Geetha A; Zanirati, Gabriele; Kodali, Maheedhar; Shetty, Ashok K

    2016-01-01

    Grafting of neural stem cells (NSCs) or GABA-ergic progenitor cells (GPCs) into the hippocampus could offer an alternative therapy to hippocampal resection in patients with drug-resistant chronic epilepsy, which afflicts >30% of temporal lobe epilepsy (TLE) cases. Multipotent, self-renewing NSCs could be expanded from multiple regions of the developing and adult brain, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). On the other hand, GPCs could be generated from the medial and lateral ganglionic eminences of the embryonic brain and from hESCs and hiPSCs. To provide comprehensive methodologies involved in testing the efficacy of transplantation of NSCs and GPCs in a rat model of chronic TLE, NSCs derived from the rat medial ganglionic eminence (MGE) and MGE-like GPCs derived from hiPSCs are taken as examples in this unit. The topics comprise description of the required materials, reagents and equipment, methods for obtaining rat MGE-NSCs and hiPSC-derived MGE-like GPCs in culture, generation of chronically epileptic rats, intrahippocampal grafting procedure, post-grafting evaluation of the effects of grafts on spontaneous recurrent seizures and cognitive and mood impairments, analyses of the yield and the fate of graft-derived cells, and the effects of grafts on the host hippocampus. © 2016 by John Wiley & Sons, Inc. PMID:27532817

  10. Neural Stem Cell or Human Induced Pluripotent Stem Cell-Derived GABA-ergic Progenitor Cell Grafting in an Animal Model of Chronic Temporal Lobe Epilepsy.

    PubMed

    Upadhya, Dinesh; Hattiangady, Bharathi; Shetty, Geetha A; Zanirati, Gabriele; Kodali, Maheedhar; Shetty, Ashok K

    2016-08-17

    Grafting of neural stem cells (NSCs) or GABA-ergic progenitor cells (GPCs) into the hippocampus could offer an alternative therapy to hippocampal resection in patients with drug-resistant chronic epilepsy, which afflicts >30% of temporal lobe epilepsy (TLE) cases. Multipotent, self-renewing NSCs could be expanded from multiple regions of the developing and adult brain, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). On the other hand, GPCs could be generated from the medial and lateral ganglionic eminences of the embryonic brain and from hESCs and hiPSCs. To provide comprehensive methodologies involved in testing the efficacy of transplantation of NSCs and GPCs in a rat model of chronic TLE, NSCs derived from the rat medial ganglionic eminence (MGE) and MGE-like GPCs derived from hiPSCs are taken as examples in this unit. The topics comprise description of the required materials, reagents and equipment, methods for obtaining rat MGE-NSCs and hiPSC-derived MGE-like GPCs in culture, generation of chronically epileptic rats, intrahippocampal grafting procedure, post-grafting evaluation of the effects of grafts on spontaneous recurrent seizures and cognitive and mood impairments, analyses of the yield and the fate of graft-derived cells, and the effects of grafts on the host hippocampus. © 2016 by John Wiley & Sons, Inc.

  11. Mechanisms of induction and expression of long-term depression at GABAergic synapses in the neonatal rat hippocampus.

    PubMed

    Caillard, O; Ben-Ari, Y; Gaïarsa, J L

    1999-09-01

    Synaptic plasticity at excitatory glutamatergic synapses is believed to be instrumental in the maturation of neuronal networks. Using whole-cell patch-clamp recordings, we have studied the mechanisms of induction and expression of long-term depression at excitatory GABAergic synapses in the neonatal rat hippocampus (LTD(GABA-A)). We report that the induction of LTD(GABA-A) requires a GABA(A) receptor-mediated membrane depolarization, which is necessary to remove the Mg(2+) block from postsynaptic NMDA receptors. LTD(GABA-A) is associated with an increase in the coefficient of variation of evoked GABA(A) receptor-mediated synaptic currents and a decrease in the frequency, but not amplitude, of Sr(2+)-induced asynchronous GABA(A) quantal events. We conclude that LTD(GABA-A) induction requires the activation of both GABA(A) and NMDA postsynaptic receptors and that its expression is likely presynaptic.

  12. The betaine-GABA transporter (BGT1, slc6a12) is predominantly expressed in the liver and at lower levels in the kidneys and at the brain surface.

    PubMed

    Zhou, Y; Holmseth, S; Hua, R; Lehre, A C; Olofsson, A M; Poblete-Naredo, I; Kempson, S A; Danbolt, N C

    2012-02-01

    The Na(+)- and Cl(-)-dependent GABA-betaine transporter (BGT1) has received attention mostly as a protector against osmolarity changes in the kidney and as a potential controller of the neurotransmitter GABA in the brain. Nevertheless, the cellular distribution of BGT1, and its physiological importance, is not fully understood. Here we have quantified mRNA levels using TaqMan real-time PCR, produced a number of BGT1 antibodies, and used these to study BGT1 distribution in mice. BGT1 (protein and mRNA) is predominantly expressed in the liver (sinusoidal hepatocyte plasma membranes) and not in the endothelium. BGT1 is also present in the renal medulla, where it localizes to the basolateral membranes of collecting ducts (particularly at the papilla tip) and the thick ascending limbs of Henle. There is some BGT1 in the leptomeninges, but brain parenchyma, brain blood vessels, ependymal cells, the renal cortex, and the intestine are virtually BGT1 deficient in 1- to 3-mo-old mice. Labeling specificity was assured by processing tissue from BGT1-deficient littermates in parallel as negative controls. Addition of 2.5% sodium chloride to the drinking water for 48 h induced a two- to threefold upregulation of BGT1, tonicity-responsive enhancer binding protein, and sodium-myo-inositol cotransporter 1 (slc5a3) in the renal medulla, but not in the brain and barely in the liver. BGT1-deficient and wild-type mice appeared to tolerate the salt treatment equally well, possibly because betaine is one of several osmolytes. In conclusion, this study suggests that BGT1 plays its main role in the liver, thereby complementing other betaine-transporting carrier proteins (e.g., slc6a20) that are predominantly expressed in the small intestine or kidney rather than the liver. PMID:22071246

  13. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A) receptor-mediated signaling.

    PubMed

    Kakehashi, Anna; Kato, Ayumi; Ishii, Naomi; Wei, Min; Morimura, Keiichirou; Fukushima, Shoji; Wanibuchi, Hideki

    2014-01-01

    Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis) at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P(+)) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2'-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P(+) foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21(Waf1/Cip1), p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P(+) foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P(+) foci by activating GABA(A)R-mediated signaling. PMID:25419570

  14. Double isotopic method using dansyl chloride for the determination of GABA in rat C6 astrocytoma cell cultures

    SciTech Connect

    Kohl, R.L.; Quay, W.B.; Perez-Polo, J.R.

    1986-01-01

    Methods are described for the quantitative measurement of GABA in culture. The method can be adapted to any amino acid or dansyl-chloride-reactive species. The sensitivity and selectivity of the procedure result from the double isotopic design in which (/sup 14/C)-labeled internal standard was added to the samples before reaction with (3M)-labeled dansyl chloride. Values obtained by ion-exchange amino acid analysis of cultures agree closely with the values obtained by the double isotopic method. This method is sensitive enough to measure GABA intracellularly and the condition medium.

  15. Distribution of 3H-GABA uptake sites in the nematode Ascaris

    SciTech Connect

    Guastella, J.; Stretton, A.O. )

    1991-05-22

    The distribution of uptake sites for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the nematode Ascaris suum was examined by autoradiography of 3H-GABA uptake. Single neural processes in both the ventral and dorsal nerve cords were labeled with 3H-GABA. Serial section analysis identified the cells of origin of these processes as the RMEV-like and RMED-like neurons. These cells belong to a set of four neurons in the nerve ring, all of which are labeled by 3H-GABA. 3H-GABA labeling of at least two other sets of cephalic neurons was seen. One of these pairs consists of medium-sized lateral ganglia neurons, located at the level of the amphid commissure bundle. A second pair is located in the lateral ganglia at the level of the deirid commissure bundle. The position and size of these lateral ganglia cells suggest that they are the GABA-immunoreactive lateral ganglia cells frequently seen in whole-mount immunocytochemical preparations. Four neuronal cell bodies located in the retrovesicular ganglion were also labeled with 3H-GABA. These cells, which are probably cholinergic excitatory motor neurons, do not contain detectable GABA-like immunoreactivity. Heavy labeling of muscle cells was also observed. The ventral and dorsal nerve cord inhibitory motor neurons, which are known to contain GABA-like immunoreactivity, were not labeled above background with 3H-GABA. Together with the experiments reported previously, these results define three classes of GABA-associated neurons in Ascaris: (1) neurons that contain endogenous GABA and possess a GABA uptake system; (2) neurons that contain endogenous GABA, but that either lack a GABA uptake system or possess a GABA uptake system of low activity; (3) neurons that possess a GABA uptake system, but that lack endogenous GABA.

  16. Genetic association of ErbB4 and Human Cortical GABA levels in-vivo

    PubMed Central

    Marenco, Stefano; Geramita, Matthew; van der Veen, Jan Willem; Barnett, Alan S.; Kolachana, Bhaskar; Shen, Jun; Weinberger, Daniel R.; Law, Amanda J.

    2011-01-01

    NRG1-ErbB4 signaling controls inhibitory circuit development in the mammalian cortex through ErbB4 dependent regulation of GABAergic interneuron connectivity. Common genetic variation in ErbB4 (rs7598440) has been associated with ErbB4 messenger RNA levels in the human cortex and risk for schizophrenia. Recent work demonstrates that Erbb4 is expressed exclusively on inhibitory interneurons, where its presence on parvalbumin positive cells mediates the effects of NRG1 on inhibitory circuit formation in the cortex. We therefore hypothesized that genetic variation in ErbB4 at rs7598440 would impact indices of GABA concentration in the human cortex. We tested this hypothesis in 116 healthy volunteers by measuring GABA and GLX (glutamate + glutamine) with proton magnetic resonance spectroscopy (MRS) in the dorsal anterior cingulate gyrus. ErbB4 rs7598440 genotype significantly predicted cortical GABA concentration (p=0.014), but not GLX (p=0.51), with A allele carriers having higher GABA as predicted by their impact on ErbB4 expression. These data establish an association of ErbB4 and GABA in human brain and have implications for understanding the pathogenesis of schizophrenia and other psychiatric disorders. PMID:21832192

  17. Setting the time course of inhibitory synaptic currents by mixing multiple GABA(A) receptor α subunit isoforms.

    PubMed

    Eyre, Mark D; Renzi, Massimiliano; Farrant, Mark; Nusser, Zoltan

    2012-04-25

    The kinetics of IPSCs influence many neuronal processes, such as the frequencies of oscillations and the duration of shunting inhibition. The subunit composition of recombinant GABA(A) receptors (GABA(A)Rs) strongly affects the deactivation kinetics of GABA-evoked currents. However, for GABAergic synapses, the relationship between subunit composition and IPSC decay is less clear. Here we addressed this by combining whole-cell recordings of miniature IPSCs (mIPSCs) and quantitative immunolocalization of synaptic GABA(A)R subunits. In cerebellar stellate, thalamic relay, and main olfactory bulb (MOB) deep short-axon cells of Wistar rats, the only synaptic α subunit was α1, and zolpidem-sensitive mIPSCs had weighted decay time constants (τ(w)) of 4-6 ms. Nucleus reticularis thalami neurons expressed only α3 as the synaptic α subunit and exhibited slow (τ(w) = 28 ms), zolpidem-insensitive mIPSCs. By contrast, MOB external tufted cells contained two α subunit types (α1 and α3) at their synapses. Quantitative analysis of multiple immunolabeled images revealed small within-cell, but large between-cell, variability in synaptic α1/α3 ratios. This corresponded to large cell-to-cell variability in the decay (τ(w) = 3-30 ms) and zolpidem sensitivity of mIPSCs. Currents evoked by rapid application of GABA to patches excised from HEK cells expressing different mixtures of α1 and α3 subunits displayed highly variable deactivation times that correlated with the α1/α3 cDNA ratio. Our results demonstrate that diversity in the decay of IPSCs can be generated by varying the expression of different GABA(A)R subunits that alone confer different decay kinetics, allowing the time course of inhibition to be tuned to individual cellular requirements.

  18. GABA predicts visual intelligence.

    PubMed

    Cook, Emily; Hammett, Stephen T; Larsson, Jonas

    2016-10-01

    Early psychological researchers proposed a link between intelligence and low-level perceptual performance. It was recently suggested that this link is driven by individual variations in the ability to suppress irrelevant information, evidenced by the observation of strong correlations between perceptual surround suppression and cognitive performance. However, the neural mechanisms underlying such a link remain unclear. A candidate mechanism is neural inhibition by gamma-aminobutyric acid (GABA), but direct experimental support for GABA-mediated inhibition underlying suppression is inconsistent. Here we report evidence consistent with a global suppressive mechanism involving GABA underlying the link between sensory performance and intelligence. We measured visual cortical GABA concentration, visuo-spatial intelligence and visual surround suppression in a group of healthy adults. Levels of GABA were strongly predictive of both intelligence and surround suppression, with higher levels of intelligence associated with higher levels of GABA and stronger surround suppression. These results indicate that GABA-mediated neural inhibition may be a key factor determining cognitive performance and suggests a physiological mechanism linking surround suppression and intelligence. PMID:27495012

  19. Systematic analysis of γ-aminobutyric acid (GABA) metabolism and function in the social amoeba Dictyostelium discoideum.

    PubMed

    Wu, Yuantai; Janetopoulos, Chris

    2013-05-24

    While GABA has been suggested to regulate spore encapsulation in the social amoeba Dictyostelium discoideum, the metabolic profile and other potential functions of GABA during development remain unclear. In this study, we investigated the homeostasis of GABA metabolism by disrupting genes related to GABA metabolism and signaling. Extracellular levels of GABA are tightly regulated during early development, and GABA is generated by the glutamate decarboxylase, GadB, during growth and in early development. However, overexpression of the prespore-specific homologue, GadA, in the presence of GadB reduces production of extracellular GABA. Perturbation of extracellular GABA levels delays the process of aggregation. Cytosolic GABA is degraded by the GABA transaminase, GabT, in the mitochondria. Disruption of a putative vesicular GABA transporter (vGAT) homologue DdvGAT reduces secreted GABA. We identified the GABAB receptor-like family member GrlB as the major GABA receptor during early development, and either disruption or overexpression of GrlB delays aggregation. This delay is likely the result of an abolished pre-starvation response and late expression of several "early" developmental genes. Distinct genes are employed for GABA generation during sporulation. During sporulation, GadA alone is required for generating GABA and DdvGAT is likely responsible for GABA secretion. GrlE but not GrlB is the GABA receptor during late development.

  20. Modulation of spontaneous intracellular Ca²⁺ fluctuations and spontaneous cholinergic transmission in rat chromaffin cells in situ by endogenous GABA acting on GABAA receptors.

    PubMed

    Tzitzitlini, Alejandre-García; Pedro, Segura-Chama; Martha, Pérez-Armendáriz E; Rodolfo, Delgado-Lezama; Arturo, Hernández-Cruz

    2016-02-01

    Using fluorescence [Ca(2+)]i imaging in rat adrenal slices, we characterized the effects of agonists and antagonists of the GABAA receptor (GABAA-R) on resting intracellular Ca(2+) ([Ca(2+)]i) and spontaneous [Ca(2+)]i fluctuations (SCFs) in hundreds of individual chromaffin cells (CCs) recorded simultaneously in situ. Muscimol, a GABAA-R agonist (20 μM; 25 s), induced an increase of resting [Ca(2+)]i in 43 ± 3 % of CCs, a decrease in 26 ± 2 %, and no response in 30 ± 5 %. In Ca(2+)-free external medium, SCFs ceased completely and muscimol failed to elicit [Ca(2+)]i rises. All muscimol-induced [Ca(2+)]i changes were blocked by the GABAA-R antagonist bicuculline, suggesting that they result from changes in membrane potential depending on the cell's Cl(-) equilibrium potential. Unexpectedly, bicuculline increased the amplitude and frequency of SCFs in 54 % of CCs, revealing a tonic inhibition of SCFs by ambient GABA acting through GABAA-R. Mecamylamine (a specific nicotinic cholinergic blocker) decreased basal SCF activity in 18 % of CCs and inhibited bicuculline-induced SCF intensification, suggesting that spontaneous acetylcholine (ACh) release from nerve endings contributes to SCF generation in CCs in situ and that blockade of presynaptic GABAA-Rs intensifies SCFs in part through the disinhibition of spontaneous cholinergic transmission. Electrophysiological experiments confirmed that spontaneous excitatory postsynaptic currents recorded from CCs in situ were enhanced by bicuculline. To our knowledge, this is the first description of a regulatory effect of endogenous GABA on synaptic currents and SCFs of adrenal CCs. These findings denote a novel GABA-mediated presynaptic and postsynaptic regulatory mechanism of CC activity which may participate in the control of catecholamine secretion.

  1. Identification of the First Highly Subtype-Selective Inhibitor of Human GABA Transporter GAT3.

    PubMed

    Damgaard, Maria; Al-Khawaja, Anas; Vogensen, Stine B; Jurik, Andreas; Sijm, Maarten; Lie, Maria E K; Bæk, Mathias I; Rosenthal, Emil; Jensen, Anders A; Ecker, Gerhard F; Frølund, Bente; Wellendorph, Petrine; Clausen, Rasmus P

    2015-09-16

    Screening a library of small-molecule compounds using a cell line expressing human GABA transporter 3 (hGAT3) in a [(3)H]GABA uptake assay identified isatin derivatives as a new class of hGAT3 inhibitors. A subsequent structure-activity relationship (SAR) study led to the identification of hGAT3-selective inhibitors (i.e., compounds 20 and 34) that were superior to the reference hGAT3 inhibitor, (S)-SNAP-5114, in terms of potency (low micromolar IC50 values) and selectivity (>30-fold selective for hGAT3 over hGAT1/hGAT2/hBGT1). Further pharmacological characterization of compound 20 (5-(thiophen-2-yl)indoline-2,3-dione) revealed a noncompetitive mode of inhibition at hGAT3. This suggests that this compound class, which has no structural resemblance to GABA, has a binding site different from the substrate, GABA. This was supported by a molecular modeling study that suggested a unique binding site that matched the observed selectivity, inhibition kinetics, and SAR of the compound series. These compounds are the most potent GAT3 inhibitors reported to date that provide selectivity for GAT3 over other GABA transporter subtypes.

  2. Single-Cell Gene Expression Analysis of Cholinergic Neurons in the Arcuate Nucleus of the Hypothalamus

    PubMed Central

    Chua, Streamson; Jo, Young-Hwan

    2016-01-01

    The cholinoceptive system in the hypothalamus, in particular in the arcuate nucleus (ARC), plays a role in regulating food intake. Neurons in the ARC contain multiple neuropeptides, amines, and neurotransmitters. To study molecular and neurochemical heterogeneity of ARC neurons, we combine single-cell qRT-PCR and single-cell whole transcriptome amplification methods to analyze expression patterns of our hand-picked 60 genes in individual neurons in the ARC. Immunohistochemical and single-cell qRT-PCR analyses show choline acetyltransferase (ChAT)-expressing neurons in the ARC. Gene expression patterns are remarkably distinct in each individual cholinergic neuron. Two-thirds of cholinergic neurons express tyrosine hydroxylase (Th) mRNA. A large subset of these Th-positive cholinergic neurons is GABAergic as they express the GABA synthesizing enzyme glutamate decarboxylase and vesicular GABA transporter transcripts. Some cholinergic neurons also express the vesicular glutamate transporter transcript gene. POMC and POMC-processing enzyme transcripts are found in a subpopulation of cholinergic neurons. Despite this heterogeneity, gene expression patterns in individual cholinergic cells appear to be highly regulated in a cell-specific manner. In fact, membrane receptor transcripts are clustered with their respective intracellular signaling and downstream targets. This novel population of cholinergic neurons may be part of the neural circuitries that detect homeostatic need for food and control the drive to eat. PMID:27611685

  3. Single-Cell Gene Expression Analysis of Cholinergic Neurons in the Arcuate Nucleus of the Hypothalamus.

    PubMed

    Jeong, Jae Hoon; Woo, Young Jae; Chua, Streamson; Jo, Young-Hwan

    2016-01-01

    The cholinoceptive system in the hypothalamus, in particular in the arcuate nucleus (ARC), plays a role in regulating food intake. Neurons in the ARC contain multiple neuropeptides, amines, and neurotransmitters. To study molecular and neurochemical heterogeneity of ARC neurons, we combine single-cell qRT-PCR and single-cell whole transcriptome amplification methods to analyze expression patterns of our hand-picked 60 genes in individual neurons in the ARC. Immunohistochemical and single-cell qRT-PCR analyses show choline acetyltransferase (ChAT)-expressing neurons in the ARC. Gene expression patterns are remarkably distinct in each individual cholinergic neuron. Two-thirds of cholinergic neurons express tyrosine hydroxylase (Th) mRNA. A large subset of these Th-positive cholinergic neurons is GABAergic as they express the GABA synthesizing enzyme glutamate decarboxylase and vesicular GABA transporter transcripts. Some cholinergic neurons also express the vesicular glutamate transporter transcript gene. POMC and POMC-processing enzyme transcripts are found in a subpopulation of cholinergic neurons. Despite this heterogeneity, gene expression patterns in individual cholinergic cells appear to be highly regulated in a cell-specific manner. In fact, membrane receptor transcripts are clustered with their respective intracellular signaling and downstream targets. This novel population of cholinergic neurons may be part of the neural circuitries that detect homeostatic need for food and control the drive to eat. PMID:27611685

  4. Gene expression analysis of CL-20-induced reversible neurotoxicity reveals GABA(A) receptors as potential targets in the earthworm Eisenia fetida.

    PubMed

    Gong, Ping; Guan, Xin; Pirooznia, Mehdi; Liang, Chun; Perkins, Edward J

    2012-01-17

    The earthworm Eisenia fetida is one of the most used species in standardized soil ecotoxicity tests. End points such as survival, growth, and reproduction are eco-toxicologically relevant but provide little mechanistic insight into toxicity pathways, especially at the molecular level. Here we apply a toxicogenomic approach to investigate the mode of action underlying the reversible neurotoxicity of hexanitrohexaazaisowurtzitane (CL-20), a cyclic nitroamine explosives compound. We developed an E. fetida-specific shotgun microarray targeting 15119 unique E. fetida transcripts. Using this array we profiled gene expression in E. fetida in response to exposure to CL-20. Eighteen earthworms were exposed for 6 days to 0.2 μg/cm(2) of CL-20 on filter paper, half of which were allowed to recover in a clean environment for 7 days. Nine vehicle control earthworms were sacrificed at days 6 and 13, separately. Electrophysiological measurements indicated that the conduction velocity of earthworm medial giant nerve fiber decreased significantly after 6-day exposure to CL-20, but was restored after 7 days of recovery. Total RNA was isolated from the four treatment groups including 6-day control, 6-day exposed, 13-day control, and 13-day exposed (i.e., 6-day exposure followed by 7-day recovery), and was hybridized to the 15K shotgun oligo array. Statistical and bioinformatic analyses suggest that CL-20 initiated neurotoxicity by noncompetitively blocking the ligand-gated GABA(A) receptor ion channel, leading to altered expression of genes involved in GABAergic, cholinergic, and Agrin-MuSK pathways. In the recovery phase, expression of affected genes returned to normality, possibly as a result of autophagy and CL-20 dissociation/metabolism. This study provides significant insights into potential mechanisms of CL-20-induced neurotoxicity and the recovery of earthworms from transient neurotoxicity stress.

  5. Gene expression analysis of CL-20-induced reversible neurotoxicity reveals GABA(A) receptors as potential targets in the earthworm Eisenia fetida.

    PubMed

    Gong, Ping; Guan, Xin; Pirooznia, Mehdi; Liang, Chun; Perkins, Edward J

    2012-01-17

    The earthworm Eisenia fetida is one of the most used species in standardized soil ecotoxicity tests. End points such as survival, growth, and reproduction are eco-toxicologically relevant but provide little mechanistic insight into toxicity pathways, especially at the molecular level. Here we apply a toxicogenomic approach to investigate the mode of action underlying the reversible neurotoxicity of hexanitrohexaazaisowurtzitane (CL-20), a cyclic nitroamine explosives compound. We developed an E. fetida-specific shotgun microarray targeting 15119 unique E. fetida transcripts. Using this array we profiled gene expression in E. fetida in response to exposure to CL-20. Eighteen earthworms were exposed for 6 days to 0.2 μg/cm(2) of CL-20 on filter paper, half of which were allowed to recover in a clean environment for 7 days. Nine vehicle control earthworms were sacrificed at days 6 and 13, separately. Electrophysiological measurements indicated that the conduction velocity of earthworm medial giant nerve fiber decreased significantly after 6-day exposure to CL-20, but was restored after 7 days of recovery. Total RNA was isolated from the four treatment groups including 6-day control, 6-day exposed, 13-day control, and 13-day exposed (i.e., 6-day exposure followed by 7-day recovery), and was hybridized to the 15K shotgun oligo array. Statistical and bioinformatic analyses suggest that CL-20 initiated neurotoxicity by noncompetitively blocking the ligand-gated GABA(A) receptor ion channel, leading to altered expression of genes involved in GABAergic, cholinergic, and Agrin-MuSK pathways. In the recovery phase, expression of affected genes returned to normality, possibly as a result of autophagy and CL-20 dissociation/metabolism. This study provides significant insights into potential mechanisms of CL-20-induced neurotoxicity and the recovery of earthworms from transient neurotoxicity stress. PMID:22191394

  6. Accumulation of GABAergic Neurons, Causing a Focal Ambient GABA Gradient, and Downregulation of KCC2 Are Induced During Microgyrus Formation in a Mouse Model of Polymicrogyria

    PubMed Central

    Wang, Tianying; Kumada, Tatsuro; Morishima, Toshitaka; Iwata, Satomi; Kaneko, Takeshi; Yanagawa, Yuchio; Yoshida, Sachiko; Fukuda, Atsuo

    2014-01-01

    Although focal cortical malformations are considered neuronal migration disorders, their formation mechanisms remain unknown. We addressed how the γ-aminobutyric acid (GABA)ergic system affects the GABAergic and glutamatergic neuronal migration underlying such malformations. A focal freeze-lesion (FFL) of the postnatal day zero (P0) glutamic acid decarboxylase–green fluorescent protein knock-in mouse neocortex produced a 3- or 4-layered microgyrus at P7. GABAergic interneurons accumulated around the necrosis including the superficial region during microgyrus formation at P4, whereas E17.5-born, Cux1-positive pyramidal neurons outlined the GABAergic neurons and were absent from the superficial layer, forming cell-dense areas in layer 2 of the P7 microgyrus. GABA imaging showed that an extracellular GABA level temporally increased in the GABAergic neuron-positive area, including the necrotic center, at P4. The expression of the Cl– transporter KCC2 was downregulated in the microgyrus-forming GABAergic and E17.5-born glutamatergic neurons at P4; these cells may need a high intracellular Cl– concentration to induce depolarizing GABA effects. Bicuculline decreased the frequency of spontaneous Ca2+ oscillations in these microgyrus-forming cells. Thus, neonatal FFL causes specific neuronal accumulation, preceded by an increase in ambient GABA during microgyrus formation. This GABA increase induces GABAA receptor-mediated Ca2+ oscillation in KCC2-downregulated microgyrus-forming cells, as seen in migrating cells during early neocortical development. PMID:23246779

  7. Enhancement by GABA of the association rate of picrotoxin and tert-butylbicyclophosphorothionate to the rat cloned alpha 1 beta 2 gamma 2 GABAA receptor subtype.

    PubMed Central

    Dillon, G. H.; Im, W. B.; Carter, D. B.; McKinley, D. D.

    1995-01-01

    1. We examined how gamma-aminobutyric acid (GABA) influences interaction of picrotoxin and tert-butylbicyclophosphorothionate (TBPS) with recombinant rat alpha 1 beta 2 gamma 2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl- currents measured by the whole-cell patch clamp technique. 2. During application of GABA (5 microM) for 15 s, picrotoxin and TBPS dose-dependently accelerated the decay of inward GABA-induced currents (a holding potential of -60 mV under a symmetrical Cl- gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration-dependent manner. This indicates their interaction with both GABA-bound and resting receptors. 3. The half maximal inhibitory concentration for picrotoxin and TBPS at the beginning of a 15 s GABA (5 microM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 microM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the alpha 1 beta 2 gamma 2 subtype, produced no appreciable change in potency of picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U-93631, a novel ligand to the picrotoxin site, which has higher affinity to both mono- and multiliganded receptors than resting receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582470

  8. Co-Localization of GABA Shunt Enzymes for the Efficient Production of Gamma-Aminobutyric Acid via GABA Shunt Pathway in Escherichia coli.

    PubMed

    Pham, Van Dung; Somasundaram, Sivachandiran; Park, Si Jae; Lee, Seung Hwan; Hong, Soon Ho

    2016-04-28

    Gamma-aminobutyric acid (GABA) is a non-protein amino acid, which is an important inhibitor of neurotransmission in the human brain. GABA is also used as the precursor of biopolymer Nylon-4 production. In this study, the carbon flux from the tricarboxylic acid cycle was directed to the GABA shunt pathway for the production of GABA from glucose. The GABA shunt enzymes succinate-semialdehyde dehydrogenase (GabD) and GABA aminotransferase (GabT) were co-localized along with the GABA transporter (GadC) by using a synthetic scaffold complex. The co-localized enzyme scaffold complex produced 0.71 g/l of GABA from 10 g/l of glucose. Inactivation of competing metabolic pathways in mutant E. coli strains XBM1 and XBM6 increased GABA production 13% to reach 0.80 g/l GABA by the enzymes co-localized and expressed in the mutant strains. The recombinant E. coli system developed in this study demonstrated the possibility of the pathway of the GABA shunt as a novel GABA production pathway.

  9. Colocalization of serotonin and GABA in retinal neurons of Ichthyophis kohtaoensis (amphibia; Gymnophiona).

    PubMed

    Dünker, N

    1998-01-01

    Ichthyophis kohtaoensis, a member of the limbless Gymnophiona, has a specialized subterranean burrowing mode of life and a predominantly olfactory-guided orientation. The only visually guided behavior seems to be negative phototaxis. As these animals possess extremely small eyes (only 540 microm in diameter in adults), functional investigations of single retinal cells by electrophysiological methods have so far failed. Therefore, the content and distribution of retinal transmitters have been investigated as indications of a functioning sense organ in an animal that is supposed to be blind. Previous immunohistochemical investigation of the retinal transmitter system revealed immunoreactivity for gamma-aminobutyric acid (GABA), serotonin, dopamine and tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthetic pathway. The present studies have been performed in order to determine a possible colocalization of serotonin and GABA in retinal neurons of the caecilian retina. Therefore retinal cryostat sections of various developmental stages have been investigated by the indirect fluorescence method. In single-label preparations, serotonin is localized to cells in the inner nuclear layer and the ganglion cell layer. GABA immunocytochemistry labels a variety of cell types in the inner nuclear layer as well as cell bodies in the ganglion cell layer. In double-label preparations, some of the serotonergic cells are found to express GABA immunoreactivity and some GABAergic neurons also label for serotonin immunocytochemistry. Thus, despite the fact that caecilians mainly rely on olfaction and are believed to have a reduced visual system, their retina exhibits a surprisingly "normal" distribution of neurotransmitters and neuromodulators, also typical of other anamniotes with a well-developed visual system, including the partial colocalization of serotonin and GABA at all developmental stages of I. kohtaoensis. These results indicate that a functional system

  10. Age-related Hearing Loss: GABA, Nicotinic Acetylcholine and NMDA Receptor Expression Changes in Spiral Ganglion Neurons of the Mouse

    PubMed Central

    Tang, Xiaolan; Zhu, Xiaoxia; Ding, Bo; Walton, Joseph P.; Frisina, Robert D.; Su, Jiping

    2014-01-01

    Age-related hearing loss – presbycusis – is the number one communication disorder and most prevalent neurodegenerative condition of our aged population. Although speech understanding in background noise is quite difficult for those with presbycusis, there are currently no biomedical treatments to prevent, delay or reverse this condition. A better understanding of the cochlear mechanisms underlying presbycusis will help lead to future treatments. Objectives of the present study were to investigate gamma-amino butyric acid A (GABAA) receptor subunit α1, nicotinic acetylcholine (nACh) receptor subunit β2, and N-methyl-D-aspartate (NMDA) receptor subunit NR1 mRNA and protein expression changes in spiral ganglion neurons of the CBA/CaJ mouse cochlea, that occur in age-related hearing loss, utilizing quantitative immunohistochemistry and semi-quantitative RT-PCR techniques. We found that auditory brainstem response (ABR) thresholds shifted over 40 dB from 3–48 kHz in old mice compared to young adults. DPOAE thresholds also shifted over 40 dB from 6–49 kHz in old mice, and their amplitudes were significantly decreased or absent in the same frequency range. Spiral ganglion neuron (SGN) density decreased with age in basal, middle and apical turns, and SGN density of the basal turn declined the most. A positive correlation was observed between SGN density and ABR wave 1 amplitude. mRNA and protein expression of GABAAR α1 and AChR β2 decreased with age in SGNs in the old mouse cochlea. mRNA and protein expression of NMDAR NR1 increased with age in SGNs of the old mice. These findings demonstrate that there are functionally-relevant age-related changes of GABAAR, nAChR, NMDAR expression in CBA mouse SGNs reflecting their degeneration, which may be related to functional changes in cochlear synaptic transmission with age, suggesting biological mechanisms for peripheral age-related hearing loss. PMID:24316061

  11. Memantine treatment reduces the expression of the K(+)/Cl(-) cotransporter KCC2 in the hippocampus and cerebral cortex, and attenuates behavioural responses mediated by GABA(A) receptor activation in mice.

    PubMed

    Molinaro, Gemma; Battaglia, Giuseppe; Riozzi, Barbara; Di Menna, Luisa; Rampello, Liborio; Bruno, Valeria; Nicoletti, Ferdinando

    2009-04-10

    A 7-day treatment with memantine (25 mg/kg, i.p.), a drug that is currently prescribed for the treatment of Alzheimer's disease, increased the levels of brain-derived neurotrophic factor (BDNF) and reduced the expression of the neuron-specific K(+)/Cl(-) co-transporter, KCC2, in the hippocampus and cerebral cortex of mice. Knowing that KCC2 maintains low intracellular Cl(-) concentrations, which drive Cl(-) influx in response to GABA(A) receptor activation, we monitored the behavioural response to the GABA(A) receptor enhancer, diazepam, in mice pre-treated for 7 days with saline or 25 mg/kg of memantine. Memantine treatment substantially attenuated motor impairment induced by an acute challenge with diazepam (6 mg/kg, i.p.), as assessed by the rotarod test and the horizontal wire test. We suggest that a prolonged treatment with memantine induces changes in the activity of GABA(A) receptors that might contribute to the therapeutic and/or toxic effects of the drug.

  12. Functional expression of γ-amino butyric acid transporter 2 in human and guinea pig airway epithelium and smooth muscle.

    PubMed

    Zaidi, Sarah; Gallos, George; Yim, Peter D; Xu, Dingbang; Sonett, Joshua R; Panettieri, Reynold A; Gerthoffer, William; Emala, Charles W

    2011-08-01

    γ-Amino butyric acid (GABA) is a primary inhibitory neurotransmitter in the central nervous system, and is classically released by fusion of synaptic vesicles with the plasma membrane or by egress via GABA transporters (GATs). Recently, a GABAergic system comprised of GABA(A) and GABA(B) receptors has been identified on airway epithelial and smooth muscle cells that regulate mucus secretion and contractile tone of airway smooth muscle (ASM). In addition, the enzyme that synthesizes GABA, glutamic acid decarboxylase, has been identified in airway epithelial cells; however, the mechanism(s) by which this synthesized GABA is released from epithelial intracellular stores is unknown. We questioned whether any of the four known isoforms of GATs are functionally expressed in ASM or epithelial cells. We detected mRNA and protein expression of GAT2 and -4, and isoforms of glutamic acid decarboxylase in native and cultured human ASM and epithelial cells. In contrast, mRNA encoding vesicular GAT (VGAT), the neuronal GABA transporter, was not detected. Functional inhibition of (3)H-GABA uptake was demonstrated using GAT2 and GAT4/betaine-GABA transporter 1 (BGT1) inhibitors in both human ASM and epithelial cells. These results demonstrate that two isoforms of GATs, but not VGAT, are expressed in both airway epithelial and smooth muscle cells. They also provide a mechanism by which locally synthesized GABA can be released from these cells into the airway to activate GABA(A) channels and GABA(B) receptors, with subsequent autocrine and/or paracrine signaling effects on airway epithelium and ASM. PMID:21057105

  13. Comparison of Steroid Modulation of Spontaneous Inhibitory Postsynaptic Currents in Cultured Hippocampal Neurons and Steady-State Single-Channel Currents from Heterologously Expressed α1β2γ2L GABA(A) Receptors.

    PubMed

    Chakrabarti, Sampurna; Qian, Mingxing; Krishnan, Kathiresan; Covey, Douglas F; Mennerick, Steven; Akk, Gustav

    2016-04-01

    Neuroactive steroids are efficacious modulators of γ-aminobutyric acid type A receptor (GABA(A)) receptor function. The effects of steroids on the GABA(A) receptor are typically determined by comparing steady-state single-channel open probability or macroscopic peak responses elicited by GABA in the absence and presence of a steroid. Due to differences in activation conditions (exposure duration, concentration of agonist), it is not obvious whether modulation measured using typical experimental protocols can be used to accurately predict the effect of a modulator on native receptors under physiologic conditions. In the present study, we examined the effects of 14 neuroactive steroids and analogs on the properties of spontaneous inhibitory postsynaptic currents (sIPSCs) in cultured rat hippocampal neurons. The goal was to determine whether the magnitude of modulation of the decay time course of sIPSCs correlates with the extent of modulation and kinetic properties of potentiation as determined in previous single-channel studies. The steroids were selected to cover a wide range of efficacy on heterologously expressed rat α1β2γ2L GABA(A) receptors, ranging from essentially inert to highly efficacious (strong potentiators of single-channel and macroscopic peak responses). The data indicate a strong correlation between prolongation of the decay time course of sIPSCs and potentiation of single-channel open probability. Furthermore, changes in intracluster closed time distributions were the single best predictor of prolongation of sIPSCs. We infer that the information obtained in steady-state single-channel recordings can be used to forecast modulation of synaptic currents.

  14. Role of the alpha subunit in the modulation of GABA(A) receptors by anabolic androgenic steroids.

    PubMed

    Yang, Paul; Jones, Brian L; Henderson, Leslie P

    2005-09-01

    Neural transmission mediated by circuits expressing alpha2 subunit-containing gamma-aminobutyric acid type A (GABA(A)) receptors is critical for the expression of behaviors known to be altered by anabolic androgenic steroids (AAS). Here we show that micromolar concentrations of AAS, which reflect levels found in steroid abusers, induce positive modulation of currents from alpha2beta3 gamma2L recombinant receptors elicited by pulses of GABA that mimic synaptic conditions in a manner that is mechanistically distinct from modulation induced at alpha1beta3 gamma2L receptors. Specifically, at alpha2-containing receptors, the AAS, 17alpha-methyltestosterone (17alpha-MeT) enhanced peak current, slowed deactivation, diminished desensitization, and promoted entry of receptors into more distal states along the activation pathway. Analysis of GABA(A) receptor-mediated synaptic currents in primary cortical neurons followed by single cell real-time RT-PCR demonstrated that 17alpha-MeT enhancement of synaptic currents is proportional to the ratio of alpha2 to alpha1 subunit mRNA. Finally, we show that the modulation elicited by AAS is not comparable to that produced by micromolar concentrations of other positive allosteric modulators at alpha2-containing receptors. In sum, these data indicate that AAS elicit effects on GABA(A) receptor function that depend significantly on alpha subunit composition and that the mechanism of AAS modulation of GABA(A) receptors is distinct from that of other positive allosteric modulators.

  15. GABA depolarizes immature neurons and inhibits network activity in the neonatal neocortex in vivo.

    PubMed

    Kirmse, Knut; Kummer, Michael; Kovalchuk, Yury; Witte, Otto W; Garaschuk, Olga; Holthoff, Knut

    2015-01-01

    A large body of evidence from in vitro studies suggests that GABA is depolarizing during early postnatal development. However, the mode of GABA action in the intact developing brain is unknown. Here we examine the in vivo effects of GABA in cells of the upper cortical plate using a combination of electrophysiological and Ca(2+)-imaging techniques. We report that at postnatal days (P) 3-4, GABA depolarizes the majority of immature neurons in the occipital cortex of anaesthetized mice. At the same time, GABA does not efficiently activate voltage-gated Ca(2+) channels and fails to induce action potential firing. Blocking GABA(A) receptors disinhibits spontaneous network activity, whereas allosteric activation of GABA(A) receptors has the opposite effect. In summary, our data provide evidence that in vivo GABA acts as a depolarizing neurotransmitter imposing an inhibitory control on network activity in the neonatal (P3-4) neocortex. PMID:26177896

  16. A mitochondrial GABA permease connects the GABA shunt and the TCA cycle, and is essential for normal carbon metabolism.

    PubMed

    Michaeli, Simon; Fait, Aaron; Lagor, Kelly; Nunes-Nesi, Adriano; Grillich, Nicole; Yellin, Ayelet; Bar, Dana; Khan, Munziba; Fernie, Alisdair R; Turano, Frank J; Fromm, Hillel

    2011-08-01

    In plants, γ-aminobutyric acid (GABA) accumulates in the cytosol in response to a variety of stresses. GABA is transported into mitochondria, where it is catabolized into TCA cycle or other intermediates. Although there is circumstantial evidence for mitochondrial GABA transporters in eukaryotes, none have yet been identified. Described here is an Arabidopsis protein similar in sequence and topology to unicellular GABA transporters. The expression of this protein complements a GABA-transport-deficient yeast mutant. Thus the protein was termed AtGABP to indicate GABA-permease activity. In vivo localization of GABP fused to GFP and immunobloting of subcellular fractions demonstrate its mitochondrial localization. Direct [(3) H]GABA uptake measurements into isolated mitochondria revealed impaired uptake into mitochondria of a gabp mutant compared with wild-type (WT) mitochondria, implicating AtGABP as a major mitochondrial GABA carrier. Measurements of CO(2) release, derived from radiolabeled substrates in whole seedlings and in isolated mitochondria, demonstrate impaired GABA-derived input into the TCA cycle, and a compensatory increase in TCA cycle activity in gabp mutants. Finally, growth abnormalities of gabp mutants under limited carbon availability on artificial media, and in soil under low light intensity, combined with their metabolite profiles, suggest an important role for AtGABP in primary carbon metabolism and plant growth. Thus, AtGABP-mediated transport of GABA from the cytosol into mitochondria is important to ensure proper GABA-mediated respiration and carbon metabolism. This function is particularly essential for plant growth under conditions of limited carbon.

  17. SNAP25 Expression in Mammalian Retinal Horizontal Cells

    PubMed Central

    Hirano, Arlene A.; Brandstätter, Johann Helmut; Morgans, Catherine W.; Brecha, Nicholas C.

    2014-01-01

    Horizontal cells mediate inhibitory feedforward and feedback lateral interactions in the outer retina at photoreceptor terminals and bipolar cell dendrites; however, the mechanisms that underlie synaptic transmission from mammalian horizontal cells are poorly understood. The localization of a vesicular γ-aminobutyric acid (GABA) transporter (VGAT) to horizontal cell processes in primate and rodent retinae suggested that mammalian horizontal cells release transmitter in a vesicular manner. Toward determining whether the molecular machinery for vesicular transmitter release is present in horizontal cells, we investigated the expression of SNAP25 (synaptosomal-associated protein of 25 kDa), a key SNARE protein, by immunocytochemistry with cell type-specific markers in the retinae of mouse, rat, rabbit, and monkey. Different commercial antibodies to SNAP25 were tested on vertical sections of retina. We report the robust expression of SNAP25 in both plexiform layers. Double labeling with SNAP25 and calbindin antibodies demonstrated that horizontal cell processes and their endings in photoreceptor triad synapses were strongly labeled for both proteins in mouse, rat, rabbit, and monkey retinae. Double labeling with parvalbumin antibodies in monkey retina verified SNAP25 immunoreactivity in all horizontal cells. Pre-embedding immunoelectron microscopy in rabbit retina confirmed expression of SNAP25 in lateral elements within photoreceptor triad synapses. The SNAP25 immunoreactivity in the plexiform layers and outer nuclear layer fell into at least three patterns depending on the antibody, suggesting a differential distribution of SNAP25 isoforms. The presence of SNAP25a and SNAP25b isoforms in mouse retina was established by reverse transcriptase-polymerase chain reaction. SNAP25 expression in mammalian horizontal cells along with other SNARE proteins is consistent with vesicular exocytosis. PMID:21280047

  18. Attenuation of γ-aminobutyric acid (GABA) transaminase activity contributes to GABA increase in the cerebral cortex of mice exposed to β-cypermethrin.

    PubMed

    Han, Y; Cao, D; Li, X; Zhang, R; Yu, F; Ren, Y; An, L

    2014-03-01

    The current study investigated the γ-aminobutyric acid (GABA) levels and GABA metabolic enzymes (GABA transaminase (GABA(T)) and glutamate decarboxylase (GAD)) activities at 2 and 4 h after treatment, using a high-performance liquid chromatography with ultraviolet detectors and colorimetric assay, in the cerebral cortex of mice treated with 20, 40 or 80 mg/kg β-cypermethrin by a single oral gavage, with corn oil as vehicle control. In addition, GABA protein (4 h after treatment), GABA(T) protein (2 h after treatment) and GABA receptors messenger RNA (mRNA) expression were detected by immunohistochemistry, Western blot and real-time quantitative reverse transcriptase polymerase chain reaction, respectively. β-Cypermethrin (80 mg/kg) significantly increased GABA levels in the cerebral cortex of mice, at both 2 and 4 h after treatment, compared with the control. Also, GABA immunohistochemistry results suggested that the number of positive granules was increased in the cerebral cortex of mice 4 h after exposure to 80 mg/kg β-cypermethrin when compared with the control. Furthermore, the results also showed that GABA(T) activity detected was significantly decreased in the cerebral cortex of mice 2 h after β-cypermethrin administration (40 or 80 mg/kg). No significant changes were found in GAD activity, or the expression of GABA(T) protein and GABAB receptors mRNA, in the cerebral cortex of mice, except that 80 mg/kg β-cypermethrin caused a significant decrease, compared with the vehicle control, in GABAA receptors mRNA expression 4 h after administration. These results suggested that attenuated GABA(T) activity induced by β-cypermethrin contributed to increased GABA levels in the mouse brain. The downregulated GABAA receptors mRNA expression is most likely a downstream event.

  19. A novel GABA-mediated corticotropin-releasing hormone secretory mechanism in the median eminence

    PubMed Central

    Kakizawa, Keisuke; Watanabe, Miho; Mutoh, Hiroki; Okawa, Yuta; Yamashita, Miho; Yanagawa, Yuchio; Itoi, Keiichi; Suda, Takafumi; Oki, Yutaka; Fukuda, Atsuo

    2016-01-01

    Corticotropin-releasing hormone (CRH), which is synthesized in the paraventricular nucleus (PVN) of the hypothalamus, plays an important role in the endocrine stress response. The excitability of CRH neurons is regulated by γ-aminobutyric acid (GABA)–containing neurons projecting to the PVN. We investigated the role of GABA in the regulation of CRH release. The release of CRH was impaired, accumulating in the cell bodies of CRH neurons in heterozygous GAD67-GFP (green fluorescent protein) knock-in mice (GAD67+/GFP), which exhibited decreased GABA content. The GABAA receptor (GABAAR) and the Na+-K+-2Cl− cotransporter (NKCC1), but not the K+-Cl− cotransporter (KCC2), were expressed in the terminals of the CRH neurons at the median eminence (ME). In contrast, CRH neuronal somata were enriched with KCC2 but not with NKCC1. Thus, intracellular Cl− concentrations ([Cl−]i) may be increased at the terminals of CRH neurons compared with concentrations in the cell body. Moreover, GABAergic terminals projecting from the arcuate nucleus were present in close proximity to CRH-positive nerve terminals. Furthermore, a GABAAR agonist increased the intracellular calcium (Ca2+) levels in the CRH neuron terminals but decreased the Ca2+ levels in their somata. In addition, the increases in Ca2+ concentrations were prevented by an NKCC1 inhibitor. We propose a novel mechanism by which the excitatory action of GABA maintains a steady-state CRH release from axon terminals in the ME. PMID:27540587

  20. A novel GABA-mediated corticotropin-releasing hormone secretory mechanism in the median eminence.

    PubMed

    Kakizawa, Keisuke; Watanabe, Miho; Mutoh, Hiroki; Okawa, Yuta; Yamashita, Miho; Yanagawa, Yuchio; Itoi, Keiichi; Suda, Takafumi; Oki, Yutaka; Fukuda, Atsuo

    2016-08-01

    Corticotropin-releasing hormone (CRH), which is synthesized in the paraventricular nucleus (PVN) of the hypothalamus, plays an important role in the endocrine stress response. The excitability of CRH neurons is regulated by γ-aminobutyric acid (GABA)-containing neurons projecting to the PVN. We investigated the role of GABA in the regulation of CRH release. The release of CRH was impaired, accumulating in the cell bodies of CRH neurons in heterozygous GAD67-GFP (green fluorescent protein) knock-in mice (GAD67(+/GFP)), which exhibited decreased GABA content. The GABAA receptor (GABAAR) and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), but not the K(+)-Cl(-) cotransporter (KCC2), were expressed in the terminals of the CRH neurons at the median eminence (ME). In contrast, CRH neuronal somata were enriched with KCC2 but not with NKCC1. Thus, intracellular Cl(-) concentrations ([Cl(-)]i) may be increased at the terminals of CRH neurons compared with concentrations in the cell body. Moreover, GABAergic terminals projecting from the arcuate nucleus were present in close proximity to CRH-positive nerve terminals. Furthermore, a GABAAR agonist increased the intracellular calcium (Ca(2+)) levels in the CRH neuron terminals but decreased the Ca(2+) levels in their somata. In addition, the increases in Ca(2+) concentrations were prevented by an NKCC1 inhibitor. We propose a novel mechanism by which the excitatory action of GABA maintains a steady-state CRH release from axon terminals in the ME. PMID:27540587

  1. Co-localization of glycine and gaba immunoreactivity in interneurons in Macaca monkey cerebellar cortex.

    PubMed

    Crook, J; Hendrickson, A; Robinson, F R

    2006-09-15

    Previous work demonstrates that the cerebellum uses glycine as a fast inhibitory neurotransmitter [Ottersen OP, Davanger S, Storm-Mathisen J (1987) Glycine-like immunoreactivity in the cerebellum of rat and Senegalese baboon, Papio papio: a comparison with the distribution of GABA-like immunoreactivity and with [3H]glycine and [3H]GABA uptake. Exp Brain Res 66(1):211-221; Ottersen OP, Storm-Mathisen J, Somogyi P (1988) Colocalization of glycine-like and GABA-like immunoreactivities in Golgi cell terminals in the rat cerebellum: a postembedding light and electron microscopic study. Brain Res 450(1-2):342-353; Dieudonne S (1995) Glycinergic synaptic currents in Golgi cells of the rat cerebellum. Proc Natl Acad Sci U S A 92:1441-1445; Dumoulin A, Triller A, Dieudonne S (2001) IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells. J Neurosci 21(16):6045-6057; Dugue GP, Dumoulin A, Triller A, Dieudonne S (2005) Target-dependent use of coreleased inhibitory transmitters at central synapses. J Neurosci 25(28):6490-6498; Zeilhofer HU, Studler B, Arabadzisz D, Schweizer C, Ahmadi S, Layh B, Bosl MR, Fritschy JM (2005) Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J Comp Neurol 482(2):123-141]. In the rat cerebellum glycine is not released by itself but is released together with GABA by Lugaro cells onto Golgi cells [Dumoulin A, Triller A, Dieudonne S (2001) IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells. J Neurosci 21(16):6045-6057] and by Golgi cells onto unipolar brush and granule cells [Dugue GP, Dumoulin A, Triller A, Dieudonne S (2005) Target-dependent use of coreleased inhibitory transmitters at central synapses. J Neurosci 25(28):6490-6498]. Here we report, from immunolabeling evidence in Macaca cerebellum, that interneurons in the granular cell layer are glycine+ at a density

  2. Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABA(A) receptor δ subunit in cerebellar granule neurons and delays motor development in rats.

    PubMed

    Diaz, Marvin R; Vollmer, Cyndel C; Zamudio-Bulcock, Paula A; Vollmer, William; Blomquist, Samantha L; Morton, Russell A; Everett, Julie C; Zurek, Agnieszka A; Yu, Jieying; Orser, Beverley A; Valenzuela, C Fernando

    2014-04-01

    Exposure to ethanol (EtOH) during fetal development can lead to long-lasting alterations, including deficits in fine motor skills and motor learning. Studies suggest that these are, in part, a consequence of cerebellar damage. Cerebellar granule neurons (CGNs) are the gateway of information into the cerebellar cortex. Functionally, CGNs are heavily regulated by phasic and tonic GABAergic inhibition from Golgi cell interneurons; however, the effect of EtOH exposure on the development of GABAergic transmission in immature CGNs has not been investigated. To model EtOH exposure during the 3rd trimester-equivalent of human pregnancy, neonatal pups were exposed intermittently to high levels of vaporized EtOH from postnatal day (P) 2 to P12. This exposure gradually increased pup serum EtOH concentrations (SECs) to ∼60 mM (∼0.28 g/dl) during the 4 h of exposure. EtOH levels gradually decreased to baseline 8 h after the end of exposure. Surprisingly, basal tonic and phasic GABAergic currents in CGNs were not significantly affected by postnatal alcohol exposure (PAE). However, PAE increased δ subunit expression at P28 as detected by immunohistochemical and western blot analyses. Also, electrophysiological studies with an agonist that is highly selective for δ-containing GABA(A) receptors, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THIP), showed an increase in THIP-induced tonic current. Behavioral studies of PAE rats did not reveal any deficits in motor coordination, except for a delay in the acquisition of the mid-air righting reflex that was apparent at P15 to P18. These findings demonstrate that repeated intermittent exposure to high levels of EtOH during the equivalent of the last trimester of human pregnancy has significant but relatively subtle effects on motor coordination and GABAergic transmission in CGNs in rats.

  3. Corelease of acetylcholine and GABA from cholinergic forebrain neurons

    PubMed Central

    Saunders, Arpiar; Granger, Adam J; Sabatini, Bernardo L

    2015-01-01

    Neurotransmitter corelease is emerging as a common theme of central neuromodulatory systems. Though corelease of glutamate or GABA with acetylcholine has been reported within the cholinergic system, the full extent is unknown. To explore synaptic signaling of cholinergic forebrain neurons, we activated choline acetyltransferase expressing neurons using channelrhodopsin while recording post-synaptic currents (PSCs) in layer 1 interneurons. Surprisingly, we observed PSCs mediated by GABAA receptors in addition to nicotinic acetylcholine receptors. Based on PSC latency and pharmacological sensitivity, our results suggest monosynaptic release of both GABA and ACh. Anatomical analysis showed that forebrain cholinergic neurons express the GABA synthetic enzyme Gad2 and the vesicular GABA transporter (Slc32a1). We confirmed the direct release of GABA by knocking out Slc32a1 from cholinergic neurons. Our results identify GABA as an overlooked fast neurotransmitter utilized throughout the forebrain cholinergic system. GABA/ACh corelease may have major implications for modulation of cortical function by cholinergic neurons. DOI: http://dx.doi.org/10.7554/eLife.06412.001 PMID:25723967

  4. The non-anaesthetic propofol analogue 2,6-di-tert-butylphenol fails to modulate GABA(A) receptor function.

    PubMed

    Ahrens, Jörg; Leuwer, Martin; de la Roche, Jeanne; Foadi, Nilufar; Krampfl, Klaus; Haeseler, Gertrud

    2009-01-01

    Modulation of inhibitory synaptic transmission within the central nervous system contributes considerably to the anaesthetic effects of propofol and its analogues in vivo. We have studied the effects of the non-anaesthetic propofol analogue 2,6-di-tert-butylphenol on rat alpha(1)beta(2)gamma(2) GABA(A) receptors expressed in a mammalian expression system (HEK 293 cells) using the whole-cell patch clamp technique. Our experiments showed that 2,6-di-tert-butylphenol completely lacks co-activation and direct activation of the inhibitory GABA(A) receptor. Our results support the assumption that modulation of inhibitory GABA(A) receptor function is responsible for the anaesthetic effects of propofol in vivo.

  5. A Sensitive Period of Mice Inhibitory System to Neonatal GABA Enhancement by Vigabatrin is Brain Region Dependent

    PubMed Central

    Levav-Rabkin, Tamar; Melamed, Osnat; Clarke, Gerard; Farber, Malca; Cryan, John F; Dinan, Timothy G; Grossman, Yoram; Golan, Hava M

    2010-01-01

    Neurodevelopmental disorders, such as schizophrenia and autism, have been associated with disturbances of the GABAergic system in the brain. We examined immediate and long-lasting influences of exposure to the GABA-potentiating drug vigabatrin (GVG) on the GABAergic system in the hippocampus and cerebral cortex, before and during the developmental switch in GABA function (postnatal days P1–7 and P4–14). GVG induced a transient elevation of GABA levels. A feedback response to GABA enhancement was evident by a short-term decrease in glutamate decarboxylase (GAD) 65 and 67 levels. However, the number of GAD65/67-immunoreactive (IR) cells was greater in 2-week-old GVG-treated mice. A long-term increase in GAD65 and GAD67 levels was dependent on brain region and treatment period. Vesicular GABA transporter was insensitive to GVG. The overall effect of GVG on the Cl− co-transporters NKCC1 and KCC2 was an enhancement of their synthesis, which was dependent on the treatment period and brain region studied. In addition, a short-term increase was followed by a long-term decrease in KCC2 oligomerization in the cell membrane of P4–14 hippocampi and cerebral cortices. Analysis of the Ca2+ binding proteins expressed in subpopulations of GABAergic cells, parvalbumin and calbindin, showed region-specific effects of GVG during P4–14 on parvalbumin-IR cell density. Moreover, calbindin levels were elevated in GVG mice compared to controls during this period. Cumulatively, these results suggest a particular susceptibility of the hippocampus to GVG when exposed during days P4–14. In conclusion, our studies have identified modifications of key components in the inhibitory system during a critical developmental period. These findings provide novel insights into the deleterious consequences observed in children following prenatal and neonatal exposure to GABA-potentiating drugs. PMID:20043003

  6. Effect of the alpha subunit subtype on the macroscopic kinetic properties of recombinant GABA(A) receptors.

    PubMed

    Picton, Amber J; Fisher, Janet L

    2007-08-24

    The GABA(A) receptors (GABARs) are chloride-permeable ligand-gated ion channels responsible for fast inhibitory neurotransmission. These receptors are structurally heterogeneous, and in mammals can be formed from a combination of sixteen different subunit subtypes. Much of this variety comes from the six different alpha subunit subtypes. All neuronal GABARs contain an alpha subunit, and the identity of the alpha subtype affects the pharmacological properties of the receptors. The expression of each of the different alpha subtypes is regulated developmentally and regionally and changes with both normal physiological processes such development and synaptic plasticity, and pathological conditions such as epilepsy. In order to understand the functional significance of this structural heterogeneity, we examined the effect of the alpha subtype on the receptor's response to GABA. Each of the six alpha subtypes was transiently co-expressed with the beta3 and gamma2L subunits in mammalian cells. The sensitivity to GABA was measured with whole-cell recordings. We also determined the activation, deactivation, desensitization, and recovery kinetics for the six isoforms using rapid application recordings from excised macropatches. We found unique characteristics associated with each alpha subunit subtype. These properties would be expected to influence the post-synaptic response to GABA, creating functional diversity among neurons expressing different alpha subunits.

  7. Context-dependent modulation of alphabetagamma and alphabetadelta GABA A receptors by penicillin: implications for phasic and tonic inhibition.

    PubMed

    Feng, Hua-Jun; Botzolakis, Emmanuel J; Macdonald, Robert L

    2009-01-01

    Penicillin, an open-channel blocker of GABA(A) receptors, was recently reported to inhibit phasic, but not tonic, currents in hippocampal neurons. To distinguish between isoform-specific and context-dependent modulation as possible explanations for this selectivity, the effects of penicillin were evaluated on recombinant GABA(A) receptors expressed in HEK293T cells. When co-applied with saturating GABA, penicillin decreased peak amplitude, induced rebound, and prolonged deactivation of currents evoked from both synaptic and extrasynaptic receptor isoforms. However, penicillin had isoform-specific effects on the extent of desensitization, reflecting its ability to differentially modulate peak (non-equilibrium) and residual (near-equilibrium) currents. This suggested that the context of activation could determine the apparent sensitivity of a given receptor isoform to penicillin. To test this hypothesis, we explored the ability of penicillin to modulate synaptic and extrasynaptic isoform currents that were activated under more physiologically relevant conditions. Interestingly, while currents evoked from synaptic isoforms under phasic conditions (transient activation by a saturating concentration of GABA) were substantially inhibited by penicillin, currents evoked from extrasynaptic isoforms under tonic conditions (prolonged application by a sub-saturating concentration of GABA) were minimally affected. We therefore concluded that the reported inability of penicillin to modulate tonic currents could not simply be attributed to insensitivity of extrasynaptic receptors, but rather, reflected an inability to modulate these receptors in their native context of activation.

  8. The endogenous GABA bioactivity of camel, bovine, goat and human milks.

    PubMed

    Limon, Agenor; Gallegos-Perez, Jose-Luis; Reyes-Ruiz, Jorge M; Aljohi, Mohammad A; Alshanqeeti, Ali S; Miledi, Ricardo

    2014-02-15

    GABA orally administered has several beneficial effects on health, including the regulation of hyperglycaemic states in humans. Those effects are similar to the effects reported for camel milk (CMk); however, it is not known whether compounds with GABAergic activity are present in milk from camels or other species. We determined CMk free-GABA concentration by LS/MS and its bioactivity on human GABA receptors. We found that camel and goat milks have significantly more bioavailable GABA than cow and human milks and are able to activate GABAρ receptors. The relationship between GABA and taurine concentrations suggests that whole camel milk may be more efficient to activate GABAρ1 receptors than goat milk. Because GABAρ receptors are normally found in enteroendocrine cells in the lumen of the digestive tract, these results suggest that GABA in camel and goat milk may participate in GABA-modulated functions of enteroendocrine cells in the GI lumen.

  9. Estimation of ambient GABA levels in layer I of the mouse neonatal cortex in brain slices.

    PubMed

    Dvorzhak, Anton; Myakhar, Olga; Unichenko, Petr; Kirmse, Knut; Kirischuk, Sergei

    2010-07-01

    GABAergic synapses on Cajal-Retzius neurons in layer I of the murine neocortex experience GABA(B) receptor (GABA(B)R)-mediated tonic inhibition. Extracellular GABA concentration ([GABA](o)) that determines the strength of GABA(B)R-mediated inhibition is controlled by GABA transporters (GATs). In this study, we hypothesized that the strength of presynaptic GABA(B)R activation reflects [GABA](o) in the vicinity of synaptic contacts. Slices obtained from two age groups were used, namely postnatal days (P)2-3 and P5-7. GABAergic postsynaptic currents (IPSCs) were recorded using the whole-cell patch-clamp technique. Minimal electrical stimulation in layer I was applied to elicit evoked IPSCs (eIPSCs) using a paired-pulse protocol. Three parameters were selected for comparison: the mean eIPSC amplitude, paired-pulse ratio, and failure rate. When GAT-1 and GAT-2/3 were blocked by NO-711 (10 microM) and SNAP-5114 (40 microM), respectively, no tonic GABA(B)R-mediated inhibition was observed. In order to restore the control levels of GABA(B)R-mediated inhibition, 250 and 125 nm exogenous GABA was required at P2-3 and P5-7, respectively. Addition of 3-mercaptopropionic acid, a glutamate decarboxylase inhibitor, did not significantly change the obtained values arguing against the suggestion that a mechanism different from GATs contributes to [GABA](o) control. We conclude that juxtasynaptic [GABA](o) is higher (about 250 nM) at P2-3 than at P5-7 (about 125 nM). As both radial cell migration and corticogenesis in general are strongly dependent on [GABA](o) and the formation of the last layer 2/3 is finished by P4 in rodents, the observed [GABA](o) reduction in layer I might reflect this crucial event in the cortical development. PMID:20421290

  10. GABA-A receptor inhibition of local calcium signaling in spines and dendrites.

    PubMed

    Marlin, Joseph J; Carter, Adam G

    2014-11-26

    Cortical interneurons activate GABA-A receptors to rapidly control electrical and biochemical signaling at pyramidal neurons. Different populations of interneurons are known to uniquely target the soma and dendrites of pyramidal neurons. However, the ability of these interneurons to inhibit Ca(2+) signaling at spines and dendrites is largely unexplored. Here we use whole-cell recordings, two-photon microscopy, GABA uncaging and optogenetics to study dendritic inhibition at layer 5 (L5) pyramidal neurons in slices of mouse PFC. We first show that GABA-A receptors strongly inhibit action potential (AP)-evoked Ca(2+) signals at both spines and dendrites. We find robust inhibition over tens of milliseconds that spreads along the dendritic branch. However, we observe no difference in the amount of inhibition at neighboring spines and dendrites. We then examine the influence of interneurons expressing parvalbumin (PV), somatostatin (SOM), or 5HT3a receptors. We determine that these populations of interneurons make unique contacts onto the apical and basal dendrites of L5 pyramidal neurons. We also show that SOM and 5HT3a but not PV interneurons potently inhibit AP Ca(2+) signals via GABA-A receptors at both spines and dendrites. These findings reveal how multiple interneurons regulate local Ca(2+) signaling in pyramidal neurons, with implications for cortical function and disease.

  11. Are presynaptic GABA-Cρ2 receptors involved in anti-nociception?

    PubMed

    Tadavarty, R; Hwang, J; Rajput, P S; Soja, P J; Kumar, U; Sastry, B R

    2015-10-01

    We investigated the anti-nociceptive effects of GABA-C receptors in the central nervous system. Intracisternal injection of CACA, a GABA-C receptor agonist or isoguvacine, a GABA-A receptor agonist, significantly increased the tail-withdrawal latency. TPMPA, a GABA-C receptor antagonist blocked the effects of CACA but not isoguvacine indicating that GABA-C receptors are involved in regulating pain. Further, double-labelled immunofluorescence studies revealed that GABA-Cρ2 receptors are expressed presynaptically in the spinal dorsal horn, especially, substantia gelatinosa, a region that has been previously implicated in analgesia by regulating nociceptive inflow. These data provide a provenance for future work looking at presynaptic spinal GABA-C receptors in the control of nociception.

  12. GABA transporter subtype 1 and GABA transporter subtype 3 modulate glutamatergic transmission via activation of presynaptic GABA(B) receptors in the rat globus pallidus.

    PubMed

    Jin, Xiao-Tao; Paré, Jean-Francois; Smith, Yoland

    2012-08-01

    The intra-pallidal application of γ-aminobutyric acid (GABA) transporter subtype 1 (GAT-1) or GABA transporter subtype 3 (GAT-3) transporter blockers [1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride (SKF 89976A) or 1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-(S)-3-piperidinecarboxylic acid (SNAP 5114)] reduces the activity of pallidal neurons in monkey. This effect could be mediated through the activation of presynaptic GABA(B) heteroreceptors in glutamatergic terminals by GABA spillover following GABA transporter (GAT) blockade. To test this hypothesis, we applied the whole-cell recording technique to study the effects of SKF 89976A and SNAP 5114 on evoked excitatory postsynaptic currents (eEPSCs) in the presence of gabazine, a GABA(A) receptor antagonist, in rat globus pallidus slice preparations. Under the condition of postsynaptic GABA(B) receptor blockade by the intra-cellular application of N-(2,6-dimethylphenylcarbamoylmethyl)-triethylammonium bromide (OX314), bath application of SKF 89976A (10 μM) or SNAP 5114 (10 μM) decreased the amplitude of eEPSCs, without a significant effect on its holding current and whole cell input resistance. The inhibitory effect of GAT blockade on eEPSCs was blocked by (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid, a GABA(B) receptor antagonist. The paired-pulse ratio of eEPSCs was increased, whereas the frequency, but not the amplitude, of miniature excitatory postsynaptic currents was reduced in the presence of either GAT blocker, demonstrating a presynaptic effect. These results suggest that synaptically released GABA can inhibit glutamatergic transmission through the activation of presynaptic GABA(B) heteroreceptors following GAT-1 or GAT-3 blockade. In conclusion, our findings demonstrate that presynaptic GABA(B) heteroreceptors in putative glutamatergic subthalamic afferents to the globus pallidus are sensitive to increases in extracellular GABA induced

  13. Structure of alpha6 beta3 delta GABA(A) receptors and their lack of ethanol sensitivity.

    PubMed

    Baur, Roland; Kaur, Kuldeep H; Sigel, Erwin

    2009-12-01

    Delta (delta) subunit containing GABA(A) receptors are expressed extra-synaptically and mediate tonic inhibition. In cerebellar granule cells, they often form a receptor together with alpha(6) subunits. We were interested to determine the architecture of these receptors. We predefined the subunit arrangement of 24 different GABA(A) receptor pentamers by subunit concatenation. These receptors (composed of alpha(6), beta(3) and delta subunits) were expressed in Xenopus oocytes and their electrophysiological properties analyzed. Currents elicited in response to GABA were determined in presence and absence of 3alpha, 21-dihydroxy-5alpha-pregnan-20-one and to 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol. alpha(6)-beta(3)-alpha(6)/delta receptors showed a substantial response to GABA alone. Three receptors, beta(3)-alpha(6)-delta/alpha(6)-beta(3), alpha(6)-beta(3)-alpha(6)/beta(3)-delta and beta(3)-delta-beta(3)/alpha(6)-beta(3), were only uncovered in the combined presence of the neurosteroid 3alpha, 21-dihydroxy-5alpha-pregnan-20-one with GABA. All four receptors were activated by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol. None of the functional receptors was modulated by physiological concentrations (up to 30 mM) of ethanol. GABA concentration response curves indicated that the delta subunit can contribute to the formation of an agonist site. We conclude from the investigated receptors that the delta subunit can assume multiple positions in a receptor pentamer composed of alpha(6), beta(3) and delta subunits.

  14. GAD67-mediated GABA synthesis and signaling regulate inhibitory synaptic innervation in the visual cortex.

    PubMed

    Chattopadhyaya, Bidisha; Di Cristo, Graziella; Wu, Cai Zhi; Knott, Graham; Kuhlman, Sandra; Fu, Yu; Palmiter, Richard D; Huang, Z Josh

    2007-06-21

    The development of GABAergic inhibitory circuits is shaped by neural activity, but the underlying mechanisms are unclear. Here, we demonstrate a novel function of GABA in regulating GABAergic innervation in the adolescent brain, when GABA is mainly known as an inhibitory transmitter. Conditional knockdown of the rate-limiting synthetic enzyme GAD67 in basket interneurons in adolescent visual cortex resulted in cell autonomous deficits in axon branching, perisomatic synapse formation around pyramidal neurons, and complexity of the innervation fields; the same manipulation had little influence on the subsequent maintenance of perisomatic synapses. These effects of GABA deficiency were rescued by suppressing GABA reuptake and by GABA receptor agonists. Germline knockdown of GAD67 but not GAD65 showed similar deficits, suggesting a specific role of GAD67 in the maturation of perisomatic innervation. Since intracellular GABA levels are modulated by neuronal activity, our results implicate GAD67-mediated GABA synthesis in activity-dependent regulation of inhibitory innervation patterns.

  15. A Gut Feeling about GABA: Focus on GABAB Receptors

    PubMed Central

    Hyland, Niall P.; Cryan, John F.

    2010-01-01

    γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the body and hence GABA-mediated neurotransmission regulates many physiological functions, including those in the gastrointestinal (GI) tract. GABA is located throughout the GI tract and is found in enteric nerves as well as in endocrine-like cells, implicating GABA as both a neurotransmitter and an endocrine mediator influencing GI function. GABA mediates its effects via GABA receptors which are either ionotropic GABAA or metabotropic GABAB. The latter which respond to the agonist baclofen have been least characterized, however accumulating data suggest that they play a key role in GI function in health and disease. Like GABA, GABAB receptors have been detected throughout the gut of several species in the enteric nervous system, muscle, epithelial layers as well as on endocrine-like cells. Such widespread distribution of this metabotropic GABA receptor is consistent with its significant modulatory role over intestinal motility, gastric emptying, gastric acid secretion, transient lower esophageal sphincter relaxation and visceral sensation of painful colonic stimuli. More intriguing findings, the mechanisms underlying which have yet to be determined, suggest GABAB receptors inhibit GI carcinogenesis and tumor growth. Therefore, the diversity of GI functions regulated by GABAB receptors makes it a potentially useful target in the treatment of several GI disorders. In light of the development of novel compounds such as peripherally acting GABAB receptor agonists, positive allosteric modulators of the GABAB receptor and GABA producing enteric bacteria, we review and summarize current knowledge on the function of GABAB receptors within the GI tract. PMID:21833169

  16. GABA-independent GABAA Receptor Openings Maintain Tonic Currents

    PubMed Central

    Wlodarczyk, Agnieszka I.; Sylantyev, Sergiy; Herd, Murray B.; Kersanté, Flavie; Lambert, Jeremy J.; Rusakov, Dmitri A.; Linthorst, Astrid C.E.; Semyanov, Alexey; Belelli, Delia; Pavlov, Ivan; Walker, Matthew C.

    2013-01-01

    Activation of GABAA receptors (GABAARs) produces two forms of inhibition: ‘phasic’ inhibition generated by the rapid, transient activation of synaptic GABAARs by presynaptic GABA release, and tonic inhibition generated by the persistent activation of peri- or extrasynaptic GABAARs which can detect extracellular GABA. Such tonic GABAAR-mediated currents are particularly evident in dentate granule cells in which they play a major role in regulating cell excitability. Here we show that in rat dentate granule cells in ex-vivo hippocampal slices, tonic currents are predominantly generated by GABA-independent GABAA receptor openings. This tonic GABAAR conductance is resistant to the competitive GABAAR antagonist SR95531, which at high concentrations acts as a partial agonist, but can be blocked by an open channel blocker picrotoxin. When slices are perfused with 200 nM GABA, a concentration that is comparable to cerebrospinal fluid concentrations but is twice that measured by us in the hippocampus in vivo using zero-net-flux microdialysis, negligible GABA is detected by dentate granule cells. Spontaneously opening GABAARs, therefore, maintain dentate granule cell tonic currents in the face of low extracellular GABA concentrations. PMID:23447601

  17. Assessment of subunit-dependent direct gating and allosteric modulatory effects of carisoprodol at GABA(A) receptors.

    PubMed

    Kumar, Manoj; González, Lorie A; Dillon, Glenn H

    2015-10-01

    Carisoprodol is a widely prescribed muscle relaxant, abuse of which has grown considerably in recent years. It directly activates and allosterically modulates α1β2γ2 GABAARs, although the site(s) of action are unknown. To gain insight into the actions of carisoprodol, subunit-dependent effects of this drug were assessed. Whole-cell patch clamp recordings were obtained from HEK293 cells expressing α1β2, α1β3 or αxβzγ2 (where x = 1-6 and z = 1-3) GABAARs, and in receptors incorporating the δ subunit (modeling extrasynaptic receptors). The ability to directly gate and allosterically potentiate GABA-gated currents was observed for all configurations. Presence or absence of the γ2 subunit did not affect the ability of carisoprodol to directly gate or allosterically modulate the receptor. Presence of the β1 subunit conferred highest efficacy for direct activation relative to maximum GABA currents, while presence of the β2 subunit conferred highest efficacy for allosteric modulation of the GABA response. With regard to α subunits, carisoprodol was most efficacious at enhancing the actions of GABA in receptors incorporating the α1 subunit. The ability to directly gate the receptor was generally comparable regardless of the α subunit isoform, although receptors incorporating the α3 subunit showed significantly reduced direct gating efficacy and affinity. In extrasynaptic (α1β3δ and α4β3δ) receptors, carisoprodol had greater efficacy than GABA as a direct gating agonist. In addition, carisoprodol allosterically potentiated both EC20 and saturating GABA concentrations in these receptors. In assessing voltage-dependence, we found direct gating and inhibitory effects were insensitive to membrane voltage, whereas allosteric modulatory effects were affected by membrane voltage. Our findings demonstrate direct and allosteric effects of carisoprodol at synaptic and extrasynpatic GABAARs and that subunit isoform influences these effects.

  18. Functioning of the dimeric GABA(B) receptor extracellular domain revealed by glycan wedge scanning.

    PubMed

    Rondard, Philippe; Huang, Siluo; Monnier, Carine; Tu, Haijun; Blanchard, Bertrand; Oueslati, Nadia; Malhaire, Fanny; Li, Ying; Trinquet, Eric; Labesse, Gilles; Pin, Jean-Philippe; Liu, Jianfeng

    2008-05-01

    The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABA(B1) and GABA(B2). GABA(B1) binds agonists, whereas GABA(B2) is required for trafficking GABA(B1) to the cell surface, increasing agonist affinity to GABA(B1), and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABA(B1) VFT leads to GABA(B2) 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABA(B) VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N-glycan at this interface prevents the association of the two subunits and abolishes all activities of GABA(B2), including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N-glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation.

  19. Non-neuronal release of gamma-aminobutyric Acid by embryonic pluripotent stem cells.

    PubMed

    Teng, Lin; Tang, Ya-Bin; Sun, Fan; An, Shi-Min; Zhang, Chun; Yang, Xin-Jie; Lv, Hao-Yu; Lu, Qin; Cui, Yong-Yao; Hu, Jin-Jia; Zhu, Liang; Chen, Hong-Zhuan

    2013-11-15

    γ-Aminobutyric acid (GABA), the principle inhibitory transmitter in the mature central nervous system, is also involved in activities outside the nervous system. Recent studies have shown that functional GABA receptors are expressed in embryonic stem (ES) cells and these receptors control ES cell proliferation. However, it is not clear whether ES cells have their own GABAergic transmission output machinery that can fulfill GABA release or whether the cells merely process the GABA receptors by receiving and responding to the diffused GABA released elsewhere. To get further insight into this unresolved problem, we detected the repertoire of components for GABA synthesis, storage, reaction, and termination in ES and embryonal carcinoma stem cells by biological assays, and then directly quantified released GABA in the intercellular milieu from these pluripotent stem (PS) cells by an analytical chemical assay based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). We found that embryonic PS cells processed a GABAergic circuit machinery and spontaneously released GABA, which suggests the potential that embryonic PS cells could autonomously establish a GABA niche via release of the transmitter.

  20. [GABA-Receptors in Modulation of Fear Memory Extinction].

    PubMed

    Dubrovina, N I

    2016-01-01

    GABA is the major inhibitory neurotransmitter in the central nervous system determining the efficacy of neuronal interaction. GABA-receptors play a key role in different aspects of fear memory--acquisition and consolidation, retention, reconsolidation and extinction. Extinction is an important behavioural phenomenon which allows organism to adapt its behavior to a changing environment. Extinction of fear memory is a form of new inhibitory learning which interferes with expression of the initial acquired fear conditioning. Resistance to extinction is symptom of depression and posttraumatic stress disorder. The aim of the present review was to summarize own and literary data about GABAergic modulation of fear extinction and pharmacological correction of extinction impairment at influences on GABA(A)- and GABA(B)- receptors. PMID:27538279

  1. Extent of colocalization of serotonin and GABA in neurons of the ventral medulla oblongata in rat.

    PubMed

    Millhorn, D E; Hökfelt, T; Seroogy, K; Verhofstad, A A

    1988-09-27

    The colocalization of serotonin (5-hydroxytryptamine; 5-HT) and gamma-aminobutyric acid (GABA) in the ventral aspect of the rat medulla oblongata was studied using antibodies directed against 5-HT and GABA. Although 5-HT- and GABA-immunoreactive cell bodies were observed over the entire rostral-caudal extent of the ventral medulla, the colocalization of these two classical neurotransmitters in single cells was, for the most part, limited to a region that corresponds anatomically to nucleus raphe magnus/nucleus paragigantocellularis. Schematic drawings showing the distribution of 5-HT/GABA cell bodies in the ventral medulla are provided. PMID:3066433

  2. Cl− uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1

    PubMed Central

    Yamada, Junko; Okabe, Akihito; Toyoda, Hiroki; Kilb, Werner; Luhmann, Heiko J; Fukuda, Atsuo

    2004-01-01

    GABA is the principal inhibitory neurotransmitter in the mature brain, but during early postnatal development the elevated [Cl−]i in immature neocortical neurones causes GABAA receptor activation to be depolarizing. The molecular mechanisms underlying this intracellular Cl− accumulation remain controversial. Therefore, the GABA reversal potential (EGABA) or [Cl−]i in early postnatal rat neocortical neurones was measured by the gramicidin-perforated patch-clamp method, and the relative expression levels of the cation−Cl− cotransporter mRNAs (in the same cells) were examined by semiquantitative single-cell multiplex RT-PCR to look for statistical correlations with [Cl−]i. The mRNA expression levels were positively (the Cl− accumulating Na+,K+−2Cl− cotransporter NKCC1) or negatively (the Cl− extruding K+−Cl− cotransporter KCC2) correlated with [Cl−]i. NKCC1 mRNA expression was high in early postnatal days, but decreased during postnatal development, whereas KCC2 mRNA expression displayed the opposite pattern. [Cl−]i and NKCC1 mRNA expression were each higher in cortical plate (CP) neurones than in the presumably older layer V/VI pyramidal neurones in a given slice. The pharmacological effects of bumetanide on EGABA were consistent with the different expression levels of NKCC1 mRNA. These data suggest that NKCC1 may play a pivotal role in the generation of GABA-mediated depolarization in immature CP cells, while KCC2 promotes the later maturation of GABAergic inhibition in the rat neocortex. PMID:15090604

  3. Identification and selective inhibition of the channel mode of the neuronal GABA transporter 1.

    PubMed

    Krause, Stephan; Schwarz, Wolfgang

    2005-12-01

    The function of GAT1, the transporter for the inhibitory neurotransmitter GABA, is characterized by expression in Xenopus laevis oocytes and measurements of GABA-induced uptake of [3H]GABA, 22Na+, and 36Cl-, and GABA-evoked currents under voltage-clamp conditions. N-[4,4-Diphenyl-3-butenyl]-nipecotic acid (SKF-89976-A), a specific inhibitor of GAT1, is used in our system as a pharmacological tool. The GABA-evoked current can be decomposed into a transport current, which is coupled to the GABA uptake, and a transmitter-gated current, which is uncoupled from the GABA uptake. The transport current results from a fixed stoichiometry of 1 GABA/2 Na+/1 Cl- transported during each cycle, as determined by radioactive tracer flux measurements. The transmitter-gated current is mediated by an Na+-conductance pathway. As a competitive inhibitor for GABA uptake, SKF-89976-A can separate the two current components. The GABA uptake is blocked with a K(I) value of approximately 7 microM, whereas the uncoupled transmitter-gated current is inhibited with a K(I) value of approximately 0.03 microM. Thus, the results of this study not only identify the transport mode and the channel mode of GAT1 but also raise the possibility of separating these components in a physiological environment.

  4. Stable expression of cloned rat GABAA receptor subunits in a human kidney cell line.

    PubMed

    Hamilton, B J; Lennon, D J; Im, H K; Im, W B; Seeburg, P H; Carter, D B

    1993-04-30

    A predominant form of the GABAA/benzodiazepine receptor-Cl- channel complex is believed to consist of three different 48-55 kDa subunits (alpha, beta, gamma) with unknown stoichiometry. Plasmids containing the rat GABAA receptor cDNAs coding for alpha 1, beta 2, and gamma 2 were co-transfected, along with a plasmid encoding G418 resistance, into human embryonic kidney cells previously transformed with Adenovirus 5 (HEK-293) [J. Gen. Virol., 36 (1977) 59-72]. Four percent of the G418 resistant colonies were found to express mRNA for all three of the GABAA subunits constitutively. A single cell clone derived from one of the alpha 1 beta 2 gamma 2 expressors has demonstrated stable electrophysiological characteristics over 25 passages. The GABA-activated Cl- current in this cell line is blocked by picrotoxin and bicuculline, and is modulated by a variety of agonist and inverse agonist ligands including diazepam, Ro 154513, zolpidem, and beta-CCE. The cell line has been used successfully over a 12-month period as a screen for novel drugs modulating GABA-mediated polarization of neuronal cells. PMID:7687050

  5. Extrasynaptic release of GABA and dopamine by retinal dopaminergic neurons

    PubMed Central

    Hirasawa, Hajime; Contini, Massimo; Raviola, Elio

    2015-01-01

    In the mouse retina, dopaminergic amacrine (DA) cells synthesize both dopamine and GABA. Both transmitters are released extrasynaptically and act on neighbouring and distant retinal neurons by volume transmission. In simultaneous recordings of dopamine and GABA release from isolated perikarya of DA cells, a proportion of the events of dopamine and GABA exocytosis were simultaneous, suggesting co-release. In addition, DA cells establish GABAergic synapses onto AII amacrine cells, the neurons that transfer rod bipolar signals to cone bipolars. GABAA but not dopamine receptors are clustered in the postsynaptic membrane. Therefore, dopamine, irrespective of its site of release—synaptic or extrasynaptic—exclusively acts by volume transmission. Dopamine is released upon illumination and sets the gain of retinal neurons for vision in bright light. The GABA released at DA cells' synapses probably prevents signals from the saturated rods from entering the cone pathway when the dark-adapted retina is exposed to bright illumination. The GABA released extrasynaptically by DA and other amacrine cells may set a ‘GABAergic tone’ in the inner plexiform layer and thus counteract the effects of a spillover of glutamate released at the bipolar cell synapses of adjacent OFF and ON strata, thus preserving segregation of signals between ON and OFF pathways. PMID:26009765

  6. Cytoskeletal rearrangement and Src and PI-3K-dependent Akt activation control GABA(B)R-mediated chemotaxis.

    PubMed

    Barati, Michelle T; Lukenbill, Janice; Wu, Rui; Rane, Madhavi J; Klein, Jon B

    2015-06-01

    The γ-amino butyric acid (GABA) type B receptors (GABA(B)R) function as chemoattractant receptors in response to GABA(B)R agonists in human neutrophils. The goal of this study was to define signaling mechanisms regulating GABA(B)R-mediated chemotaxis and cytoskeletal rearrangement. In a proteomic study we identified serine/threonine kinase Akt, tyrosine kinases Src and Pyk2, microtubule regulator kinesin and microtubule affinity-regulating kinase (MARK) co-immunoprecipitating with GABA(B)R. To define the contributions of these candidate signaling events in GABA(B)R-mediated chemotaxis, we used rat basophilic leukemic cells (RBL-2H3 cells) stably transfected with human GABA(B1b) and GABA(B2) receptors. The GABA(B)R agonist baclofen induced Akt phosphorylation and chemotaxis by binding to its specific GABA(B)R since pretreatment of cells with CGP52432, a GABA(B)R antagonist, blocked such effects. Moreover, baclofen induced Akt phosphorylation was shown to be dependent upon PI-3K and Src kinases. Baclofen failed to stimulate actin polymerization in suspended RBL cells unless exposed to a baclofen gradient. However, baclofen stimulated both actin and tubulin polymerization in adherent RBL-GABA(B)R cells. Blockade of actin and tubulin polymerization by treatment of cells with cytochalasin D or nocodazole respectively, abolished baclofen-mediated chemotaxis. Furthermore, baclofen stimulated Pyk2 and STAT3 phosphorylation, both known regulators of cell migration. In conclusion, GABA(B)R stimulation promotes chemotaxis in RBL cells which is dependent on signaling via PI3-K/Akt, Src kinases and on rearrangement of both microtubules and actin cytoskeleton. These data define mechanisms of GABA(B)R-mediated chemotaxis which may potentially be used to therapeutically regulate cellular response to injury and disease.

  7. Gene expression changes in serotonin, GABA-A receptors, neuropeptides and ion channels in the dorsal raphe nucleus of adolescent alcohol-preferring (P) rats following binge-like alcohol drinking.

    PubMed

    McClintick, Jeanette N; McBride, William J; Bell, Richard L; Ding, Zheng-Ming; Liu, Yunlong; Xuei, Xiaoling; Edenberg, Howard J

    2015-02-01

    Alcohol binge-drinking during adolescence is a serious public health concern with long-term consequences. We used RNA sequencing to assess the effects of excessive adolescent ethanol binge-drinking on gene expression in the dorsal raphe nucleus (DRN) of alcohol preferring (P) rats. Repeated binges across adolescence (three 1h sessions across the dark-cycle per day, 5 days per week for 3 weeks starting at 28 days of age; ethanol intakes of 2.5-3 g/kg/session) significantly altered the expression of approximately one-third of the detected genes. Multiple neurotransmitter systems were altered, with the largest changes in the serotonin system (21 of 23 serotonin-related genes showed decreased expression) and GABA-A receptors (8 decreased and 2 increased). Multiple neuropeptide systems were also altered, with changes in the neuropeptide Y and corticotropin-releasing hormone systems similar to those associated with increased drinking and decreased resistance to stress. There was increased expression of 21 of 32 genes for potassium channels. Expression of downstream targets of CREB signaling was increased. There were also changes in expression of genes involved in inflammatory processes, axonal guidance, growth factors, transcription factors, and several intracellular signaling pathways. These widespread changes indicate that excessive binge drinking during adolescence alters the functioning of the DRN and likely its modulation of many regions of the central nervous system, including the mesocorticolimbic system.

  8. Gene expression changes in serotonin, GABA-A receptors, neuropeptides and ion channels in the dorsal raphe nucleus of adolescent alcohol-preferring (P) rats following binge-like alcohol drinking

    PubMed Central

    McClintick, Jeanette N.; McBride, William J.; Bell, Richard L.; Ding, Zheng-Ming; Liu, Yunlong; Xuei, Xiaoling; Edenberg, Howard J.

    2014-01-01

    Alcohol binge-drinking during adolescence is a serious public health concern with long-term consequences. We used RNA sequencing to assess the effects of excessive adolescent ethanol binge-drinking on gene expression in the dorsal raphe nucleus (DRN) of alcohol preferring (P) rats. Repeated binges across adolescence (three 1h sessions across the dark-cycle per day, 5 days per week for 3 weeks starting at 28 days of age; ethanol intakes of 2.5 – 3 g/kg/session) significantly altered the expression of approximately one-third of the detected genes. Multiple neurotransmitter systems were altered, with the largest changes in the serotonin system (21 of 23 serotonin-related genes showed decreased expression) and GABA-A receptors (8 decreased and 2 increased). Multiple neuropeptide systems were also altered, with changes in the neuropeptide Y and corticotropin-releasing hormone systems similar to those associated with increased drinking and decreased resistance to stress. There was increased expression of 21 of 32 genes for potassium channels. Expression of downstream targets of CREB signaling was increased. There were also changes in expression of genes involved in inflammatory processes, axonal guidance, growth factors, transcription factors, and several intracellular signaling pathways. These widespread changes indicate that excessive binge drinking during adolescence alters the functioning of the DRN and likely its modulation of many regions of the central nervous system, including the mesocorticolimbic system. PMID:25542586

  9. Conditional Knock-Out of Vesicular GABA Transporter Gene from Starburst Amacrine Cells Reveals the Contributions of Multiple Synaptic Mechanisms Underlying Direction Selectivity in the Retina

    PubMed Central

    Pei, Zhe; Chen, Qiang; Koren, David; Giammarinaro, Benno; Acaron Ledesma, Hector

    2015-01-01

    Direction selectivity of direction-selective ganglion cells (DSGCs) in the retina results from patterned excitatory and inhibitory inputs onto DSGCs during motion stimuli. The inhibitory inputs onto DSGCs are directionally tuned to the antipreferred (null) direction and therefore potently suppress spiking during motion in the null direction. However, whether direction-selective inhibition is indispensable for direction selectivity is unclear. Here, we selectively eliminated the directional tuning of inhibitory inputs onto DSGCs by disrupting GABA release from the presynaptic interneuron starburst amacrine cell in the mouse retina. We found that, even without directionally tuned inhibition, direction selectivity can still be implemented in a subset of On-Off DSGCs by direction-selective excitation and a temporal offset between excitation and isotropic inhibition. Our results therefore demonstrate the concerted action of multiple synaptic mechanisms for robust direction selectivity in the retina. SIGNIFICANCE STATEMENT The direction-selective circuit in the retina has been a classic model to study neural computations by the brain. An important but unresolved question is how direction selectivity is implemented by directionally tuned excitatory and inhibitory mechanisms. Here we specifically removed the direction tuning of inhibition from the circuit. We found that direction tuning of inhibition is important but not indispensable for direction selectivity of DSGCs' spiking activity, and that the residual direction selectivity is implemented by direction-selective excitation and temporal offset between excitation and inhibition. Our results highlight the concerted actions of synaptic excitation and inhibition required for robust direction selectivity in the retina and provide critical insights into how patterned excitation and inhibition collectively implement sensory processing. PMID:26400950

  10. Conditional Knock-Out of Vesicular GABA Transporter Gene from Starburst Amacrine Cells Reveals the Contributions of Multiple Synaptic Mechanisms Underlying Direction Selectivity in the Retina.

    PubMed

    Pei, Zhe; Chen, Qiang; Koren, David; Giammarinaro, Benno; Acaron Ledesma, Hector; Wei, Wei

    2015-09-23

    Direction selectivity of direction-selective ganglion cells (DSGCs) in the retina results from patterned excitatory and inhibitory inputs onto DSGCs during motion stimuli. The inhibitory inputs onto DSGCs are directionally tuned to the antipreferred (null) direction and therefore potently suppress spiking during motion in the null direction. However, whether direction-selective inhibition is indispensable for direction selectivity is unclear. Here, we selectively eliminated the directional tuning of inhibitory inputs onto DSGCs by disrupting GABA release from the presynaptic interneuron starburst amacrine cell in the mouse retina. We found that, even without directionally tuned inhibition, direction selectivity can still be implemented in a subset of On-Off DSGCs by direction-selective excitation and a temporal offset between excitation and isotropic inhibition. Our results therefore demonstrate the concerted action of multiple synaptic mechanisms for robust direction selectivity in the retina. Significance statement: The direction-selective circuit in the retina has been a classic model to study neural computations by the brain. An important but unresolved question is how direction selectivity is implemented by directionally tuned excitatory and inhibitory mechanisms. Here we specifically removed the direction tuning of inhibition from the circuit. We found that direction tuning of inhibition is important but not indispensable for direction selectivity of DSGCs' spiking activity, and that the residual direction selectivity is implemented by direction-selective excitation and temporal offset between excitation and inhibition. Our results highlight the concerted actions of synaptic excitation and inhibition required for robust direction selectivity in the retina and provide critical insights into how patterned excitation and inhibition collectively implement sensory processing.

  11. Response kinetics and pharmacological properties of heteromeric receptors formed by coassembly of GABA rho- and gamma 2-subunits.

    PubMed

    Qian, H; Ripps, H

    1999-12-01

    Two of the gamma-aminobutyric acid (GABA) receptors, GABAA and GABAC, are ligand-gated chloride channels expressed by neurons in the retina and throughout the central nervous system. The different subunit composition of these two classes of GABA receptor result in very different physiological and pharmacological properties. Although little is known at the molecular level as to the subunit composition of any native GABA receptor, it is thought that GABAC receptors are homomeric assemblies of rho-subunits. However, we found that the kinetic and pharmacological properties of homomeric receptors formed by each of the rho-subunits cloned from perch retina did not resemble those of the GABAC receptors on perch bipolar cells. Because both GABAA and GABAC receptors are present on retinal bipolar cells, we attempted to determine whether subunits of these two receptor classes are capable of interacting with each other. We report here that, when coexpressed in Xenopus oocytes, heteromeric (rho 1B gamma 2) receptors formed by coassembly of the rho 1B-subunit with the gamma 2-subunit of the GABAA receptor displayed response properties very similar to those obtained with current recordings from bipolar cells. In addition to being unresponsive to bicuculline and diazepam, the time-constant of deactivation, and the sensitivities to GABA, picrotoxin and zinc closely approximated the values obtained from the native GABAC receptors on bipolar cells. These results provide the first direct evidence of interaction between GABA rho and GABAA receptor subunits. It seems highly likely that coassembly of GABAA and rho-subunits contributes to the molecular organization of GABAC receptors in the retina and perhaps throughout the nervous system. PMID:10643085

  12. GABA deficiency in NF1

    PubMed Central

    Patricio, Miguel; Bernardino, Inês; Rebola, José; Abrunhosa, Antero J.; Ferreira, Nuno; Castelo-Branco, Miguel

    2016-01-01

    Objective: To provide a comprehensive investigation of the γ-aminobutyric acid (GABA) system in patients with neurofibromatosis type 1 (NF1) that allows understanding the nature of the GABA imbalance in humans at pre- and postsynaptic levels. Methods: In this cross-sectional study, we employed multimodal imaging and spectroscopy measures to investigate GABA type A (GABAA) receptor binding, using [11C]-flumazenil PET, and GABA concentration, using magnetic resonance spectroscopy (MRS). Fourteen adult patients with NF1 and 13 matched controls were included in the study. MRS was performed in the occipital cortex and in a frontal region centered in the functionally localized frontal eye fields. PET and MRS acquisitions were performed in the same day. Results: Patients with NF1 have reduced concentration of GABA+ in the occipital cortex (p = 0.004) and frontal eye fields (p = 0.026). PET results showed decreased binding of GABAA receptors in patients in the parieto-occipital cortex, midbrain, and thalamus, which are not explained by decreased gray matter levels. Conclusions: Abnormalities in the GABA system in NF1 involve both GABA concentration and GABAA receptor density suggestive of neurodevelopmental synaptopathy with both pre- and postsynaptic involvement. PMID:27473134

  13. GABA neurotransmission in the cerebellar interposed nuclei: involvement in classically conditioned eyeblinks and neuronal activity.

    PubMed

    Aksenov, D; Serdyukova, N; Irwin, K; Bracha, V

    2004-02-01

    The cerebellar interposed nuclei (IN) are an essential part of circuits that control classically conditioned eyeblinks in the rabbit. The function of the IN is under the control of GABAergic projections from Purkinje cells of the cerebellar cortex. The exact involvement of cerebellar cortical input into the IN during eyeblink expression is not clear. While it is known that the application of gamma-aminobutyric acid-A (GABA(A)) agonists and antagonists affects the performance of classically conditioned eyeblinks, the effects of these drugs on IN neurons in vivo are not known. The purpose of the present study was to measure the effects of muscimol and picrotoxin on the expression of conditioned eyeblinks and the activity of IN cells simultaneously. Injections of muscimol abolished conditioned responses and either silenced or diminished the activity of IN cells. Two injections were administered in each picrotoxin experiment. The first injection of picrotoxin slightly modified the timing and amplitude of the eyeblink, produced mild tonic eyelid closure, increased tonic activity of IN cells, and reduced the amplitude of the neural responses. The second injection of picrotoxin abolished conditioned responses, further increased tonic eyelid closure, dramatically elevated the tonic activity of IN cells, and in most cases, abolished neuronal responses. These results demonstrate that both GABA(A)-mediated inactivation and tonic up-regulation of IN cells can interrupt the expression of conditioned eyeblinks and that this behavioral effect is accompanied by the suppression of the neuronal activity correlates of the conditioned stimulus and response.

  14. Nonvesicular inhibitory neurotransmission via reversal of the GABA transporter GAT-1.

    PubMed

    Wu, Yuanming; Wang, Wengang; Díez-Sampedro, Ana; Richerson, George B

    2007-12-01

    GABA transporters play an important but poorly understood role in neuronal inhibition. They can reverse, but this is widely thought to occur only under pathological conditions. Here we use a heterologous expression system to show that the reversal potential of GAT-1 under physiologically relevant conditions is near the normal resting potential of neurons and that reversal can occur rapidly enough to release GABA during simulated action potentials. We then use paired recordings from cultured hippocampal neurons and show that GABAergic transmission is not prevented by four methods widely used to block vesicular release. This nonvesicular neurotransmission was potently blocked by GAT-1 antagonists and was enhanced by agents that increase cytosolic [GABA] or [Na(+)] (which would increase GAT-1 reversal). We conclude that GAT-1 regulates tonic inhibition by clamping ambient [GABA] at a level high enough to activate high-affinity GABA(A) receptors and that transporter-mediated GABA release can contribute to phasic inhibition.

  15. Early depolarizing GABA controls critical period plasticity in the rat visual cortex

    PubMed Central

    Deidda, Gabriele; Allegra, Manuela; Cerri, Chiara; Naskar, Shovan; Bony, Guillaume; Zunino, Giulia; Bozzi, Yuri; Caleo, Matteo; Cancedda, Laura

    2014-01-01

    SUMMARY Hyperpolarizing and inhibitory GABA regulates “critical periods” for plasticity in sensory cortices. Here, we examine the role of early, depolarizing GABA in controlling plasticity mechanisms. We report that brief interference with depolarizing GABA during early development prolonged critical period plasticity in visual cortical circuits, without affecting overall development of the visual system. The effects on plasticity were accompanied by dampened inhibitory neurotransmission, down-regulation of BDNF expression, and reduced density of extracellular matrix-perineuronal nets. Early interference with depolarizing GABA decreased perinatal BDNF signaling, and pharmacological increase of BDNF signaling during GABA interference rescued the effects on plasticity and its regulators later in life. We conclude that depolarizing GABA exerts a long-lasting, selective modulation of plasticity of cortical circuits by a strong crosstalk with BDNF. PMID:25485756

  16. The effect of chronic administration of corticosterone on anxiety- and depression-like behavior and the expression of GABA-A receptor alpha-2 subunits in brain structures of low- and high-anxiety rats.

    PubMed

    Skórzewska, Anna; Lehner, Małgorzata; Wisłowska-Stanek, Aleksandra; Krząścik, Paweł; Ziemba, Andrzej; Płaźnik, Adam

    2014-01-01

    The aim of this study was to examine changes in rat emotional behavior and determine differences in the expression of GABA-A receptor alpha-2 subunits in brain structures of low- (LR) and high-anxiety (HR) rats after the repeated corticosterone administration. The animals were divided into LR and HR groups based on the duration of their conditioned freezing in a contextual fear test. Repeated daily administration of corticosterone (20 mg/kg) for 21 days decreased activity in a forced swim test, reduced body weight and decreased prefrontal cortex corticosterone concentration in both the LR and HR groups. These effects of corticosterone administration were stronger in the HR group in comparison with the appropriate control group, and compared to LR treated and LR control animals. Moreover, in the HR group, chronic corticosterone administration increased anxiety-like behavior in the open field and elevated plus maze tests. The behavioral effects in HR rats were accompanied by a decrease in alpha-2 subunit density in the medial prefrontal cortex (prelimbic cortex and frontal association cortex) and by an increase in the expression of alpha-2 subunits in the basolateral amygdala. These studies have shown that HR rats are more susceptible to anxiogenic and depressive effects of chronic corticosterone administration, which are associated with modification of GABA-A receptor function in the medial prefrontal cortex and basolateral amygdala. The current data may help to better understand the neurobiological mechanisms responsible for individual differences in changes in mood and emotions induced by repeated administration of high doses of glucocorticoids or by elevated levels of these hormones associated with chronic stress or affective pathology.

  17. Parkinson's Disease and Neurodegeneration: GABA-Collapse Hypothesis.

    PubMed

    Błaszczyk, Janusz W

    2016-01-01

    Neurodegenerative diseases constitute a heterogeneous group of age-related disorders that are characterized by a slow but irreversible deterioration of brain functions. Evidence accumulated over more than two decades has implicated calcium-related homeostatic mechanisms, giving rise to the Ca(2+) hypothesis of brain aging and, ultimately, cell death. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter within the central (CNS), peripheral and enteric nervous systems. It appears to be involved in a wide variety of physiological functions within and outside the nervous system, that are maintained through a complex interaction between GABA and calcium-dependent neurotransmission and cellular metabolic functions. Within CNS the Ca(2+)/GABA mechanism stabilizes neuronal activity both at cellular and systemic levels. Decline in the Ca(2+)/GABA control initiates several cascading processes leading to both weakened protective barriers (in particular the blood-brain barrier) and accumulations of intracellular deposits of calcium and Lewy bodies. Linking such a vital mechanism of synaptic transmission with metabolism (both at cellular and tissue level) by means of a common reciprocal Ca(2+)/GABA inhibition results in a fragile balance, which is prone to destabilization and auto-destruction. The GABA decline etiology proposed here appears to apply to all human neurodegenerative processes initiated by abnormal intracellular calcium levels. Therefore, the original description of Parkinson's disease (PD) as due to the selective damage of dopaminergic neurons in the mesencephalon should be updated into the concept of a severe multisystemic neurodegenerative disorder of the nervous system, whose clinical symptoms reflect the localization and progression of the most advanced GABA pathology. A future and more complete therapeutic approach to PD should be aimed first at slowing (or stopping) the progression of Ca(2+)/GABA functional decline. PMID:27375426

  18. Parkinson's Disease and Neurodegeneration: GABA-Collapse Hypothesis

    PubMed Central

    Błaszczyk, Janusz W.

    2016-01-01

    Neurodegenerative diseases constitute a heterogeneous group of age-related disorders that are characterized by a slow but irreversible deterioration of brain functions. Evidence accumulated over more than two decades has implicated calcium-related homeostatic mechanisms, giving rise to the Ca2+ hypothesis of brain aging and, ultimately, cell death. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter within the central (CNS), peripheral and enteric nervous systems. It appears to be involved in a wide variety of physiological functions within and outside the nervous system, that are maintained through a complex interaction between GABA and calcium-dependent neurotransmission and cellular metabolic functions. Within CNS the Ca2+/GABA mechanism stabilizes neuronal activity both at cellular and systemic levels. Decline in the Ca2+/GABA control initiates several cascading processes leading to both weakened protective barriers (in particular the blood-brain barrier) and accumulations of intracellular deposits of calcium and Lewy bodies. Linking such a vital mechanism of synaptic transmission with metabolism (both at cellular and tissue level) by means of a common reciprocal Ca2+/GABA inhibition results in a fragile balance, which is prone to destabilization and auto-destruction. The GABA decline etiology proposed here appears to apply to all human neurodegenerative processes initiated by abnormal intracellular calcium levels. Therefore, the original description of Parkinson's disease (PD) as due to the selective damage of dopaminergic neurons in the mesencephalon should be updated into the concept of a severe multisystemic neurodegenerative disorder of the nervous system, whose clinical symptoms reflect the localization and progression of the most advanced GABA pathology. A future and more complete therapeutic approach to PD should be aimed first at slowing (or stopping) the progression of Ca2+/GABA functional decline. PMID:27375426

  19. Control of cortical neuronal migration by glutamate and GABA

    PubMed Central

    Luhmann, Heiko J.; Fukuda, A.; Kilb, W.

    2015-01-01

    Neuronal migration in the cortex is controlled by the paracrine action of the classical neurotransmitters glutamate and GABA. Glutamate controls radial migration of pyramidal neurons by acting primarily on NMDA receptors and regulates tangential migration of inhibitory interneurons by activating non-NMDA and NMDA receptors. GABA, acting on ionotropic GABAA-rho and GABAA receptors, has a dichotomic action on radially migrating neurons by acting as a GO signal in lower layers and as a STOP signal in upper cortical plate (CP), respectively. Metabotropic GABAB receptors promote radial migration into the CP and tangential migration of interneurons. Besides GABA, the endogenous GABAergic agonist taurine is a relevant agonist controlling radial migration. To a smaller extent glycine receptor activation can also influence radial and tangential migration. Activation of glutamate and GABA receptors causes increases in intracellular Ca2+ transients, which promote neuronal migration by acting on the cytoskeleton. Pharmacological or genetic manipulation of glutamate or GABA receptors during early corticogenesis induce heterotopic cell clusters in upper layers and loss of cortical lamination, i.e., neuronal migration disorders which can be associated with neurological or neuropsychiatric diseases. The pivotal role of NMDA and ionotropic GABA receptors in cortical neuronal migration is of major clinical relevance, since a number of drugs acting on these receptors (e.g., anti-epileptics, anesthetics, alcohol) may disturb the normal migration pattern when present during early corticogenesis. PMID:25688185

  20. Control of cortical neuronal migration by glutamate and GABA.

    PubMed

    Luhmann, Heiko J; Fukuda, A; Kilb, W

    2015-01-01

    Neuronal migration in the cortex is controlled by the paracrine action of the classical neurotransmitters glutamate and GABA. Glutamate controls radial migration of pyramidal neurons by acting primarily on NMDA receptors and regulates tangential migration of inhibitory interneurons by activating non-NMDA and NMDA receptors. GABA, acting on ionotropic GABAA-rho and GABAA receptors, has a dichotomic action on radially migrating neurons by acting as a GO signal in lower layers and as a STOP signal in upper cortical plate (CP), respectively. Metabotropic GABAB receptors promote radial migration into the CP and tangential migration of interneurons. Besides GABA, the endogenous GABAergic agonist taurine is a relevant agonist controlling radial migration. To a smaller extent glycine receptor activation can also influence radial and tangential migration. Activation of glutamate and GABA receptors causes increases in intracellular Ca(2+) transients, which promote neuronal migration by acting on the cytoskeleton. Pharmacological or genetic manipulation of glutamate or GABA receptors during early corticogenesis induce heterotopic cell clusters in upper layers and loss of cortical lamination, i.e., neuronal migration disorders which can be associated with neurological or neuropsychiatric diseases. The pivotal role of NMDA and ionotropic GABA receptors in cortical neuronal migration is of major clinical relevance, since a number of drugs acting on these receptors (e.g., anti-epileptics, anesthetics, alcohol) may disturb the normal migration pattern when present during early corticogenesis.

  1. Identification and functional characterization of a dual GABA/taurine transporter in the bullfrog retinal pigment epithelium

    PubMed Central

    1995-01-01

    Intracellular microelectrodes, fluorescence imaging, and radiotracer flux techniques were used to investigate the physiological response of the retinal pigment epithelium (RPE) to the major retinal inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). GABA is released tonically in the dark by amphibian horizontal cells, but is not taken up by the nearby Muller cells. Addition of GABA to the apical bath produced voltage responses in the bullfrog RPE that were not blocked nor mimicked by any of the major GABA-receptor antagonists or agonists. Nipecotic acid, a substrate for GABA transport, inhibited the voltage effects of GABA. GABA and nipecotic acid also inhibited the voltage effects of taurine, suggesting that the previously characterized beta- alanine sensitive taurine carrier also takes up GABA. The voltage responses of GABA, taurine, nipecotic acid, and beta-alanine all showed first-order saturable kinetics with the following Km's: GABA (Km = 160 microM), beta-alanine (Km = 250 microM), nipecotic acid (Km = 420 microM), and taurine (Km = 850 microM). This low affinity GABA transporter is dependent on external Na, partially dependent on external Cl, and is stimulated in low [K]o, which approximates subretinal space [K]o during light onset. Apical GABA also produced a significant conductance increase at the basolateral membrane. These GABA-induced conductance changes were blocked by basal Ba2+, suggesting that GABA decreased basolateral membrane K conductance. In addition, the apical membrane Na/K ATPase was stimulated in the presence of GABA. A model for the interaction between the GABA transporter, the Na/K ATPase, and the basolateral membrane K conductance accounts for the electrical effects of GABA. Net apical-to-basal flux of [3H]-GABA was also observed in radioactive flux experiments. The present study shows that a high capacity GABA uptake mechanism with unique pharmacological properties is located at the RPE apical membrane and could play an

  2. Luteinizing hormone releasing hormone (LHRH) neurons maintained in nasal explants decrease LHRH messenger ribonucleic acid levels after activation of GABA(A) receptors.

    PubMed

    Fueshko, S M; Key, S; Wray, S

    1998-06-01

    Inhibition of the LHRH system appears to play an important role in preventing precocious activation of the hypothalamic-pituitary-gonadal axis. Evidence points to gamma-aminobutyric acid (GABA) as the major negative regulator of postnatal LHRH neuronal activity. Changes in LHRH messenger RNA (mRNA) levels after alterations of GABAergic activity have been reported in vivo. However, the extent to which GABA acts directly on LHRH neurons to effect LHRH mRNA levels has been difficult to ascertain. The present work evaluates the effect of GABAergic activity, via GABA(A) receptors, on LHRH neuropeptide gene expression in LHRH neurons maintained in olfactory explants generated from E11.5 mouse embryos. These explants maintain large numbers of primary LHRH neurons that migrate from bilateral olfactory pits in a directed manner. Using in situ hybridization histochemistry and single cell analysis, we report dramatic alterations in LHRH mRNA levels. Inhibition of spontaneous synaptic activity by GABA(A) antagonists, bicuculline (10(-5) M) or picrotoxin (10(-4) M), or of electrical activity by tetrodotoxin (TTX, 10(-6) M) significantly increased LHRH mRNA levels. In contrast, LHRH mRNA levels decreased in explants cultured with the GABA(A) receptor agonist, muscimol (10(-4) M), or KCl (50 mM). The observed responses suggest that LHRH neurons possess functional pathways linking GABA(A) receptors to repression of neuropeptide gene expression and indicate that gene expression in embryonic LHRH neurons, outside the CNS, is highly responsive to alterations in neuronal activity.

  3. Synaptic-Type α1β2γ2L GABAA Receptors Produce Large Persistent Currents in the Presence of Ambient GABA and Anesthetic Drugs

    PubMed Central

    Li, Ping

    2015-01-01

    Synaptic GABAA receptors respond to synaptically released GABA and are considered to be unaffected by the low levels of ambient transmitter in the brain. We show that synaptic-type α1β2γ2L GABAA receptors expressed in HEK293 cells respond with large steady-state currents to combinations of a low concentration (0.5 μM) of GABA and clinically used GABAergic modulators propofol, etomidate, or pentobarbital or the steroid alphaxalone. At a maximally effective concentration of modulator, the current levels at the end of 2-minute applications of drug combinations were >10% of the peak response to saturating GABA. In the absence of modulators, 0.5 μM GABA generated a steady-state response of 1% of the peak response to saturating GABA. The concentration-response curves for enhancement of steady-state currents by propofol, etomidate, pentobarbital, or alphaxalone were at similar or lower drug concentrations compared with concentration-response relationships for enhancement of peak responses. We propose that modulation of tonically activated synaptic-type GABAA receptors contributes to the clinical actions of sedative drugs. PMID:25667223

  4. Neuronal transmembrane chloride electrochemical gradient: a key player in GABA A receptor activation physiological effect.

    PubMed

    Cupello, A

    2003-06-01

    It has long been accepted that GABA is the main inhibitory neurotransmitter in the mammalian brain, acting via GABA(A) or GABA(B) receptors. However, new evidences have shown that it may work as an excitatory transmitter, especially in the brain of newly-born animals and acting via GABA(A) receptors. The difference in the end results of GABA(A) receptors activation in the two cases is not due to the receptor associated channels, which in both cases are chloride channels. The different physiological effect in the two cases is due to different electrochemical gradients for chloride. When GABA acting via GABA(A) receptors is inhibitory, either there is no transmembrane electrochemical gradient for chloride or there is one forcing such negative ions into the nerve cell, once chloride channels are open. Viceversa, GABA is excitatory when the electrochemical gradient is such to make chloride ions flow outside the cell, upon opening of the GABA activated chloride channels.In this review this concept is discussed in details and evidence in the scientific literature for the existence of different types of chloride pumps (either internalizing or extruding chloride) is compiled.

  5. Invasive ability of human renal cell carcinoma cell line Caki-2 is accelerated by gamma-aminobutyric acid, via sustained activation of ERK1/2 inducible matrix metalloproteinases.

    PubMed

    Inamoto, Teruo; Azuma, Haruhito; Sakamoto, Takeshi; Kiyama, Satoshi; Ubai, Takanobu; Kotake, Yatsugu; Watanabe, Masahito; Katsuoka, Yoji

    2007-10-01

    Gamma-aminobutyric acid (GABA) was first discovered as an inhibitory neurotransmitter in the central nervous system (CNS) and has been reported to have a variety of functions, including regulation of cell division, cell differentiation and maturation, and to be involved in the development of certain cancers outside the CNS. In the present study, using the human renal cell carcinoma cell line Caki-2, we demonstrated that GABA stimulation significantly increased the expression of MMP-2 and -9 and subsequently increased the invasive activity of the cancer cells. Because MAPK signaling is one of the key regulators of MMP expression, we further evaluated MAPK signaling after stimulation with GABA. It was found that GABA stimulation promoted the phosphorylation of MAPKs, including ERK1/2, JNK, and p38. ERK1/2 phosphorylation was sustained for up to 12 h, while phosphorylation of JNK and p38 returned to the endogenous level by 30 min. It was noteworthy that the ras/raf/MEK/ERK pathway inhibitor PD98059 attenuated GABA-induced MMP-9 expression and that both PD98059 and MMP inhibitors attenuated the GABA-induced invasive activity of Caki-2 cells. Moreover, data obtained by depletion of the MEK/ERK pathway using interfering RNA transfection of Caki-2 cells clearly corroborated the above results, as both MMP-9 expression and GABA-induced invasive ability were decreased significantly. We also demonstrated that the GABA-induced increase in invasive ability via ERK1/2 up-regulation was mediated mainly through the GABA-B receptor. These results indicate that GABA stimulation promotes cancer cell invasion and that the effect is partly due to ERK1/2-dependent up-regulation of MMPs.

  6. Mutation of the Drosophila vesicular GABA transporter disrupts visual figure detection

    PubMed Central

    Fei, Hao; Chow, Dawnis M.; Chen, Audrey; Romero-Calderón, Rafael; Ong, Wei S.; Ackerson, Larry C.; Maidment, Nigel T.; Simpson, Julie H.; Frye, Mark A.; Krantz, David E.

    2010-01-01

    The role of gamma amino butyric acid (GABA) release and inhibitory neurotransmission in regulating most behaviors remains unclear. The vesicular GABA transporter (VGAT) is required for the storage of GABA in synaptic vesicles and provides a potentially useful probe for inhibitory circuits. However, specific pharmacologic agents for VGAT are not available, and VGAT knockout mice are embryonically lethal, thus precluding behavioral studies. We have identified the Drosophila ortholog of the vesicular GABA transporter gene (which we refer to as dVGAT), immunocytologically mapped dVGAT protein expression in the larva and adult and characterized a dVGATminos mutant allele. dVGAT is embryonically lethal and we do not detect residual dVGAT expression, suggesting that it is either a strong hypomorph or a null. To investigate the function of VGAT and GABA signaling in adult visual flight behavior, we have selectively rescued the dVGAT mutant during development. We show that reduced GABA release does not compromise the active optomotor control of wide-field pattern motion. Conversely, reduced dVGAT expression disrupts normal object tracking and figure–ground discrimination. These results demonstrate that visual behaviors are segregated by the level of GABA signaling in flies, and more generally establish dVGAT as a model to study the contribution of GABA release to other complex behaviors. PMID:20435823

  7. Dual action of isoflurane on the gamma-aminobutyric acid (GABA)-mediated currents through recombinant alpha(1)beta(2)gamma(2L)-GABA(A)-receptor channels.

    PubMed

    Neumahr, S; Hapfelmeier, G; Scheller, M; Schneck, H; Franke, C; Kochs, E

    2000-05-01

    Isoflurane (ISO) increased the agonist-induced chloride flux through the gamma-aminobutyric acid A receptor (GABA(A)R). This may reflect an anesthetic-induced increase in the apparent agonist affinity. A dual effect of anesthetics was postulated for both the nicotinic acetylcholine receptor (nAChR) and the GABA(A)R. We tested the hypothesis that, in addition to a blocking effect, ISO increases gamma-aminobutyric acid (GABA)-gated currents through recombinant GABA(A)R channels. HEK293 cells were transfected with rat cDNA for alpha(1),beta(2),gamma(2L) subunits. Currents elicited by 1 mM or 0. 01 mM GABA, respectively, alone, or with increasing concentrations of ISO, were recorded by using standard patch clamp techniques. ISO reduced the peak current elicited by 1 mM GABA. Currents induced by 0.01 mM GABA were potentiated by small ISO (twofold at 0.5 mM ISO) and inhibited by larger concentrations. Withdrawal of ISO and GABA induced rebound currents, suggesting an open-channel block by ISO. These currents increased with increasing concentrations of ISO. At large concentrations of ISO, the inhibitory effect predominated and was caused by, at least partly, an open-channel block. At small concentrations of ISO, potentiation of the GABA-gated currents was more prominent. This dual action of ISO indicates different binding sites at the GABA(A)R. The balance between potentiation and block depends on the concentrations of both ISO and GABA.

  8. Molecular analysis of the A322D mutation in the GABA receptor alpha-subunit causing juvenile myoclonic epilepsy.

    PubMed

    Krampfl, Klaus; Maljevic, Snezana; Cossette, Patrick; Ziegler, Elke; Rouleau, Guy A; Lerche, Holger; Bufler, Johannes

    2005-07-01

    Juvenile myoclonic epilepsy (JME) belongs to the most common forms of hereditary epilepsy, the idiopathic generalized epilepsies. Although the mode of inheritance is usually complex, mutations in single genes have been shown to cause the disease in some families with autosomal dominant inheritance. The first mutation in a multigeneration JME family has been recently found in the alpha1-subunit of the GABAA receptor (GABRA1), predicting the single amino acid substitution A322D. We further characterized the functional consequences of this mutation by coexpressing alpha1-, beta2- and gamma2-subunits in human embryonic kidney (HEK293) cells. By using an ultrafast application system, mutant receptors have shown reduced macroscopic current amplitudes at saturating GABA concentrations and a highly reduced affinity to GABA compared to the wild-type (WT). Dose-response curves for current amplitudes, activation kinetics, and GABA-dependent desensitization parameters showed a parallel shift towards 30- to 40-fold higher GABA concentrations. Both deactivation and resensitization kinetics were considerably accelerated in mutant channels. In addition, mutant receptors labelled with enhanced green fluorescent protein (EGFP) were not integrated in the cell membrane, in contrast to WT receptors. Therefore, the A322D mutation leads to a severe loss-of-function of the human GABAA receptor by several mechanisms, including reduced surface expression, reduced GABA-sensitivity, and accelerated deactivation. These molecular defects could decrease and shorten the resulting inhibitory postsynaptic currents (IPSCs) in vivo, which can induce a hyperexcitability of the postsynaptic membrane and explain the occurrence of epileptic seizures.

  9. Cannabinoids increase type 1 cannabinoid receptor expression in a cell culture model of striatal neurons: implications for Huntington's disease.

    PubMed

    Laprairie, Robert B; Kelly, Melanie E M; Denovan-Wright, Eileen M

    2013-09-01

    The type 1 cannabinoid receptor (CB1) is a G protein-coupled receptor that is expressed at high levels in the striatum. Activation of CB1 increases expression of neuronal trophic factors and inhibits neurotransmitter release from GABA-ergic striatal neurons. CB1 mRNA levels can be elevated by treatment with cannabinoids in non-neuronal cells. We wanted to determine whether cannabinoid treatment could induce CB1 expression in a cell culture model of striatal neurons and, if possible, determine the molecular mechanism by which this occurred. We found that treatment of STHdh(7/7) cells with the cannabinoids ACEA, mAEA, and AEA produced a CB1receptor-dependent increase in CB1 promoter activity, mRNA, and protein expression. This response was Akt- and NF-κB-dependent. Because decreased CB1 expression is thought to contribute to the pathogenesis of Huntington's disease (HD), we wanted to determine whether cannabinoids could increase CB1 expression in STHdh(7/111) and (111/111) cells expressing the mutant huntingtin protein. We observed that cannabinoid treatment increased CB1 mRNA levels approximately 10-fold in STHdh(7/111) and (111/111) cells, compared to vehicle treatment. Importantly, cannabinoid treatment improved ATP production, increased the expression of the trophic factor BDNF-2, and the mitochondrial regulator PGC1α, and reduced spontaneous GABA release, in HD cells. Therefore, cannabinoid-mediated increases in CB1 levels could reduce the severity of some molecular pathologies observed in HD.

  10. Strategies for multigene expression in eukaryotic cells.

    PubMed

    Mansouri, Maysam; Berger, Philipp

    2014-09-01

    Multigene delivery systems for heterologous multiprotein expression in mammalian cells are a key technology in contemporary biological research. Multiprotein expression is essential for a variety of applications, including multiparameter analysis of living cells in vitro, changing the fate of stem cells, or production of multiprotein complexes for structural biology. Depending on the application, these expression systems have to fulfill different requirements. For some applications, homogenous expression in all cells with defined stoichiometry is necessary, whereas other applications need long term expression or require that the proteins are not modified at the N- and C-terminus. Here we summarize available multiprotein expression systems and discuss their advantages and disadvantages.

  11. Strategies for multigene expression in eukaryotic cells.

    PubMed

    Mansouri, Maysam; Berger, Philipp

    2014-09-01

    Multigene delivery systems for heterologous multiprotein expression in mammalian cells are a key technology in contemporary biological research. Multiprotein expression is essential for a variety of applications, including multiparameter analysis of living cells in vitro, changing the fate of stem cells, or production of multiprotein complexes for structural biology. Depending on the application, these expression systems have to fulfill different requirements. For some applications, homogenous expression in all cells with defined stoichiometry is necessary, whereas other applications need long term expression or require that the proteins are not modified at the N- and C-terminus. Here we summarize available multiprotein expression systems and discuss their advantages and disadvantages. PMID:25034976

  12. Propofol depresses cerebellar Purkinje cell activity via activation of GABA(A) and glycine receptors in vivo in mice.

    PubMed

    Jin, Ri; Liu, Heng; Jin, Wen-Zhe; Shi, Jin-Di; Jin, Qing-Hua; Chu, Chun-Ping; Qiu, De-Lai

    2015-10-01

    Propofol is an intravenous sedative-hypnotic agen, which causes rapid and reliable loss of consciousness. Under in vitro conditions, propofol activates GABAA and glycine receptors in spinal cord, hippocampus and hypothalamus neurons. However, the effects of propofol on the cerebellar neuronal activity under in vivo conditions are currently unclear. In the present study, we examined the effects of propofol on the spontaneous activity of Purkinje cells (PCs) in urethane-anesthetized mice by cell-attached recording and pharmacological methods. Our results showed that cerebellar surface perfusion of propofol (10-1000 μM) induced depression of the PC simple spike (SS) firing rate in a dose-dependent manner, but without significantly changing the properties of complex spikes (CS). The IC50 of propofol for inhibiting SS firing of PCs was 144.5 μM. Application of GABAA receptor antagonist, SR95531 (40 μM) or GABAB receptor antagonist, saclofen (20 μM), as well as glycine receptor antagonist, strychnine (10 μM) alone failed to prevent the propofol-induced inhibition of PCs spontaneous activity. However, application the mixture of SR95531 (40 μM) and strychnine (10 μM) completely blocked the propofol-induced inhibition of PC SS firing. These data indicated that cerebellar surface application of propofol depressed PC SS firing rate via facilitation of GABAA and functional glycine receptors activity in adult cerebellar PCs under in vivo conditions. Our present results provide a new insight of the anesthetic action of propofol in cerebellar cortex, suggesting that propofol depresses the SS outputs of cerebellar PCs which is involved in both GABAA and glycine receptors activity.

  13. Two-photon imaging reveals somatodendritic chloride gradient in retinal ON-type bipolar cells expressing the biosensor Clomeleon.

    PubMed

    Duebel, Jens; Haverkamp, Silke; Schleich, Wolfram; Feng, Guoping; Augustine, George J; Kuner, Thomas; Euler, Thomas

    2006-01-01

    A somatodendritic gradient of Cl(-) concentration ([Cl(-)](i)) has been postulated to generate GABA-evoked responses of different polarity in retinal bipolar cells, hyperpolarizing in OFF cells with low dendritic [Cl(-)](i), and depolarizing in ON cells with high dendritic [Cl(-)](i). As glutamate released by the photoreceptors depolarizes OFF cells and hyperpolarizes ON cells, the bipolars' antagonistic receptive field (RF) could be computed by simply integrating glutamatergic inputs from the RF center and GABAergic inputs from horizontal cells in the RF surround. Using ratiometric two-photon imaging of Clomeleon, a Cl(-) indicator transgenically expressed in ON bipolar cells, we found that dendritic [Cl(-)](i) exceeds somatic [Cl(-)](i) by up to 20 mM and that GABA application can lead to Cl(-) efflux (depolarization) in these dendrites. Blockers of Cl(-) transporters reduced the somatodendritic [Cl(-)](i) gradient. Hence, our results support the idea that ON bipolar cells employ a somatodendritic [Cl(-)](i) gradient to invert GABAergic horizontal cell input.

  14. Vitamin E-Induced Changes in Glutamate and GABA Metabolizing Enzymes of Chick Embryo Cerebrum

    PubMed Central

    Dessai, Shanti N.; Pinto, Annaliza

    2013-01-01

    Vitamin E exists in eight different forms, four tocopherols and four tocotrienols. It forms an important component of our antioxidant system. The structure of Vitamin E makes it unique and indispensable in protecting cell membranes. α-tocopherol, one of the forms of Vitamin E, is also known to regulate signal transduction pathways by mechanisms that are independent of its antioxidant properties. Vitamin E compounds reduce the production of inflammatory compounds such as prostaglandins. Swollen, dystrophic axons are considered as the hallmark of Vitamin E deficiency in the brains of rats, monkeys, and humans. The present work aimed to study the Vitamin E- (α-tochopherol acetate-) induced alterations of enzymes involved in metabolism of Glutamate and GABA during developmental neurogenesis of cerebrum. Therefore, cytosolic and crude mitochondrial enzyme activities of glutamine synthetase, aspartate transaminase, alanine transaminase, GABA transaminase, succinic Semialdehyde dehydrogenase, glutamic dehydrogenase, and α-Ketoglutarate dehydrogenase were analysed. Vitamin E induced significant changes in these enzymes thus altering the normal levels of glutamate and GABA during developmental neurogenesis. Such changes are surely to disturb the expression and/or intensity of neurotransmitter signaling during critical periods of brain development. PMID:23984094

  15. Potentiation of the ionotropic GABA receptor response by whiskey fragrance.

    PubMed

    Hossain, Sheikh Julfikar; Aoshima, Hitoshi; Koda, Hirofumi; Kiso, Yoshinobu

    2002-11-01

    It is well-known that the target of most mood-defining compounds is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activity in the human brain. To study the effects of whiskey fragrance on the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting rat whole brain mRNA or cRNA prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors. Most whiskey components such as phenol, ethoxy, and lactone derivatives potentiated the electrical responses of GABA(A) receptors, especially ethyl phenylpropanoate (EPP), which strongly potentiated the response. When this compound was applied to mice through respiration, the convulsions induced by pentetrazole were delayed, suggesting that EPP was absorbed by the brain, where it could potentiate the GABA(A) receptor responses. The extract of other alcoholic drinks such as wine, sake, brandy, and shochu also potentiated the responses to varying degrees. Although these fragrant components are present in alcoholic drinks at low concentrations (extremely small quantities compared with ethanol), they may also modulate the mood or consciousness of the human through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic fragrant compounds are easily absorbed into the brain through the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response.

  16. Potentiation of the ionotropic GABA receptor response by whiskey fragrance.

    PubMed

    Hossain, Sheikh Julfikar; Aoshima, Hitoshi; Koda, Hirofumi; Kiso, Yoshinobu

    2002-11-01

    It is well-known that the target of most mood-defining compounds is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activity in the human brain. To study the effects of whiskey fragrance on the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting rat whole brain mRNA or cRNA prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors. Most whiskey components such as phenol, ethoxy, and lactone derivatives potentiated the electrical responses of GABA(A) receptors, especially ethyl phenylpropanoate (EPP), which strongly potentiated the response. When this compound was applied to mice through respiration, the convulsions induced by pentetrazole were delayed, suggesting that EPP was absorbed by the brain, where it could potentiate the GABA(A) receptor responses. The extract of other alcoholic drinks such as wine, sake, brandy, and shochu also potentiated the responses to varying degrees. Although these fragrant components are present in alcoholic drinks at low concentrations (extremely small quantities compared with ethanol), they may also modulate the mood or consciousness of the human through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic fragrant compounds are easily absorbed into the brain through the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response. PMID:12405783

  17. Regulation of Local Ambient GABA Levels via Transporter-Mediated GABA Import and Export for Subliminal Learning.

    PubMed

    Hoshino, Osamu

    2015-06-01

    Perception of supraliminal stimuli might in general be reflected in bursts of action potentials (spikes), and their memory traces could be formed through spike-timing-dependent plasticity (STDP). Memory traces for subliminal stimuli might be formed in a different manner, because subliminal stimulation evokes a fraction (but not a burst) of spikes. Simulations of a cortical neural network model showed that a subliminal stimulus that was too brief (10 msec) to perceive transiently (more than about 500 msec) depolarized stimulus-relevant principal cells and hyperpolarized stimulus-irrelevant principal cells in a subthreshold manner. This led to a small increase or decrease in ongoing-spontaneous spiking activity frequency (less than 1 Hz). Synaptic modification based on STDP during this period effectively enhanced relevant synaptic weights, by which subliminal learning was improved. GABA transporters on GABAergic interneurons modulated local levels of ambient GABA. Ambient GABA molecules acted on extrasynaptic receptors, provided principal cells with tonic inhibitory currents, and contributed to achieving the subthreshold neuronal state. We suggest that ongoing-spontaneous synaptic alteration through STDP following subliminal stimulation may be a possible neuronal mechanism for leaving its memory trace in cortical circuitry. Regulation of local ambient GABA levels by transporter-mediated GABA import and export may be crucial for subliminal learning. PMID:25774546

  18. Distinct roles for GABA across multiple timescales in mammalian circadian timekeeping

    PubMed Central

    DeWoskin, Daniel; Myung, Jihwan; Belle, Mino D. C.; Piggins, Hugh D.; Takumi, Toru; Forger, Daniel B.

    2015-01-01

    The suprachiasmatic nuclei (SCN), the central circadian pacemakers in mammals, comprise a multiscale neuronal system that times daily events. We use recent advances in graphics processing unit computing to generate a multiscale model for the SCN that resolves cellular electrical activity down to the timescale of individual action potentials and the intracellular molecular events that generate circadian rhythms. We use the model to study the role of the neurotransmitter GABA in synchronizing circadian rhythms among individual SCN neurons, a topic of much debate in the circadian community. The model predicts that GABA signaling has two components: phasic (fast) and tonic (slow). Phasic GABA postsynaptic currents are released after action potentials, and can both increase or decrease firing rate, depending on their timing in the interspike interval, a modeling hypothesis we experimentally validate; this allows flexibility in the timing of circadian output signals. Phasic GABA, however, does not significantly affect molecular timekeeping. The tonic GABA signal is released when cells become very excited and depolarized; it changes the excitability of neurons in the network, can shift molecular rhythms, and affects SCN synchrony. We measure which neurons are excited or inhibited by GABA across the day and find GABA-excited neurons are synchronized by—and GABA-inhibited neurons repelled from—this tonic GABA signal, which modulates the synchrony in the SCN provided by other signaling molecules. Our mathematical model also provides an important tool for circadian research, and a model computational system for the many multiscale projects currently studying brain function. PMID:26130805

  19. Stoichiometry of expressed alpha(4)beta(2)delta gamma-aminobutyric acid type A receptors depends on the ratio of subunit cDNA transfected.

    PubMed

    Wagoner, Kelly R; Czajkowski, Cynthia

    2010-05-01

    The gamma-aminobutyric acid type A receptor (GABA(A)R) is the target of many depressants, including benzodiazepines, anesthetics, and alcohol. Although the highly prevalent alphabetagamma GABA(A)R subtype mediates the majority of fast synaptic inhibition in the brain, receptors containing delta subunits also play a key role, mediating tonic inhibition and the actions of endogenous neurosteroids and alcohol. However, the fundamental properties of delta-containing GABA(A)Rs, such as subunit stoichiometry, are not well established. To determine subunit stoichiometry of expressed delta-containing GABA(A)Rs, we inserted the alpha-bungarotoxin binding site tag in the alpha(4), beta(2), and delta subunit N termini. An enhanced green fluorescent protein tag was also inserted into the beta(2) subunit to shift its molecular weight, allowing us to separate subunits using SDS-PAGE. Tagged alpha(4)beta(2)delta GABA(A)Rs were expressed in HEK293T cells using various ratios of subunit cDNA, and receptor subunit stoichiometry was determined by quantitating fluorescent alpha-bungarotoxin bound to each subunit on Western blots of surface immunopurified tagged GABA(A)Rs. The results demonstrate that the subunit stoichiometry of alpha(4)beta(2)delta GABA(A)Rs is regulated by the ratio of subunit cDNAs transfected. Increasing the ratio of delta subunit cDNA transfected increased delta subunit incorporation into surface receptors with a concomitant decrease in beta(2) subunit incorporation. Because receptor subunit stoichiometry can directly influence GABA(A)R pharmacological and functional properties, considering how the transfection protocols used affect subunit stoichiometry is essential when studying heterologously expressed alpha(4)beta(2)delta GABA(A)Rs. Successful bungarotoxin binding site tagging of GABA(A)R subunits is a novel tool with which to accurately quantitate subunit stoichiometry and will be useful for monitoring GABA(A)R trafficking in live cells.

  20. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site.

    PubMed

    Snell, Heather D; Gonzales, Eric B

    2015-06-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride's positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound's potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ.

  1. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site

    PubMed Central

    Snell, Heather D.

    2015-01-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride’s positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound’s potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ. PMID:25829529

  2. Connexin expression in renin-producing cells.

    PubMed

    Kurtz, Lisa; Janssen-Bienhold, Ulrike; Kurtz, Armin; Wagner, Charlotte

    2009-03-01

    Absence of connexin 40 (Cx40) leads to ectopic juxtaglomerular renin expression and abrogates recruitment of renin-expressing cells in the adult kidney but does not disturb renin expression during kidney development. To find an explanation for these observations, we aimed to analyze the expression pattern of major vascular Cxs in normal juxtaglomerular epithelioid cells, in recruited renin-expressing cells, and in fetal renin-expressing cells. We found that during kidney development, the appearance of renin-producing cells paralleled the expression of Cx40 and, to a lesser extent, Cx45 but not other Cxs. In the adult kidney, juxtaglomerular epithelioid cells expressed Cx40 and lesser amounts of Cx37 and Cx43 but not Cx45, which localized to arteriolar smooth muscle cells. Recruitment of renin-producing cells in adult kidneys in response to long-term salt deprivation of mice correlated with the reappearance of only Cx40. Cx40-null renin-producing cells did not express Cx37, Cx43, or Cx45. These findings suggest that Cx40 expression is a characteristic of renin-producing cells in the kidney, and it seems to be essential in the recruitment of renin-producing cells in the adult but not the fetal kidney. PMID:19073828

  3. Segregation of Acetylcholine and GABA in the Rat Superior Cervical Ganglia: Functional Correlation

    PubMed Central

    Elinos, Diana; Rodríguez, Raúl; Martínez, Luis Andres; Zetina, María Elena; Cifuentes, Fredy; Morales, Miguel Angel

    2016-01-01

    Sympathetic neurons have the capability to segregate their neurotransmitters (NTs) and co-transmitters to separate varicosities of single axons; furthermore, in culture, these neurons can even segregate classical transmitters. In vivo sympathetic neurons employ acetylcholine (ACh) and other classical NTs such as gamma aminobutyric acid (GABA). Herein, we explore whether these neurons in vivo segregate these classical NTs in the superior cervical ganglia of the rat. We determined the topographical distribution of GABAergic varicosities, somatic GABAA receptor, as well as the regional distribution of the segregation of ACh and GABA. We evaluated possible regional differences in efficacy of ganglionic synaptic transmission, in the sensitivity of GABAA receptor to GABA and to the competitive antagonist picrotoxin (PTX). We found that sympathetic preganglionic neurons in vivo do segregate ACh and GABA. GABAergic varicosities and GABAA receptor expression showed a rostro-caudal gradient along ganglia; in contrast, segregation exhibited a caudo-rostral gradient. These uneven regional distributions in expression of GABA, GABAA receptors, and level of segregation correlate with stronger synaptic transmission found in the caudal region. Accordingly, GABAA receptors of rostral region showed larger sensitivity to GABA and PTX. These results suggest the presence of different types of GABAA receptors in each region that result in a different regional levels of endogenous GABA inhibition. Finally, we discuss a possible correlation of these different levels of GABA modulation and the function of the target organs innervated by rostral and caudal ganglionic neurons. PMID:27092054

  4. Photoreceptor cells constitutively express functional TLR4

    PubMed Central

    Tu, Zhidan; Portillo, Jose-Andres; Howell, Scott; Bu, Hong; Subauste, Carlos S.; Al-Ubaidi, Muayyad R; Pearlman, Eric; Lin, Feng

    2010-01-01

    Toll-like receptor 4 (TLR4) is expressed on a number of cells including neurons in the brain. However, it has yet to be determined if TLR4 is expressed on photoreceptor cells in the retina. In this report, we examined primary photoreceptor cells and an established photoreceptor cell line (661W). We found that functional TLR4 is constitutively expressed on photoreceptor cells, and can be activated by LPS. We conclude that TLR4 on photoreceptor cells could directly contribute to retinal inflammatory diseases and photoreceptor cell survival. PMID:20801528

  5. Simultaneous optical recording in multiple cells by digital holographic microscopy of chloride current associated to activation of the ligand-gated chloride channel GABA(A) receptor.

    PubMed

    Jourdain, Pascal; Boss, Daniel; Rappaz, Benjamin; Moratal, Corinne; Hernandez, Maria-Clemencia; Depeursinge, Christian; Magistretti, Pierre Julius; Marquet, Pierre

    2012-01-01

    Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABA(A) mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABA(A) receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABA(A) receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM.

  6. [Effect of retroauricular galvanic stimulation on the central vestibular system--immunohistochemical evaluation of GABA].

    PubMed

    Okami, K; Sekitani, T

    1990-03-01

    The changes of the neurotransmitter (GABA) distribution in the brain stem of the rats by retroauricular galvanic stimulation were investigated using immunohistochemical method. Twenty-one rats were divided into two groups: the control group which received no galvanic stimulation, and the galvanically stimulated group which received anodal galvanic stimulation (unipolar monoauricular, 5 mA in intensity, 500 msec of duration, 1 Hz in frequency) for 30 minutes. The specimens obtained as usual strict procedure for histological investigation were stained immunohistochemically using antisera against GABA. The results were as follows: 1. In the control group, GABA-like immunoreactivity was observed in all four main vestibular nuclei. In the superior, medial, and descending vestibular nuclei GABA-like immunoreactivity was found in the small cells and the terminals. Giant cells in the lateral vestibular nucleus were surrounded by GABA immunoreactive terminals. 2. In the galvanically stimulated group GABA-like immunoreactivity showed recognizable laterality in the lateral vestibular nucleus where GABA-like immunoreactivity surrounding giant cells showed more intensive on the side ipsilateral to the stimulation compared with the opposite side. On the other hand GABA-like immunoreactivity showed no laterality in the superior, medial, and descending vestibular nuclei. 3. It can be concluded that the retroauricular galvanic stimulation cause some changes in the inhibitory activity of the lateral vestibulo-spinal tract and of the spinal motor neuron.

  7. Meta-diamide insecticides acting on distinct sites of RDL GABA receptor from those for conventional noncompetitive antagonists.

    PubMed

    Nakao, Toshifumi; Banba, Shinich; Nomura, Michikazu; Hirase, Kangetsu

    2013-04-01

    The RDL GABA receptor is an attractive target of insecticides. Here we demonstrate that meta-diamides [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamides] are a distinct class of RDL GABA receptor antagonists showing high insecticidal activity against Spodoptera litura. We also suggest that the mode of action of the meta-diamides is distinct from that of conventional noncompetitive antagonists (NCAs), such as fipronil, picrotoxin, lindane, dieldrin, and α-endosulfan. Using a membrane potential assay, we examined the effects of the meta-diamide 3-benzamido-N-(2-bromo-4-(perfluoropropan-2-yl)-6-(trifluoromethyl)phenyl)-2-fluorobenzamide (meta-diamide 7) and NCAs on mutant Drosophila RDL GABA receptors expressed in Drosophila Mel-2 cells. NCAs had little or no inhibitory activity against at least one of the three mutant receptors (A2'S, A2'G, and A2'N), which were reported to confer resistance to NCAs. In contrast, meta-diamide 7 inhibited all three A2' mutant receptors, at levels comparable to its activity with the wild-type receptor. Furthermore, the A2'S·T6'V mutation almost abolished the inhibitory effects of all NCAs. However, meta-diamide 7 inhibited the A2'S・T6'S mutant receptor at the same level as its activity with the wild-type receptor. In contrast, a G336M mutation in the third transmembrane domain of the RDL GABA receptor abolished the inhibitory activities of meta-diamide 7, although the G336M mutation had little effect on the inhibitory activities of conventional NCAs. Molecular modeling studies also suggested that the binding site of meta-diamides was different from those of NCAs. Meta-diamide insecticides are expected to be prominent insecticides effective against A2' mutant RDL GABA receptors with a different mode of action.

  8. Normal sleep homeostasis and lack of epilepsy phenotype in GABA A receptor alpha3 subunit-knockout mice.

    PubMed

    Winsky-Sommerer, R; Knapman, A; Fedele, D E; Schofield, C M; Vyazovskiy, V V; Rudolph, U; Huguenard, J R; Fritschy, J-M; Tobler, I

    2008-06-23

    Thalamo-cortical networks generate specific patterns of oscillations during distinct vigilance states and epilepsy, well characterized by electroencephalography (EEG). Oscillations depend on recurrent synaptic loops, which are controlled by GABAergic transmission. In particular, GABA A receptors containing the alpha3 subunit are expressed predominantly in cortical layer VI and thalamic reticular nucleus (nRT) and regulate the activity and firing pattern of neurons in relay nuclei. Therefore, ablation of these receptors by gene targeting might profoundly affect thalamo-cortical oscillations. Here, we investigated the role of alpha3-GABA A receptors in regulating vigilance states and seizure activity by analyzing chronic EEG recordings in alpha3 subunit-knockout (alpha3-KO) mice. The presence of postsynaptic alpha3-GABA A receptors/gephyrin clusters in the nRT and GABA A-mediated synaptic currents in acute thalamic slices was also examined. EEG spectral analysis showed no difference between genotypes during non rapid-eye movement (NREM) sleep or at waking-NREM sleep transitions. EEG power in the spindle frequency range (10-15 Hz) was significantly lower at NREM-REM sleep transitions in mutant compared with wild-type mice. Enhancement of sleep pressure by 6 h sleep deprivation did not reveal any differences in the regulation of EEG activities between genotypes. Finally, the waking EEG showed a slightly larger power in the 11-13-Hz band in alpha3-KO mice. However, neither behavior nor the waking EEG showed alterations suggestive of absence seizures. Furthermore, alpha3-KO mice did not differ in seizure susceptibility in a model of temporal lobe epilepsy. Strikingly, despite the disruption of postsynaptic gephyrin clusters, whole-cell patch clamp recordings revealed intact inhibitory synaptic transmission in the nRT of alpha3-KO mice. These findings show that the lack of alpha3-GABA(A) receptors is extensively compensated for to preserve the integrity of thalamo

  9. Gestational changes of GABA levels and GABA binding in the human uterus

    SciTech Connect

    Erdoe, S.L.; Villanyi, P.; Laszlo, A.

    1989-01-01

    The concentrations of gamma-aminobutyric acid (GABA), the activities of L-glutamate decarboxylase and GABA-transaminase, and the nature of the sodium-independent binding of GABA were examined in uterine tissue pieces obtained surgically from pregnant and non-pregnant women. GABA concentrations were reduced, while the activity of GABA-transaminase and the specific binding of (/sup 3/H)GABA significantly increased in specimens from pregnant subjects. These findings suggest some gestation-related functional role for the GABA system in the human uterus.

  10. Development of the GABA-ergic signaling system and its role in larval swimming in sea urchin.

    PubMed

    Katow, Hideki; Abe, Kouki; Katow, Tomoko; Zamani, Alemeh; Abe, Hirokazu

    2013-05-01

    The present study aimed to elucidate the development and γ-amino butyric acid (GABA)-ergic regulation of larval swimming in the sea urchin Hemicentrotus pulcherrimus by cloning glutamate decarboxylase (Hp-gad), GABAA receptor (Hp-gabrA) and GABAA receptor-associated protein (Hp-gabarap), and by performing immunohistochemistry. The regulation of larval swimming was increasingly dependent on the GABAergic system, which was active from the 2 days post-fertilization (d.p.f.) pluteus stage onwards. GABA-immunoreactive cells were detected as a subpopulation of secondary mesenchyme cells during gastrulation and eventually constituted the ciliary band and a subpopulation of blastocoelar cells during the pluteus stage. Hp-gad transcription was detected by RT-PCR during the period when Hp-Gad-positive cells were seen as a subpopulation of blastocoelar cells and on the apical side of the ciliary band from the 2 d.p.f. pluteus stage. Consistent with these observations, inhibition of GAD with 3-mercaptopropioninc acid inhibited GABA immunoreactivity and larval swimming dose dependently. Hp-gabrA amplimers were detected weakly in unfertilized eggs and 4 d.p.f. plutei but strongly from fertilized eggs to 2 d.p.f. plutei, and Hp-GabrA, together with GABA, was localized at the ciliary band in association with dopamine receptor D1 from the two-arm pluteus stage. Hp-gabarap transcription and protein expression were detected from the swimming blastula stage. Inhibition of the GABAA receptor by bicuculline inhibited larval swimming dose dependently. Inhibition of larval swimming by either 3-mercaptopropionic acid or bicuculline was more severe in older larvae (17 and 34 d.p.f. plutei) than in younger ones (1 d.p.f. prism larvae). PMID:23307803

  11. Gene expression as a sensitive endpoint to evaluate cell differentiation and maturation of the developing central nervous system in primary cultures of rat cerebellar granule cells (CGCs) exposed to pesticides

    SciTech Connect

    Hogberg, Helena T.; Kinsner-Ovaskainen, Agnieszka; Hartung, Thomas; Coecke, Sandra; Bal-Price, Anna K.

    2009-03-15

    The major advantage of primary neuronal cultures for developmental neurotoxicity (DNT) testing is their ability to replicate the crucial stages of neurodevelopment. In our studies using primary culture of cerebellar granule cells (CGCs) we have evaluated whether the gene expression relevant to the most critical developmental processes such as neuronal differentiation (NF-68 and NF-200) and functional maturation (NMDA and GABA{sub A} receptors), proliferation and differentiation of astrocytes (GFAP and S100{beta}) as well as the presence of neural precursor cells (nestin and Sox10) could be used as an endpoint for in vitro DNT. The expression of these genes was assessed after exposure to various pesticides (paraquat parathion, dichlorvos, pentachlorophenol and cycloheximide) that could induce developmental neurotoxicity through different mechanisms. All studied pesticides significantly modified the expression of selected genes, related to the different stages of neuronal and/or glial cell development and maturation. The most significant changes were observed after exposure to paraquat and parathion (i.e. down-regulation of mRNA expression of NF-68 and NF-200, NMDA and GABA{sub A} receptors). Similarly, dichlorvos affected mainly neurons (decreased mRNA expression of NF-68 and GABA{sub A} receptors) whereas cycloheximide had an effect on neurons and astrocytes, as significant decreases in the mRNA expression of both neurofilaments (NF-68 and NF-200) and the astrocyte marker (S100{beta}) were observed. Our results suggest that toxicity induced by pesticides that target multiple pathways of neurodevelopment can be identified by studying expression of genes that are involved in different stages of cell development and maturation, and that gene expression could be used as a sensitive endpoint for initial screening to identify the compounds with the potential to cause developmental neurotoxicity.

  12. Hyperglycosylation and reduced GABA currents of mutated GABRB3 polypeptide in remitting childhood absence epilepsy.

    PubMed

    Tanaka, Miyabi; Olsen, Richard W; Medina, Marco T; Schwartz, Emily; Alonso, Maria Elisa; Duron, Reyna M; Castro-Ortega, Ramon; Martinez-Juarez, Iris E; Pascual-Castroviejo, Ignacio; Machado-Salas, Jesus; Silva, Rene; Bailey, Julia N; Bai, Dongsheng; Ochoa, Adriana; Jara-Prado, Aurelio; Pineda, Gregorio; Macdonald, Robert L; Delgado-Escueta, Antonio V

    2008-06-01

    Childhood absence epilepsy (CAE) accounts for 10% to 12% of epilepsy in children under 16 years of age. We screened for mutations in the GABA(A) receptor (GABAR) beta 3 subunit gene (GABRB3) in 48 probands and families with remitting CAE. We found that four out of 48 families (8%) had mutations in GABRB3. One heterozygous missense mutation (P11S) in exon 1a segregated with four CAE-affected persons in one multiplex, two-generation Mexican family. P11S was also found in a singleton from Mexico. Another heterozygous missense mutation (S15F) was present in a singleton from Honduras. An exon 2 heterozygous missense mutation (G32R) was present in two CAE-affected persons and two persons affected with EEG-recorded spike and/or sharp wave in a two-generation Honduran family. All mutations were absent in 630 controls. We studied functions and possible pathogenicity by expressing mutations in HeLa cells with the use of Western blots and an in vitro translation and translocation system. Expression levels did not differ from those of controls, but all mutations showed hyperglycosylation in the in vitro translation and translocation system with canine microsomes. Functional analysis of human GABA(A) receptors (alpha 1 beta 3-v2 gamma 2S, alpha 1 beta 3-v2[P11S]gamma 2S, alpha 1 beta 3-v2[S15F]gamma 2S, and alpha 1 beta 3-v2[G32R]gamma 2S) transiently expressed in HEK293T cells with the use of rapid agonist application showed that each amino acid transversion in the beta 3-v2 subunit (P11S, S15F, and G32R) reduced GABA-evoked current density from whole cells. Mutated beta 3 subunit protein could thus cause absence seizures through a gain in glycosylation of mutated exon 1a and exon 2, affecting maturation and trafficking of GABAR from endoplasmic reticulum to cell surface and resulting in reduced GABA-evoked currents.

  13. Genetic manipulation of the γ-aminobutyric acid (GABA) shunt in rice: overexpression of truncated glutamate decarboxylase (GAD2) and knockdown of γ-aminobutyric acid transaminase (GABA-T) lead to sustained and high levels of GABA accumulation in rice kernels.

    PubMed

    Shimajiri, Yasuka; Oonishi, Takayuki; Ozaki, Kae; Kainou, Kumiko; Akama, Kazuhito

    2013-06-01

    Gamma-aminobutyric acid (GABA) is a non-protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA-transaminase, GABA-T), we attempted seed-specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB-1) or rice embryo globulin promoters (REG) and GABA-T-based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T₁ and T₂ generations of rice lines displayed high GABA concentrations (2-100 mg/100 g grain). In analyses of two selected lines from the T₃ generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA-T expression was relatively weak. In these two lines both with two T-DNA copies, their starch, amylose, and protein levels were slightly lower than non-transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75-350 mg/100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts.

  14. Differential effects of GABA in modulating nociceptive vs. non-nociceptive synapses.

    PubMed

    Wang, Y; Summers, T; Peterson, W; Miiller, E; Burrell, B D

    2015-07-01

    GABA (γ-amino-butyric acid) -mediated signaling is normally associated with synaptic inhibition due to ionotropic GABA receptors that gate an inward Cl(-) current, hyperpolarizing the membrane potential. However, there are also situations where ionotropic GABA receptors trigger a Cl(-) efflux that results in depolarization. The well-characterized central nervous system of the medicinal leech was used to study the functional significance of opposing effects of GABA at the synaptic circuit level. Specifically, we focused on synapses made by the nociceptive N cell and the non-nociceptive P (pressure) cell that converge onto a common postsynaptic target. It is already known that GABA hyperpolarizes the P cell, but depolarizes the N cell and that inhibition of ionotropic GABA receptors by bicuculline (BIC) has opposing effects on the synapses made by these two inputs; enhancing P cell synaptic transmission, but depressing N cell synapses. The goal of the present study was to determine whether the opposing effects of GABA were due to differences in Cl(-) homeostasis between the two presynaptic neurons. VU 0240551 (VU), an inhibitor of the Cl(-) exporter K-Cl co-transporter isoform 2 (KCC2), attenuated GABA-mediated hyperpolarization of the non-nociceptive afferent while bumetanide (BUM), an inhibitor of the Cl(-) importer Na-K-Cl co-transporter isoform 1 (NKCC1), reduced GABA-mediated depolarization of the nociceptive neuron. VU treatment also enhanced P cell synaptic signaling, similar to the previously observed effects of BIC and consistent with the idea that GABA inhibits synaptic signaling at the presynaptic level. BUM treatment depressed N cell synapses, again similar to what is observed following BIC treatment and suggests that GABA has an excitatory effect on these synapses. The opposing effects of GABA could also be observed at the behavioral level with BIC and VU increasing responsiveness to non-nociceptive stimulation while BIC and BUM decreased responsiveness

  15. Single rodent mesohabenular axons release glutamate and GABA

    PubMed Central

    Root, David H.; Mejias-Aponte, Carlos; Zhang, Shiliang; Wang, Huiling; Hoffman, Alexander F.; Lupica, Carl R.; Morales, Marisela

    2016-01-01

    The lateral habenula (LHb) is involved in reward, aversion, addiction, and depression, through descending interactions with several brain structures, including the ventral tegmental area (VTA). VTA provides reciprocal inputs to LHb, but their actions are unclear. Here we show that the majority of rat and mouse VTA neurons innervating LHb co-express markers for both glutamate-signaling (vesicular glutamate transporter 2, VGluT2) and GABA-signaling (glutamate decarboxylase, GAD; and vesicular GABA transporter, VGaT). A single axon from these mesohabenular neurons co-expresses VGluT2-protein and VGaT-protein, and surprisingly establishes symmetric and asymmetric synapses on LHb neurons. In LHb slices, light activation of mesohabenular fibers expressing channelrhodopsin-2 (ChR2) driven by VGluT2 or VGaT promoters elicits release of both glutamate and GABA onto single LHb neurons. In vivo light-activation of mesohabenular terminals inhibits or excites LHb neurons. Our findings reveal an unanticipated type of VTA neuron that co-transmits glutamate and GABA, and provides the majority of mesohabenular inputs. PMID:25242304

  16. Intracellular calcium ions decrease the affinity of the GABA receptor.

    PubMed

    Inoue, M; Oomura, Y; Yakushiji, T; Akaike, N

    Intracellular free Ca2+ [( Ca2+]i) plays a crucial role in the transduction of extracellular signals. It has been implicated in the modulation of light sensitivity in Limulus photoreceptors and in the efficacy of synaptic transmission; calcium ion fluxes are also involved in the postsynaptic facilitation of nicotinic transmission seen in sympathetic ganglia, and in activation of the acetylcholine (ACh) receptor. [Ca2+]i is also a second messenger for many biologically active substances. We recorded neuronal activities of sensory neurones from the bullfrog (Rana catesbiana), using the suction pipette method and a 'concentration clamp' technique to apply gamma-aminobutyric acid (GABA) to the cell. We report the first evidence that [Ca2+]i suppresses the GABA-activated Cl- conductance, by decreasing the apparent affinity of the GABA receptor. PMID:2431316

  17. Expression of S100 beta in sensory and secretory cells of the vertebrate inner ear

    NASA Technical Reports Server (NTRS)

    Fermin, C. D.; Martin, D. S.

    1995-01-01

    We evaluated anti-S100 beta expression in the chick (Gallus domesticus) inner ear and determined that: 1) the monomer anti-S100 beta is expressed differentially in the vestibular and auditory perikarya; 2) expression of S100 beta in the afferent nerve terminals is time-related to synapse and myelin formation; 3) the expression of the dimer anti-S100 alpha alpha beta beta and monomer anti-S100 beta overlaps in most inner ear cell types. Most S100 alpha alpha beta beta positive cells express S100 beta, but S100 beta positive cells do not always express S100 alpha alpha beta beta. 4) the expression of S100 beta is diffused over the perikaryal cytoplasm and nuclei of the acoustic ganglia but is concentrated over the nuclei of the vestibular perikarya. 6) S100 beta is expressed in secretory cells, and it is co-localized with GABA in sensory cells. 7) Color thresholding objective quantitation indicates that the amount of S100 beta was higher (mean 22, SD +/- 4) at E19 than at E9 (mean 34, SD +/- 3) in afferent axons. 8) Moreover, S100 beta was unchanged between E11-E19 in the perikaryal cytoplasm, but did change over the nuclei. At E9, 74%, and at E21, 5% of vestibular perikarya were positive. The data suggest that S100 beta may be physically associated with neuronal and ionic controlling cells of the vertebrate inner ear, where it could provide a dual ionic and neurotrophic modulatory function.

  18. Imbalance between Glutamate and GABA in Fmr1 Knockout Astrocytes Influences Neuronal Development

    PubMed Central

    Wang, Lu; Wang, Yan; Zhou, Shimeng; Yang, Liukun; Shi, Qixin; Li, Yujiao; Zhang, Kun; Yang, Le; Zhao, Minggao; Yang, Qi

    2016-01-01

    Fragile X syndrome (FXS) is a form of inherited mental retardation that results from the absence of the fragile X mental retardation protein (FMRP), the product of the Fmr1 gene. Numerous studies have shown that FMRP expression in astrocytes is important in the development of FXS. Although astrocytes affect neuronal dendrite development in Fmr1 knockout (KO) mice, the factors released by astrocytes are still unclear. We cultured wild type (WT) cortical neurons in astrocyte-conditioned medium (ACM) from WT or Fmr1 KO mice. Immunocytochemistry and Western blotting were performed to detect the dendritic growth of both WT and KO neurons. We determined glutamate and γ-aminobutyric acid (GABA) levels using high-performance liquid chromatography (HPLC). The total neuronal dendritic length was reduced when cultured in the Fmr1 KO ACM. This neurotoxicity was triggered by an imbalanced release of glutamate and GABA from Fmr1 KO astrocytes. We found increased glutaminase and GABA transaminase (GABA-T) expression and decreased monoamine oxidase B expression in Fmr1 KO astrocytes. The elevated levels of glutamate contributed to oxidative stress in the cultured neurons. Vigabatrin (VGB), a GABA-T inhibitor, reversed the changes caused by glutamate and GABA release in Fmr1 KO astrocytes and the abnormal behaviors in Fmr1 KO mice. Our results indicate that the imbalance in the astrocytic glutamate and GABA release may be involved in the neuropathology and the underlying symptoms of FXS, and provides a therapeutic target for treatment. PMID:27517961

  19. Altered expression of KCC2 in GABAergic interneuron contributes prenatal stress-induced epileptic spasms in infant rat.

    PubMed

    Baek, Hyunjung; Yi, Min-Hee; Pandit, Sudip; Park, Jin Bong; Kwon, Hyeok Hee; Zhang, Enji; Kim, Sena; Shin, Nara; Kim, Eunjee; Lee, Young Ho; Kim, Yonghyun; Kim, Dong Woon; Kang, Joon Won

    2016-07-01

    Long-term stress during pregnancy causes neurologic deficits to offspring with altered gamma-aminobutyric acid (GABA) system in the brain. However, it is not clear how prenatal stress affects the maturing GABAergic interneurons and the resulting abnormalities in infantile seizures. Here, we showed that prenatal stress alters the maturation of GABA inhibitory system using a seizure model induced by prenatal stress. Prenatal stress with betamethasone or acute immobilization stress (AIS) on gestational day 15 increased the seizure susceptibility to N-methyl-d-aspartate-triggered spasms on postnatal day 15. The expression of GABA was lower in the prenatally stressed group, which compromise the decrease of glutamate decarboxylase 67-immunopositive cells. Prenatal stress markedly decreased the expression of K(+)/Cl(-) co-transporter (KCC2) in the cortex. GABA induced membrane depolarization demonstrated prenatal stress models had significant higher membrane depolarization compared to control. GABA increased KCC2 expression in cultured cortex-containing slices. Taken together, our results showed that prenatal stress with betamethasone or AIS altered the maturation of GABAergic progenitors and resulted in the lack of GABA input, which in turn, decreased KCC2 expression and lowered seizure threshold. We conclude that delayed GABA excitatory/inhibitory shift would render the cortical neuronal circuit more susceptible to excitatory input in prenatal stress induced seizure. PMID:27180051

  20. Oxytocin regulates neurosteroid modulation of GABA(A) receptors in supraoptic nucleus around parturition.

    PubMed

    Koksma, Jan-Jurjen; van Kesteren, Ronald E; Rosahl, Thomas W; Zwart, Ruud; Smit, August B; Lüddens, Hartmut; Brussaard, Arjen B

    2003-02-01

    In this study, we investigate how neurosteroid sensitivity of GABA(A) receptors (GABA(A)Rs) is regulated. We examined this issue in neurons of the supraoptic nucleus (SON) of the rat and found that, during parturition, the GABA(A)Rs become insensitive to the neurosteroid allopregnanolone attributable to a shift in the balance between the activities of endogenous Ser/Thr phosphatase and PKC. In particular, a constitutive endogenous tone of oxytocin within the SON after parturition suppressed neurosteroid sensitivity of GABA(A)Rs via activation of PKC. Vice versa before parturition, during late pregnancy, application of exogenous oxytocin brings the GABA(A)Rs from a neurosteroid-sensitive mode toward a condition in which the receptors are not sensitive. This indicates that there may be an inverse causal relationship between the extent to which the GABA(A)R or one of its interacting proteins is phosphorylated and the neurosteroid sensitivity of the GABA(A)R. Neurosteroid sensitivity was not affected by changes in subunit composition of GABA(A)Rs known to occur concurrently in these cells.

  1. Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants.

    PubMed Central

    Baum, G; Lev-Yadun, S; Fridmann, Y; Arazi, T; Katsnelson, H; Zik, M; Fromm, H

    1996-01-01

    Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM-binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants. Images PMID:8670800

  2. Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants.

    PubMed

    Baum, G; Lev-Yadun, S; Fridmann, Y; Arazi, T; Katsnelson, H; Zik, M; Fromm, H

    1996-06-17

    Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM-binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.

  3. Vesicular GABA transporter (VGAT) transports β-alanine.

    PubMed

    Juge, Narinobu; Omote, Hiroshi; Moriyama, Yoshinori

    2013-11-01

    Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In this study, we show that VGAT recognizes β-alanine as a substrate. Proteoliposomes containing purified VGAT transport β-alanine using Δψ but not ΔpH as a driving force. The Δψ-driven β-alanine uptake requires Cl(-). VGAT also facilitates Cl(-) uptake in the presence of β-alanine. A previously described VGAT mutant (Glu213Ala) that disrupts GABA and glycine transport similarly abrogates β-alanine uptake. These findings indicated that VGAT transports β-alanine through a mechanism similar to those for GABA and glycine, and functions as a vesicular β-alanine transporter. Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In the present study, we showed that proteoliposomes containing purified VGAT transport β-alanine using Δψ as a driving force. VGAT also facilitates Cl(-) uptake. Our findings indicated that VGAT functions as a vesicular β-alanine transporter.

  4. Fluorescent labeling of both GABAergic and glycinergic neurons in vesicular GABA transporter (VGAT)-venus transgenic mouse.

    PubMed

    Wang, Y; Kakizaki, T; Sakagami, H; Saito, K; Ebihara, S; Kato, M; Hirabayashi, M; Saito, Y; Furuya, N; Yanagawa, Y

    2009-12-15

    Inhibitory neurons play important roles in a number of brain functions. They are composed of GABAergic neurons and glycinergic neurons, and vesicular GABA transporter (VGAT) is specifically expressed in these neurons. Since the inhibitory neurons are scattered around in the CNS, it is difficult to identify these cells in living brain preparations. The glutamate decarboxylase (GAD) 67-GFP knock-in mouse has been widely used for the identification of GABAergic neurons, but their GAD67 expression was decreased compared to the wild-type mice. To overcome such a problem and to highlight the function and morphology of inhibitory neurons, we generated four lines of VGAT-Venus transgenic mice (lines #04, #29, #39 and #49) expressing Venus fluorescent protein under the control of mouse VGAT promoter. We found higher expression level of Venus transcripts and proteins as well as brighter fluorescent signal in line #39 mouse brains, compared to brains of other lines examined. By Western blots and spectrofluorometric measurements of forebrain, the line #39 mouse showed stronger GFP immunoreactivity and brighter fluorescent intensity than the GAD67-GFP knock-in mouse. In addition, Venus was present not only in somata, but also in neurites in the line #39 mouse by histological studies. In situ hybridization analysis showed that the expression pattern of Venus in the line #39 mouse was similar to that of endogenous VGAT. Double immunostaining analysis in line #39 mouse showed that Venus-expressing cells are primarily immunoreactive for GABA in cerebral cortex, hippocampus and cerebellar cortex and for GABA or glycine in dorsal cochlear nucleus. These results demonstrate that the VGAT-Venus line #39 mouse should be useful for studies on function and morphology of inhibitory neurons in the CNS.

  5. Molecular and pharmacological properties of GABA-rho subunits from white perch retina.

    PubMed

    Qian, H; Dowling, J E; Ripps, H

    1998-11-01

    Five gamma-aminobutyric acid (GABA)-rho subunits were cloned from a white perch retinal cDNA library and expressed in Xenopus oocytes. The deduced amino acid sequences indicated that all are highly homologous to the GABA-rho subunits cloned from mammalian retinas; two clones (perch-rho 1A and perch-rho 1B) were in the rho 1 family, two (perch-rho 2A and perch-rho 2B) were in the rho 2 family, and one clone has been tentatively identified as a perch-rho 3 subunit. When expressed in Xenopus oocytes, all but one of the subunits (rho 3) formed functional homooligomeric receptors. However, the receptors expressed by each of the GABA-rho subunits displayed unique response properties that distinguished one from the other. For example, receptors formed by perch-rho 1B subunits were more sensitive to GABA than the receptors formed by other GABA-rho subunits, the dose-response curves for the various receptors revealed different Hill coefficients, and there were differences in the kinetics of the GABA-induced currents. In addition, the GABA-mediated current-voltage curve for rho 2 receptors was approximately linear, whereas the responses from rho 1 receptors showed outward rectification. A further division in the properties of the GABA-rho subunits was revealed in their responses to imidazole-4-acetic acid (I4AA); the drug behaved as an antagonist on A-type rho receptors and a partial agonist on the B-type rho receptors. These results suggest that there is a large diversity of GABAC receptors in the vertebrate retina, probably formed by homooligomeric and heterooligomeric combinations of GABA rho subunits, that exhibit different functional properties. PMID:9805275

  6. Chapter 2: hypothalamic neural systems controlling the female reproductive life cycle gonadotropin-releasing hormone, glutamate, and GABA.

    PubMed

    Maffucci, Jacqueline A; Gore, Andrea C

    2009-01-01

    The hypothalamic-pituitary-gonadal (HPG) axis undergoes a number of changes throughout the reproductive life cycle that are responsible for the development, puberty, adulthood, and senescence of reproductive systems. This natural progression is dictated by the neural network controlling the hypothalamus including the cells that synthesize and release gonadotropin-releasing hormone (GnRH) and their regulatory neurotransmitters. Glutamate and GABA are the primary excitatory and inhibitory neurotransmitters in the central nervous system, and as such contribute a great deal to modulating this axis throughout the lifetime via their actions on receptors in the hypothalamus, both directly on GnRH neurons as well as indirectly through other hypothalamic neural networks. Interactions among GnRH neurons, glutamate, and GABA, including the regulation of GnRH gene and protein expression, hormone release, and modulation by estrogen, are critical to age-appropriate changes in reproductive function. Here, we present evidence for the modulation of GnRH neurosecretory cells by the balance of glutamate and GABA in the hypothalamus, and the functional consequences of these interactions on reproductive physiology across the life cycle.

  7. GABA metabolism pathway genes, UGA1 and GAD1, regulate replicative lifespan in Saccharomyces cerevisiae.

    PubMed

    Kamei, Yuka; Tamura, Takayuki; Yoshida, Ryo; Ohta, Shinji; Fukusaki, Eiichiro; Mukai, Yukio

    2011-04-01

    Many of the genes involved in aging have been identified in organisms ranging from yeast to human. Our previous study showed that deletion of the UGA3 gene-which encodes a zinc-finger transcription factor necessary for γ-aminobutyric acid (GABA)-dependent induction of the UGA1 (GABA aminotransferase), UGA2 (succinate semialdehyde dehydrogenase), and UGA4 (GABA permease) genes-extends replicative lifespan in the budding yeast Saccharomyces cerevisiae. Here, we found that deletion of UGA1 lengthened the lifespan, as did deletion of UGA3; in contrast, strains with UGA2 or UGA4 deletions exhibited no lifespan extension. The Δuga1 strain cannot deaminate GABA to succinate semialdehyde. Deletion of GAD1, which encodes the glutamate decarboxylase that converts glutamate into GABA, also increased lifespan. Therefore, two genes in the GABA metabolism pathway, UGA1 and GAD1, were identified as aging genes. Unexpectedly, intracellular GABA levels in mutant cells (except for Δuga2 cells) did not differ from those in wild-type cells. Addition of GABA to culture media, which induces transcription of the UGA structural genes, had no effect on replicative lifespan of wild-type cells. Multivariate analysis of (1)H nuclear magnetic resonance spectra for the whole-cell metabolite levels demonstrated a separation between long-lived and normal-lived strains. Gas chromatography-mass spectrometry analysis of identified metabolites showed that levels of tricarboxylic acid cycle intermediates positively correlated with lifespan extension. These results strongly suggest reduced activity of the GABA-metabolizing enzymes extends lifespan by shifting carbon metabolism toward respiration, as calorie restriction does.

  8. HLA expression in hepatocellular carcinoma cell lines.

    PubMed

    Wadee, A A; Paterson, A; Coplan, K A; Reddy, S G

    1994-08-01

    The present study undertook to investigate the biological significance of human leucocyte antigen expression in hepatocellular carcinoma and to elucidate the role of potential modulating agents on human leucocyte antigen expression. These studies used several hepatic tumour-derived cell lines as in vitro model systems. The cell lines included PLC/PRF/5 (Alexander cell line), Hep3B, HepG2, TONG PHC, HA22T/VGH, HA59T/VGH and Mahlavu. The cell lines K562 and Raji were used as negative and positive controls, respectively. K562, a B lymphoid-derived cell line, was shown to express negligible amounts of human leucocyte antigens, while Raji, an erythromyeloid-derived cell line, expressed both class I and class II human leucocyte antigens as well as their respective invariant chains, beta 2-microglobulin and Ii. Using an ELISA, experiments performed on these cell lines confirmed the natural expression of class I and class II antigens by the HA22T/VGH and HA59T/VGH cell lines, whereas PLC/PRF/5 displayed class II surface antigens only. The effects of modulating agents such as interferon-gamma sodium butyrate and clofazimine on human leucocyte antigen expression were investigated using the HA22T/VGH, HA59T/VGH and TONG PHC cell lines. These agents increased class II and class II human leucocyte antigen expression on HA22T/VGH and TONG PHC cells, but had no effect on the HA59T/VGH cell line. The results suggest a potential use for these agents as modulators of human leucocyte antigen expression by human heptocellular cell lines.

  9. GABA inhibition of luminescence from lantern shark (Etmopterus spinax) photophores.

    PubMed

    Claes, Julien M; Krönström, Jenny; Holmgren, Susanne; Mallefet, Jérôme

    2011-03-01

    Photogenic organs (photophores) of the velvet belly lantern shark (Etmopterus spinax) are under hormonal control, since melatonin (MT) and prolactin (PRL) trigger luminescence while α-melanocyte-stimulating hormone (α-MSH) prevents this light to be emitted. A recent study supported, however, the presence of numerous nerve fibres in the photogenic tissue of this shark. Immunohistochemical and pharmacological results collected in this work support these nerve fibres to be inhibitory GABAergic nerves since (i) GABA immunoreactivity was detected inside the photogenic tissue, where previous labelling detected the nerve fibre structures and (ii) GABA was able to inhibit MT and PRL-induced luminescence, which was on the other hand increased by the GABA(A) antagonist bicuculline (BICU). In addition, we also demonstrated that BICU can induce light per se by provoking pigment retraction in the pigmented cells composing the iris-like structure of the photophore, attaining, however, only about 10% of hormonally induced luminescence intensity at 10(-3)mol L(-1). This strongly supports that a GABA inhibitory tonus controls photophore "aperture" in the photogenic tissue of E. spinax but also that MT and PRL have more than one target cell type in the photophores.

  10. GABA metabolism pathway genes, UGA1 and GAD1, regulate replicative lifespan in Saccharomycescerevisiae

    SciTech Connect

    Kamei, Yuka; Tamura, Takayuki; Yoshida, Ryo; Ohta, Shinji; Fukusaki, Eiichiro; Mukai, Yukio

    2011-04-01

    Highlights: {yields}We demonstrate that two genes in the yeast GABA metabolism pathway affect aging. {yields} Deletion of the UGA1 or GAD1 genes extends replicative lifespan. {yields} Addition of GABA to wild-type cultures has no effect on lifespan. {yields} Intracellular GABA levels do not differ in longevity mutants and wild-type cells. {yields} Levels of tricarboxylic acid cycle intermediates positively correlate with lifespan. -- Abstract: Many of the genes involved in aging have been identified in organisms ranging from yeast to human. Our previous study showed that deletion of the UGA3 gene-which encodes a zinc-finger transcription factor necessary for {gamma}-aminobutyric acid (GABA)-dependent induction of the UGA1 (GABA aminotransferase), UGA2 (succinate semialdehyde dehydrogenase), and UGA4 (GABA permease) genes-extends replicative lifespan in the budding yeast Saccharomycescerevisiae. Here, we found that deletion of UGA1 lengthened the lifespan, as did deletion of UGA3; in contrast, strains with UGA2 or UGA4 deletions exhibited no lifespan extension. The {Delta}uga1 strain cannot deaminate GABA to succinate semialdehyde. Deletion of GAD1, which encodes the glutamate decarboxylase that converts glutamate into GABA, also increased lifespan. Therefore, two genes in the GABA metabolism pathway, UGA1 and GAD1, were identified as aging genes. Unexpectedly, intracellular GABA levels in mutant cells (except for {Delta}uga2 cells) did not differ from those in wild-type cells. Addition of GABA to culture media, which induces transcription of the UGA structural genes, had no effect on replicative lifespan of wild-type cells. Multivariate analysis of {sup 1}H nuclear magnetic resonance spectra for the whole-cell metabolite levels demonstrated a separation between long-lived and normal-lived strains. Gas chromatography-mass spectrometry analysis of identified metabolites showed that levels of tricarboxylic acid cycle intermediates positively correlated with lifespan

  11. Daily Isoflurane Exposure Increases Barbiturate Insensitivity in Medullary Respiratory and Cortical Neurons via Expression of ε-Subunit Containing GABA ARs

    PubMed Central

    Hengen, Keith B.; Nelson, Nathan R.; Stang, Kyle M.; Johnson, Stephen M.; Smith, Stephanie M.; Watters, Jyoti J.; Mitchell, Gordon S.; Behan, Mary

    2015-01-01

    The parameters governing GABAA receptor subtype expression patterns are not well understood, although significant shifts in subunit expression may support key physiological events. For example, the respiratory control network in pregnant rats becomes relatively insensitive to barbiturates due to increased expression of ε-subunit-containing GABAARs in the ventral respiratory column. We hypothesized that this plasticity may be a compensatory response to a chronic increase in inhibitory tone caused by increased central neurosteroid levels. Thus, we tested whether increased inhibitory tone was sufficient to induce ε-subunit upregulation on respiratory and cortical neurons in adult rats. Chronic intermittent increases in inhibitory tone in male and female rats was induced via daily 5-min exposures to 3% isoflurane. After 7d of treatment, phrenic burst frequency was less sensitive to barbiturate in isoflurane-treated male and female rats in vivo. Neurons in the ventral respiratory group and cortex were less sensitive to pentobarbital in vitro following 7d and 30d of intermittent isoflurane-exposure in both male and female rats. The pentobarbital insensitivity in 7d isoflurane-treated rats was reversible after another 7d. We hypothesize that increased inhibitory tone in the respiratory control network and cortex causes a compensatory increase in ε-subunit-containing GABAARs. PMID:25748028

  12. Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA) production

    PubMed Central

    Zarei, Adel; Trobacher, Christopher P.; Shelp, Barry J.

    2016-01-01

    Polyamines represent a potential source of 4-aminobutyrate (GABA) in plants exposed to abiotic stress. Terminal catabolism of putrescine in Arabidopsis thaliana involves amine oxidase and the production of 4-aminobutanal, which is a substrate for NAD+-dependent aminoaldehyde dehydrogenase (AMADH). Here, two AMADH homologs were chosen (AtALDH10A8 and AtALDH10A9) as candidates for encoding 4-aminobutanal dehydrogenase activity for GABA synthesis. The two genes were cloned and soluble recombinant proteins were produced in Escherichia coli. The pH optima for activity and catalytic efficiency of recombinant AtALDH10A8 with 3-aminopropanal as substrate was 10.5 and 8.5, respectively, whereas the optima for AtALDH10A9 were approximately 9.5. Maximal activity and catalytic efficiency were obtained with NAD+ and 3-aminopropanal, followed by 4-aminobutanal; negligible activity was obtained with betaine aldehyde. NAD+ reduction was accompanied by the production of GABA and β-alanine, respectively, with 4-aminobutanal and 3-aminopropanal as substrates. Transient co-expression systems using Arabidopsis cell suspension protoplasts or onion epidermal cells and several organelle markers revealed that AtALDH10A9 was peroxisomal, but AtALDH10A8 was cytosolic, although the N-terminal 140 amino acid sequence of AtALDH10A8 localized to the plastid. Root growth of single loss-of-function mutants was more sensitive to salinity than wild-type plants, and this was accompanied by reduced GABA accumulation. PMID:27725774

  13. Significance of GABA(A) receptor heterogeneity: clues from developing neurons.

    PubMed

    Fritschy, Jean-Marc

    2015-01-01

    Briefly after the landmark discovery by Hanns Möhler that GABA(A) receptors (GABA(A)R) are the site of action of benzodiazepine site ligands, their distribution in the rodent CNS during development was mapped by autoradiography, demonstrating early and widespread expression of GABA(A)R in the brain and spinal cord. Ten years later, the first studies using subunit-specific antibodies revealed unsuspected heterogeneity in the subunit composition of GABA(A)R in developing brain, with striking regional specificity and rapid changes in expression and subcellular localization correlating with the maturation of neuronal circuits. These data contributed to the wealth of evidence that GABAergic neurotransmission, acting both synaptically and extrasynaptically, modulates major steps of neuronal development (proliferation, migration, differentiation, and circuit formation). In immature neurons, GABA(A)R activation leads to neuronal depolarization and activation of Ca(2+) signals, which mediate many of the developmental effects of GABA. Therefore, GABA(A)R heterogeneity might be essential to fine-tune GABA actions in the dynamics of CNS maturation. Furthermore, since a disturbance of GABAergic function during ontogeny can potentially affect many aspects of CNS maturation and contribute to the etiology of major brain disorders, GABA(A)R heterogeneity provides a substrate for the development of a tailored pharmacology targeting specific receptor subtypes. This chapter provides a brief overview of these issues with a special focus on the seminal contributions of Hanns Möhler to the emergence of these concepts of fundamental relevance in today's neuroscience research and pharmacological developments. PMID:25637436

  14. Regulation of GABA and benzodiazepine receptors following neurotoxin-induced striatal and medial forebrain bundle lesions

    SciTech Connect

    Pan, H.S.I.

    1985-01-01

    GABA, a major inhibitory transmitter, is used by many projection neurons of the striatum. To investigate the role of GABA in striatal function, the GABA receptor complex was studied after lesions of the striatum or the nigrostriatal neurons. Quantitative receptor autoradiography using thaw-mounted tissue slices was developed for the study of GABA and benzodiazepine (BDZ) receptors. With the technique established, binding to GABA and BDZ receptors after unilateral striatal kainate lesions was examined. Subsequently, changes in GABA and BDZ receptors were studied following the destruction of dopaminergic nigrostriatal cells by unilateral 6-hydroxydopamine lesion of the medial forebrain bundle. In summary, quantitative receptor autoradiography allowed the detection of GABA and BDZ receptor changes in multiple small areas in each lesioned brain. This technique made it feasible to carry out kinetic saturation, and competition studies using less than 1 mg of tissue. The data suggest that dopamine is functionally inhibitory on striatopallidal neurons but is functionally excitatory on striatoentopeduncular and striatonigral cells which in turn inhibit the thalamus. This quantitative autoradiographic technique can be generalized to study other transmitter receptors and can be combined with 2-deoxyglucose uptake studies.

  15. Discovery of a subtype selective inhibitor of the human betaine/GABA transporter 1 (BGT-1) with a non-competitive pharmacological profile.

    PubMed

    Kragholm, Bolette; Kvist, Trine; Madsen, Karsten K; Jørgensen, Lars; Vogensen, Stine B; Schousboe, Arne; Clausen, Rasmus P; Jensen, Anders A; Bräuner-Osborne, Hans

    2013-08-15

    The γ-aminobutyric acid (GABA) transporters (GATs) are essential regulators of the activity in the GABAergic system through their continuous uptake of the neurotransmitter from the synaptic cleft and extrasynaptic space. Four GAT subtypes have been identified to date, each displaying different pharmacological properties and expression patterns. The present study focus on the human betaine/GABA transporter 1 (BGT-1), which has recently emerged as a new target for treatment of epilepsy. However, the lack of selective inhibitors of this transporter has impaired the exploration of this potential considerably. With the objective of identifying novel compounds displaying selectivity for BGT-1, we performed a screening of a small compound library at cells expressing BGT-1 using a [(3)H]GABA uptake assay. The screening resulted in the identification of the compound N-(1-benzyl-4-piperidinyl)-2,4-dichlorobenzamide (BPDBA), a selective inhibitor of the human BGT-1 transporter with a non-competitive profile exhibiting no significant inhibitory activity at the other three human GAT subtypes. The selectivity profile of the compound was subsequently confirmed at cells expressing the four mouse GAT subtypes. Thus, BPDBA constitutes a potential useful pharmacological tool compound for future explorations of the function of the BGT-1 subtype.

  16. Decorin expression in quiescent myogenic cells

    SciTech Connect

    Nishimura, Takanori Nozu, Kenjiro; Kishioka, Yasuhiro; Wakamatsu, Jun-ichi; Hattori, Akihito

    2008-06-06

    Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. When satellite cells are activated by myotrauma, they proliferate, migrate, differentiate, and ultimately fuse to existing myofibers. The remainder of these cells do not differentiate, but instead return to quiescence and remain in a quiescent state until activation begins the process again. This ability to maintain their own population is important for skeletal muscle to maintain the capability to repair during postnatal life. However, the mechanisms by which satellite cells return to quiescence and maintain the quiescent state are still unclear. Here, we demonstrated that decorin mRNA expression was high in cell cultures containing a higher ratio of quiescent satellite cells when satellite cells were stimulated with various concentrations of hepatocyte growth factor. This result suggests that quiescent satellite cells express decorin at a high level compared to activated satellite cells. Furthermore, we examined the expression of decorin in reserve cells, which were undifferentiated myoblasts remaining after induction of differentiation by serum-deprivation. Decorin mRNA levels in reserve cells were higher than those in differentiated myotubes and growing myoblasts. These results suggest that decorin participates in the quiescence of myogenic cells.

  17. A tonoplast Glu/Asp/GABA exchanger that affects tomato fruit amino acid composition.

    PubMed

    Snowden, Christopher J; Thomas, Benjamin; Baxter, Charles J; Smith, J Andrew C; Sweetlove, Lee J

    2015-03-01

    Vacuolar accumulation of acidic metabolites is an important aspect of tomato fruit flavour and nutritional quality. The amino acids Asp and Glu accumulate to high concentrations during ripening, while γ-aminobutyrate (GABA) shows an approximately stoichiometric decline. Given that GABA can be catabolised to form Glu and subsequently Asp, and the requirement for the fruit to maintain osmotic homeostasis during ripening, we hypothesised the existence of a tonoplast transporter that exports GABA from the vacuole in exchange for import of either Asp or Glu. We show here that the tomato vacuolar membrane possesses such a transport property: transport of Glu across isolated tonoplast vesicle membranes was trans-stimulated in counterexchange mode by GABA, Glu and Asp. We identified SlCAT9 as a candidate protein for this exchanger using quantitative proteomics of a tonoplast-enriched membrane fraction. Transient expression of a SlCAT9-YFP fusion in tobacco confirmed a tonoplast localisation. The function of the protein was examined by overexpression of SlCAT9 in transgenic tomato plants. Tonoplast vesicles isolated from transgenic plants showed higher rates of Glu and GABA transport than wild-type (WT) only when assayed in counterexchange mode with Glu, Asp, or GABA. Moreover, there were substantial increases in the content of all three cognate amino acids in ripe fruit from the transgenic plants. We conclude that SlCAT9 is a tonoplast Glu/Asp/GABA exchanger that strongly influences the accumulation of these amino acids during fruit development.

  18. [MOLECULAR MECHANISMS OF ERK1/2 KINASES REGULATION IN THE GLUTAMATE- AND GABA-ERGIC NEURONS DURING SEIZURE EXPRESSION IN KRUSHINSKY-MOLODKINA RATS].

    PubMed

    Korotkov, A A; Glazova, M V; Nikitina, L S; Dorofeeva, N A; Kirillova, O D; Chernigovskaya, E V

    2015-10-01

    The aim of the present study was to analyze a role of the ERK1/2 signaling pathway in the regulation of excitation and inhibitory neurons in the hippocampus and the temporal cortex of Krushinsky-Molodkina rats during seizure development finalizing with ataxia. Analysis was done by Western bloting as well as by immunohistochemistry. The results demonstrated significant up-regulation of ERK1/2 activity in the hippocampus in several seconds after sound stimulation. At the same time increased ERK1/2 activity was correlated with enhanced level of SNARE protein SNAP-25 and activation of synapsin I, the proteins which regulate exocytosis machinery. Decreased level of VGLUT2 associated with activation of ERK1/2 and exocytosis proteins supposed activation of glutamate release in the hippocampus, while in the temporal cortex diminished activity of ERK1/2 and synapsin I associated with VGLUT2 up-regulation assumed inhibition of glutamatergic transmission. Our data let us supposed that decreasing of glutamate release in th& temporal cortex could be a trigger for the inhibition of hippocampal glutamatergic system and the beginning of further ataxia stage. Our data demonstrated correlation between expression and activity of exocytosis proteins and ERK1/2 mainly in the glutamategic neurons of the hippocampus and the temporal cortex that let us proposed significant role of ERK1/2 kinases as a positive regulator of glutamate release and as a result initiation of seizure expression. PMID:26827493

  19. Vertebrate Cells Express Protozoan Antigen after Hybridization

    NASA Astrophysics Data System (ADS)

    Crane, Mark St. J.; Dvorak, James A.

    1980-04-01

    Epimastigotes, the invertebrate host stage of Trypanosoma cruzi, the protozoan parasite causing Chagas' disease in man, were fused with vertebrate cells by using polyethylene glycol. Hybrid cells were selected on the basis of T. cruzi DNA complementation of biochemical deficiencies in the vertebrate cells. Some clones of the hybrid cells expressed T. cruzi-specific antigen. It might be possible to use selected antigens obtained from the hybrids as vaccines for immunodiagnosis or for elucidation of the pathogenesis of Chagas' disease.

  20. Imaging gene expression in single living cells

    PubMed Central

    Shav-Tal, Yaron; Singer, Robert H.; Darzacq, Xavier

    2016-01-01

    Technical advances in the field of live-cell imaging have introduced the cell biologist to a new, dynamic, subcellular world. The static world of molecules in fixed cells has now been extended to the time dimension. This allows the visualization and quantification of gene expression and intracellular trafficking events of the studied molecules and the associated enzymatic processes in individual cells, in real time. PMID:15459666

  1. Co-activation of VTA DA and GABA neurons mediates nicotine reinforcement.

    PubMed

    Tolu, S; Eddine, R; Marti, F; David, V; Graupner, M; Pons, S; Baudonnat, M; Husson, M; Besson, M; Reperant, C; Zemdegs, J; Pagès, C; Hay, Y A H; Lambolez, B; Caboche, J; Gutkin, B; Gardier, A M; Changeux, J-P; Faure, P; Maskos, U

    2013-03-01

    Smoking is the most important preventable cause of mortality and morbidity worldwide. This nicotine addiction is mediated through the nicotinic acetylcholine receptor (nAChR), expressed on most neurons, and also many other organs in the body. Even within the ventral tegmental area (VTA), the key brain area responsible for the reinforcing properties of all drugs of abuse, nicotine acts on several different cell types and afferents. Identifying the precise action of nicotine on this microcircuit, in vivo, is important to understand reinforcement, and finally to develop efficient smoking cessation treatments. We used a novel lentiviral system to re-express exclusively high-affinity nAChRs on either dopaminergic (DAergic) or γ-aminobutyric acid-releasing (GABAergic) neurons, or both, in the VTA. Using in vivo electrophysiology, we show that, contrary to widely accepted models, the activation of GABA neurons in the VTA plays a crucial role in the control of nicotine-elicited DAergic activity. Our results demonstrate that both positive and negative motivational values are transmitted through the dopamine (DA) neuron, but that the concerted activity of DA and GABA systems is necessary for the reinforcing actions of nicotine through burst firing of DA neurons. This work identifies the GABAergic interneuron as a potential target for smoking cessation drug development.

  2. Exogenous γ-aminobutyric acid (GABA) affects pollen tube growth via modulating putative Ca2+-permeable membrane channels and is coupled to negative regulation on glutamate decarboxylase.

    PubMed

    Yu, Guang-Hui; Zou, Jie; Feng, Jing; Peng, Xiong-Bo; Wu, Ju-You; Wu, Ying-Liang; Palanivelu, Ravishankar; Sun, Meng-Xiang

    2014-07-01

    γ-Aminobutyric acid (GABA) is implicated in pollen tube growth, but the molecular and cellular mechanisms that it mediates are largely unknown. Here, it is shown that exogenous GABA modulates putative Ca(2+)-permeable channels on the plasma membranes of tobacco pollen grains and pollen tubes. Whole-cell voltage-clamp experiments and non-invasive micromeasurement technology (NMT) revealed that the influx of Ca(2+) increases in pollen tubes in response to exogenous GABA. It is also demonstrated that glutamate decarboxylase (GAD), the rate-limiting enzyme of GABA biosynthesis, is involved in feedback controls of Ca(2+)-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca(2+)-permeable channels relays an extracellular GABA signal and integrates multiple signal pathways to modulate tobacco pollen tube growth. Thus, the data explain how GABA mediates the communication between the style and the growing pollen tubes.

  3. The Uptake of GABA in Trypanosoma cruzi.

    PubMed

    Galvez Rojas, Robert L; Ahn, Il-Young; Suárez Mantilla, Brian; Sant'Anna, Celso; Pral, Elizabeth Mieko Furusho; Silber, Ariel Mariano

    2015-01-01

    Gamma aminobutyric acid (GABA) is widely known as a neurotransmitter and signal transduction molecule found in vertebrates, plants, and some protozoan organisms. However, the presence of GABA and its role in trypanosomatids is unknown. Here, we report the presence of intracellular GABA and the biochemical characterization of its uptake in Trypanosoma cruzi, the etiological agent of Chagas' disease. Kinetic parameters indicated that GABA is taken up by a single transport system in pathogenic and nonpathogenic forms. Temperature dependence assays showed a profile similar to glutamate transport, but the effect of extracellular cations Na(+) , K(+) , and H(+) on GABA uptake differed, suggesting a different uptake mechanism. In contrast to reports for other amino acid transporters in T. cruzi, GABA uptake was Na(+) dependent and increased with pH, with a maximum activity at pH 8.5. The sensitivity to oligomycin showed that GABA uptake is dependent on ATP synthesis. These data point to a secondary active Na(+) /GABA symporter energized by Na(+) -exporting ATPase. Finally, we show that GABA occurs in the parasite's cytoplasm under normal culture conditions, indicating that it is regularly taken up from the culture medium or synthesized through an still undescribed metabolic pathway.

  4. The Uptake of GABA in Trypanosoma cruzi.

    PubMed

    Galvez Rojas, Robert L; Ahn, Il-Young; Suárez Mantilla, Brian; Sant'Anna, Celso; Pral, Elizabeth Mieko Furusho; Silber, Ariel Mariano

    2015-01-01

    Gamma aminobutyric acid (GABA) is widely known as a neurotransmitter and signal transduction molecule found in vertebrates, plants, and some protozoan organisms. However, the presence of GABA and its role in trypanosomatids is unknown. Here, we report the presence of intracellular GABA and the biochemical characterization of its uptake in Trypanosoma cruzi, the etiological agent of Chagas' disease. Kinetic parameters indicated that GABA is taken up by a single transport system in pathogenic and nonpathogenic forms. Temperature dependence assays showed a profile similar to glutamate transport, but the effect of extracellular cations Na(+) , K(+) , and H(+) on GABA uptake differed, suggesting a different uptake mechanism. In contrast to reports for other amino acid transporters in T. cruzi, GABA uptake was Na(+) dependent and increased with pH, with a maximum activity at pH 8.5. The sensitivity to oligomycin showed that GABA uptake is dependent on ATP synthesis. These data point to a secondary active Na(+) /GABA symporter energized by Na(+) -exporting ATPase. Finally, we show that GABA occurs in the parasite's cytoplasm under normal culture conditions, indicating that it is regularly taken up from the culture medium or synthesized through an still undescribed metabolic pathway. PMID:25851259

  5. Color threshold and ratio of S100 beta, MAP5, NF68/200, GABA & GAD. I. Distribution in inner ear afferents

    NASA Technical Reports Server (NTRS)

    Fermin, C. D.; Martin, D. S.; Hara, H.

    1997-01-01

    Afferents of chick embryos (Gallus domesticus) VIIIth nerve were examined at E3, E6, E9, E13, El7, and hatching (NH) for anti-S100 beta, anti-MAP5, anti-GABA, anti-GAD and anti-NF68/200 stain. Different ages were processed together to determine if the distribution of these antibodies changed during synaptogenesis and myelination. Color thresholding showed that saturation of pixels changed for S100 beta only 5%, for NF68/200 10%, and for MAP5, 10%, between E9-NH. Color ratio of NF68/200 over MAP5 was 1.00 at E13 and 0.25 at E16 and NH. S100 beta, GABA and GAD were co-expressed on nerve endings at the edge of the maculae and center of the cristae, whereas hair cells in the center of the maculae expressed either S100 beta or GABA, but not both. S100 beta/NF68/200 shared antigenic sites on the chalices, but NF68/200 expression was higher than S100 beta in the chalices at hatching. MAP5 was expressed in more neurons than NF68/200 at E11, whereas NF68/200 was more abundant than MAP5 at hatching. The results suggest that: 1) the immunoexpression of these neuronal proteins is modulated concomitantly with the establishment of afferent synapses and myelination; 2) S100 beta may serve a neurotrophic function in the chalices where it is co-expressed with the neurotransmitter GABA and its synthesizing enzyme GAD.

  6. Fundamentals of Expression in Mammalian Cells.

    PubMed

    Dyson, Michael R

    2016-01-01

    Expression of proteins in mammalian cells is a key technology important for many functional studies on human and higher eukaryotic genes. Studies include the mapping of protein interactions, solving protein structure by crystallization and X-ray diffraction or solution phase NMR and the generation of antibodies to enable a range of studies to be performed including protein detection in vivo. In addition the production of therapeutic proteins and antibodies, now a multi billion dollar industry, has driven major advances in cell line engineering for the production of grams per liter of active proteins and antibodies. Here the key factors that need to be considered for successful expression in HEK293 and CHO cells are reviewed including host cells, expression vector design, transient transfection methods, stable cell line generation and cultivation conditions. PMID:27165328

  7. Functional alpha7 nicotinic receptors are expressed on immature granule cells of the postnatal dentate gyrus.

    PubMed

    John, Danielle; Shelukhina, Irina; Yanagawa, Yuchio; Deuchars, Jim; Henderson, Zaineb

    2015-03-19

    Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The α7*nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional α7*nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 2-3 week-old Wistar rats, and 2-9 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of α7*nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric α7*nicotinic receptor modulator, which were blocked by a specific α7*nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic α7*nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating α7*nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain.

  8. Functional alpha7 nicotinic receptors are expressed on immature granule cells of the postnatal dentate gyrus

    PubMed Central

    John, Danielle; Shelukhina, Irina; Yanagawa, Yuchio; Deuchars, Jim; Henderson, Zaineb

    2015-01-01

    Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The α7⁎nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional α7⁎nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 2–3 week-old Wistar rats, and 2–9 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of α7⁎nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric α7⁎nicotinic receptor modulator, which were blocked by a specific α7⁎nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic α7⁎nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating α7⁎nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain. PMID:25553616

  9. Aging of whiskey increases the potentiation of GABA(A) receptor response.

    PubMed

    Koda, Hirofumi; Hossain, Sheikh Julfikar; Kiso, Yoshinobu; Aoshima, Hitoshi

    2003-08-27

    It is known that the target of most mood-defining compounds such as ethanol is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activities in the human brain. Because both extracts of whiskey by pentane and fragrant components in whiskey potentiate the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting cRNAs prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors in order to study the effects of whiskey itself on the GABA(A) receptor-mediated response. Whiskey itself also potentiated the electrical responses of GABA(A) receptors generally more than ethanol at the same concentration as that of the whiskey. The potentiation of the GABA(A) receptor-mediated response increased with the aging period of the whiskey. Inhalation of whiskey to mice increased the sleeping time induced by pentobarbital more than that of the same concentration of ethanol as the whiskey. These results suggest that not only ethanol but also minor components in whiskey play an important role in the potentiation of GABA(A) receptor-mediated response and possibly the sedative effect of whiskey. Although the minor components are present in extremely small quantities compared with ethanol in alcoholic beverages, they may modulate the mood or consciousness of humans through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic compounds are easily absorbed into the brain across the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response.

  10. Aging of whiskey increases the potentiation of GABA(A) receptor response.

    PubMed

    Koda, Hirofumi; Hossain, Sheikh Julfikar; Kiso, Yoshinobu; Aoshima, Hitoshi

    2003-08-27

    It is known that the target of most mood-defining compounds such as ethanol is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activities in the human brain. Because both extracts of whiskey by pentane and fragrant components in whiskey potentiate the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting cRNAs prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors in order to study the effects of whiskey itself on the GABA(A) receptor-mediated response. Whiskey itself also potentiated the electrical responses of GABA(A) receptors generally more than ethanol at the same concentration as that of the whiskey. The potentiation of the GABA(A) receptor-mediated response increased with the aging period of the whiskey. Inhalation of whiskey to mice increased the sleeping time induced by pentobarbital more than that of the same concentration of ethanol as the whiskey. These results suggest that not only ethanol but also minor components in whiskey play an important role in the potentiation of GABA(A) receptor-mediated response and possibly the sedative effect of whiskey. Although the minor components are present in extremely small quantities compared with ethanol in alcoholic beverages, they may modulate the mood or consciousness of humans through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic compounds are easily absorbed into the brain across the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response. PMID:12926865

  11. Expression cloning of human B cell immunoglobulins.

    PubMed

    Wardemann, Hedda; Kofer, Juliane

    2013-01-01

    The majority of lymphomas originate from B cells at the germinal center stage or beyond. Preferential selection of B cell clones by a limited set of antigens has been suggested to drive lymphoma development. However, little is known about the specificity of the antibodies expressed by lymphoma cells, and the role of antibody-specificity in lymphomagenesis remains elusive. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire on a single cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells, to the RT-PCR-based amplification of the expressed Igh, Igκ, and Igλ chain genes, and Ig gene expression vector cloning for the in vitro production of monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B cell lymphomas by single cell Ig gene sequencing and on the antibody reactivity of human lymphoma B cells.

  12. Leptin signaling in GABA neurons, but not glutamate neurons, is required for reproductive function.

    PubMed

    Zuure, Wieteke A; Roberts, Amy L; Quennell, Janette H; Anderson, Greg M

    2013-11-01

    The adipocyte-derived hormone leptin acts in the brain to modulate the central driver of fertility: the gonadotropin releasing hormone (GnRH) neuronal system. This effect is indirect, as GnRH neurons do not express leptin receptors (LEPRs). Here we test whether GABAergic or glutamatergic neurons provide the intermediate pathway between the site of leptin action and the GnRH neurons. Leptin receptors were deleted from GABA and glutamate neurons using Cre-Lox transgenics, and the downstream effects on puberty onset and reproduction were examined. Both mouse lines displayed the expected increase in body weight and region-specific loss of leptin signaling in the hypothalamus. The GABA neuron-specific LEPR knock-out females and males showed significantly delayed puberty onset. Adult fertility observations revealed that these knock-out animals have decreased fecundity. In contrast, glutamate neuron-specific LEPR knock-out mice displayed normal fertility. Assessment of the estrogenic hypothalamic-pituitary-gonadal axis regulation in females showed that leptin action on GABA neurons is not necessary for estradiol-mediated suppression of tonic luteinizing hormone secretion (an indirect measure of GnRH neuron activity) but is required for regulation of a full preovulatory-like luteinizing hormone surge. In conclusion, leptin signaling in GABAergic (but not glutamatergic neurons) plays a critical role in the timing of puberty onset and is involved in fertility regulation throughout adulthood in both sexes. These results form an important step in explaining the role of central leptin signaling in the reproductive system. Limiting the leptin-to-GnRH mediators to GABAergic cells will enable future research to focus on a few specific types of neurons.

  13. Distinct activities of GABA agonists at synaptic- and extrasynaptic-type GABAA receptors.

    PubMed

    Mortensen, Martin; Ebert, Bjarke; Wafford, Keith; Smart, Trevor G

    2010-04-15

    The activation characteristics of synaptic and extrasynaptic GABA(A) receptors are important for shaping the profile of phasic and tonic inhibition in the central nervous system, which will critically impact on the activity of neuronal networks. Here, we study in isolation the activity of three agonists, GABA, muscimol and 4,5,6,7-tetrahydoisoxazolo[5,4-c]pyridin-3(2H)-one (THIP), to further understand the activation profiles of alpha 1 beta 3 gamma 2, alpha 4 beta 3 gamma 2 and alpha 4 beta 3 delta receptors that typify synaptic- and extrasynaptic-type receptors expressed in the hippocampus and thalamus. The agonists display an order of potency that is invariant between the three receptors, which is reliant mostly on the agonist dissociation constant. At delta subunit-containing extrasynaptic-type GABA(A) receptors, both THIP and muscimol additionally exhibited, to different degrees, superagonist behaviour. By comparing whole-cell and single channel currents induced by the agonists, we provide a molecular explanation for their different activation profiles. For THIP at high concentrations, the unusual superagonist behaviour on alpha 4 beta 3 delta receptors is a consequence of its ability to increase the duration of longer channel openings and their frequency, resulting in longer burst durations. By contrast, for muscimol, moderate superagonist behaviour was caused by reduced desensitisation of the extrasynaptic-type receptors. The ability to specifically increase the efficacy of receptor activation, by selected exogenous agonists over that obtained with the natural transmitter, may prove to be of therapeutic benefit under circumstances when synaptic inhibition is compromised or dysfunctional.

  14. Role of human GABA(A) receptor beta3 subunit in insecticide toxicity.

    PubMed

    Ratra, G S; Kamita, S G; Casida, J E

    2001-05-01

    The gamma-aminobutyric acid type A (GABA(A)) receptor is the target for the major insecticides alpha-endosulfan, lindane, and fipronil and for many analogs. Their action as chloride channel blockers is directly measured by binding studies with [(3)H]ethynylbicycloorthobenzoate ([(3)H]EBOB). This study tests the hypothesis that GABA(A) receptor subunit composition determines the sensitivity and selectivity of insecticide toxicity. Human receptor subtypes were expressed individually (alpha1, alpha6, beta1, beta3, and gamma2) and in combination in insect Sf9 cells. Binding parameters were similar for [(3)H]EBOB in the beta3 homooligomer, alpha1beta3gamma2 heterooligomer, and native brain membranes, but toxicological profiles were very different. Surprisingly, alpha-endosulfan, lindane, and fipronil were all remarkably potent on the recombinant beta3 homooligomeric receptor (IC50 values of 0.5-2.4 nM), whereas they were similar in potency on the alpha1beta3gamma2 subtype (IC50 values of 16-33 nM) and highly selective on the native receptor (IC50 values of 7.3, 306, and 2470 nM, respectively). The selectivity order for 29 insecticides and convulsants as IC50 ratios for native/beta3 or alpha1beta3gamma2/beta3 was as follows: fipronil > lindane > 19 other insecticides including alpha-endosulfan and picrotoxinin > 4 trioxabicyclooctanes and dithianes (almost nonselective) > tetramethylenedisulfotetramine, 4-chlorophenylsilatrane, or alpha-thujone. Specificity between mammals and insects at the target site (fipronil > lindane > alpha-endosulfan) paralleled that for toxicity. Potency at the native receptor is more predictive for inhibition of GABA-stimulated chloride uptake than that at the beta3 or alpha1beta3gamma2 receptors. Therefore, the beta3 subunit contains the insecticide target and other subunits differentially modulate the binding to confer compound-dependent specificity and selective toxicity.

  15. γ-Aminobutyric acid (GABA) homeostasis regulates pollen germination and polarized growth in Picea wilsonii.

    PubMed

    Ling, Yu; Chen, Tong; Jing, Yanping; Fan, Lusheng; Wan, Yinglang; Lin, Jinxing

    2013-11-01

    γ-Aminobutyric acid (GABA) is a four-carbon non-protein amino acid found in a wide range of organisms. Recently, GABA accumulation has been shown to play a role in the stress response and cell growth in angiosperms. However, the effect of GABA deficiency on pollen tube development remains unclear. Here, we demonstrated that specific concentrations of exogenous GABA stimulated pollen tube growth in Picea wilsonii, while an overdose suppressed pollen tube elongation. The germination percentage of pollen grains and morphological variations in pollen tubes responded in a dose-dependent manner to treatment with 3-mercaptopropionic acid (3-MP), a glutamate decarboxylase inhibitor, while the inhibitory effects could be recovered in calcium-containing medium supplemented with GABA. Using immunofluorescence labeling, we found that the actin cables were disorganized in 3-MP treated cells, followed by the transition of endo/exocytosis activating sites from the apex to the whole tube shank. In addition, variations in the deposition of cell wall components were detected upon labeling with JIM5, JIM7, and aniline blue. Our results demonstrated that calcium-dependent GABA signaling regulates pollen germination and polarized tube growth in P. wilsonii by affecting actin filament patterns, vesicle trafficking, and the configuration and distribution of cell wall components.

  16. Vesicular γ-Aminobutyric Acid Transporter Expression in Amacrine and Horizontal Cells

    PubMed Central

    Cueva, Juan G.; Haverkamp, Silke; Reimer, Richard J.; Edwards, Robert; Wässle, Heinz; Brecha, Nicholas C.

    2010-01-01

    The vesicular γ-aminobutyric acid (GABA) transporter (VGAT), which transports the inhibitory amino acid transmitters GABA and glycine, is localized to synaptic vesicles in axon terminals. The localization of VGAT immunoreactivity to mouse and rat retina was evaluated with light and electron microscopy by using well-characterized VGAT antibodies. Specific VGAT immunoreactivity was localized to numerous varicose processes in all laminae of the inner plexiform layer (IPL) and to the outer plexiform layer (OPL). Amacrine cell somata characterized by weak VGAT immunoreactivity in the cytoplasm were located in the ganglion cell layer and proximal inner nuclear layer (INL) adjacent to the IPL. In rat retina, VGAT-immunoreactive cell bodies also contained GABA, glycine, or parvalbumin (PV) immunoreactivity, suggesting vesicular uptake of GABA or glycine by these cells. A few varicose VGAT-immunoreactive processes entered the OPL from the IPL. VGAT immunoreactivity in the OPL was predominantly localized to horizontal cell processes. VGAT and calcium binding protein-28K immunoreactivities (CaBP; a marker for horizontal cells) were colocalized in processes and terminals distributed to the OPL. Furthermore, VGAT immunoreactivity overlapped or was immediately adjacent to postsynaptic density-95 (PSD-95) immunoreactivity, which is prominent in photoreceptor terminals. Preem-bedding immunoelectron microscopy of mouse and rat retinae showed that VGAT immunoreactivity was localized to horizontal cell processes and their terminals. Immunoreactivity was distributed throughout the cytoplasm of the horizontal cell processes. Taken together, these findings demonstrate VGAT immunoreactivity in both amacrine and horizontal cell processes, suggesting these cells contain vesicles that accumulate GABA and glycine, possibly for vesicular release. PMID:11920703

  17. Subcellular localization and expression of multiple tomato γ-aminobutyrate transaminases that utilize both pyruvate and glyoxylate

    PubMed Central

    Clark, Shawn M.; Di Leo, Rosa; Van Cauwenberghe, Owen R.; Mullen, Robert T.; Shelp, Barry J.

    2009-01-01

    γ-Aminobutyric acid transaminase (GABA-T) catalyses the breakdown of GABA to succinic semialdehyde. In this report, three GABA-T isoforms were identified in the tomato (Solanum lycopersicum L.) plant. The deduced amino acid sequences of the three isoforms are highly similar over most of their coding regions with the exception of their N-terminal regions. Transient expression of the individual full-length GABA-T isoforms fused to the green fluorescent protein in tobacco suspension-cultured cells revealed their distinct subcellular localizations to the mitochondrion, plastid or cytosol, and that the specific targeting of the mitochondrion- and plastid-localized isoforms is mediated by their predicted N-terminal presequences. Removal of the N-terminal targeting presequences from the mitochondrion and plastid GABA-T isoforms yielded good recovery of the soluble recombinant proteins in Escherichia coli when they were co-expressed with the GroES/EL molecular chaperone complex. Activity assays indicated that all three recombinant isoforms possess both pyruvate- and glyoxylate-dependent GABA-T activities, although the mitochondrial enzyme has a specific activity that is significantly higher than that of its plastid and cytosolic counterparts. Finally, differential expression patterns of the three GABA-T isoforms in reproductive tissues, but not vegetative tissues, suggest unique roles for each enzyme in developmental processes. Overall, these findings, together with recent information about rice and pepper GABA-Ts, indicate that the subcellular distribution of GABA-T in the plant kingdom is highly variable. PMID:19470656

  18. The root-specific glutamate decarboxylase (GAD1) is essential for sustaining GABA levels in Arabidopsis.

    PubMed

    Bouché, Nicolas; Fait, Aaron; Zik, Moriyah; Fromm, Hillel

    2004-05-01

    In plants, as in most eukaryotes, glutamate decarboxylase catalyses the synthesis of GABA. The Arabidopsis genome contains five glutamate decarboxylase genes and one of these genes (glutamate decarboxylase1; i.e. GAD1 ) is expressed specifically in roots. By isolating and analyzing three gad1 T-DNA insertion alleles, derived from two ecotypes, we investigated the potential role of GAD1 in GABA production. We also analyzed a promoter region of the GAD1 gene and show that it confers root-specific expression when fused to reporter genes. Phenotypic analysis of the gad1 insertion mutants revealed that GABA levels in roots were drastically reduced compared with those in the wild type. The roots of the wild type contained about sevenfold more GABA than roots of the mutants. Disruption of the GAD1 gene also prevented the accumulation of GABA in roots in response to heat stress. Our results show that the root-specific calcium/calmodulin-regulated GAD1 plays a major role in GABA synthesis in plants under normal growth conditions and in response to stress.

  19. Gramicidin-perforated patch revealed depolarizing effect of GABA in cultured frog melanotrophs

    PubMed Central

    Le Foll, Frank; Castel, Hélène; Soriani, Olivier; Vaudry, Hubert; Cazin, Lionel

    1998-01-01

    In frog pituitary melanotrophs, GABA induces a transient stimulation followed by prolonged inhibition of hormone secretion. This biphasic effect is inconsistent with the elevation of cytosolic calcium and the inhibition of electrical activity also provoked by GABA in single melanotrophs. In the present study, standard patch-clamp configurations and gramicidin-perforated patches were used to investigate the physiological GABAA receptor-mediated response and intracellular chloride concentration ([Cl−]i) in cultured frog melanotrophs. In the gramicidin-perforated patch configuration, 1 μM GABA caused a depolarization associated with an action potential discharge and a slight fall of membrane resistance. In contrast, at a higher concentration (10 μm) GABA elicited a depolarization accompanied by a transient volley of action potentials, followed by a sustained inhibitory plateau and a marked fall of membrane resistance. Isoguvacine mimicked the GABA-evoked responses, indicating a mediation by GABAA receptors. In gramicidin-perforated cells, the depolarizing excitatory effect of 1 μm GABA was converted into a depolarizing inhibitory action when 0.4 μm allopregnanolone was added to the bath solution. After gaining the whole-cell configuration, the amplitude and/or direction of the GABA-evoked current (IGABA) rapidly changed before stabilizing. After stabilization, the reversal potential of IGABA followed the values predicted by the Nernst equation for chloride ions when [Cl−]i was varied. In gramicidin-perforated cells, the steady-state I–V relationships of 10 μm GABA- or isoguvacine-evoked currents yielded reversal potentials of −37.5 ± 1.6 (n= 17) and −38.6 ± 2.0 mV (n= 8), respectively. These values were close to those obtained by using a voltage-ramp protocol in the presence of Na+, K+ and Ca2+ channel blockers. The current evoked by 1 μm GABA also reversed at these potentials. We conclude that, in frog pituitary melanotrophs, chloride is the

  20. Pitx2 expression promotes p21 expression and cell cycle exit in neural stem cells.

    PubMed

    Heldring, Nina; Joseph, Bertrand; Hermanson, Ola; Kioussi, Chrissa

    2012-11-01

    Cortical development is a complex process that involves many events including proliferation, cell cycle exit and differentiation that need to be appropriately synchronized. Neural stem cells (NSCs) isolated from embryonic cortex are characterized by their ability of self-renewal under continued maintenance of multipotency. Cell cycle progression and arrest during development is regulated by numerous factors, including cyclins, cyclin dependent kinases and their inhibitors. In this study, we exogenously expressed the homeodomain transcription factor Pitx2, usually expressed in postmitotic progenitors and neurons of the embryonic cortex, in NSCs with low expression of endogenous Pitx2. We found that Pitx2 expression induced a rapid decrease in proliferation associated with an accumulation of NSCs in G1 phase. A search for potential cell cycle inhibitors responsible for such cell cycle exit of NSCs revealed that Pitx2 expression caused a rapid and dramatic (≉20-fold) increase in expression of the cell cycle inhibitor p21 (WAF1/Cip1). In addition, Pitx2 bound directly to the p21 promoter as assessed by chromatin immunoprecipitation (ChIP) in NSCs. Surprisingly, Pitx2 expression was not associated with an increase in differentiation markers, but instead the expression of nestin, associated with undifferentiated NSCs, was maintained. Our results suggest that Pitx2 promotes p21 expression and induces cell cycle exit in neural progenitors.

  1. The blockade of GABA mediated responses in the frog spinal cord by ammonium ions and furosemide.

    PubMed Central

    Nicoll, R A

    1978-01-01

    1. A variety of compounds which are known to block chloride transport in a variety of systems have been examined for their effects on amino acid and synaptic responses in the frog spinal cord in vitro. 2. A number of monocarboxylic aromatic acids, copper sulphate, and acetazolamide had no effect on any of the responses. 3. Ammonium ions blocked the motoneurone hyperpolarizing responses to all the neutral amino acids. In addition it selectively blocked dorsal root potentials and the action of GABA and beta-alanine on primary afferents. 5. Intracellular recording from dorsal root ganglion cells demonstrated that furosemide had little effect on the reversal potential for the GABA response. These results suggest that furosemide acts primarily by blocking the conductance increase elicited by GABA. 6. The results with furosemide provide indirect evidence that chloride ions are involved in generating the GABA depolarizations of primary afferent terminals and dorsal root potentials. PMID:722571

  2. Effects of glutamate decarboxylase and gamma-aminobutyric acid (GABA) transporter on the bioconversion of GABA in engineered Escherichia coli.

    PubMed

    Le Vo, Tam Dinh; Kim, Tae Wan; Hong, Soon Ho

    2012-05-01

    Gamma-aminobutyric acid (GABA) is a non-essential amino acid and a precursor of pyrrolidone, a monomer of nylon 4. GABA can be biosynthesized through the decarboxylation of L: -glutamate by glutamate decarboxylase. In this study, the effects of glutamate decarboxylase (gadA, gadB), glutamate/GABA antiporter (gadC) and GABA aminotransferase (gabT) on GABA production were investigated in Escherichia coli. Glutamate decarboxylase was overexpressed alone or with the glutamate/GABA antiporter to enhance GABA synthesis. GABA aminotransferase, which redirects GABA into the TCA cycle, was knock-out mutated. When gadB and gadC were co-overexpressed in the gabT mutant strain, a final GABA concentration of 5.46 g/l was obtained from 10 g/l of monosodium glutamate (MSG), which corresponded to a GABA yield of 89.5%.

  3. Expression sequences of cell adhesion molecules.

    PubMed Central

    Crossin, K L; Chuong, C M; Edelman, G M

    1985-01-01

    A reexamination of the expression of cell adhesion molecules (CAMs) during the development of the chicken embryo was carried out using more sensitive immunocytochemical techniques than had been used previously. While the previously determined sequence of CAM expression was confirmed, neural CAM (N-CAM) was also detected on endodermal structures such as the lung epithelium, gut epithelium, and pancreas and on budding structures such as the pancreatic duct and gall bladder. It was also found on ectodermal derivatives of the skin. In most of these sites, N-CAM expression was transient, but in the chicken embryo lung, the epithelium remained positive for N-CAM and liver CAM (L-CAM) into adult life. Thus, at one time or another, both of these primary CAMs can be expressed on derivatives of all three germ layers. At sites of embryonic induction, epithelial cells expressing both L-CAM and N-CAM, or L-CAM only, were apposed to mesenchymal cells expressing N-CAM. Examples included epiblast (NL) and notochord (N); endodermal epithelium (NL) and lung mesenchyme (N); Wolffian duct (NL) and mesonephric mesenchyme (N); apical ectodermal ridge (NL) and limb mesenchyme (N); and feather placode (L) and dermal condensation (N). The cumulative observations indicate that cell surface modulation of the primary CAMs at induction sites can be classified into two modes. In mode I, expression of N-CAM (or both CAMs) in mesenchyme decreases to low amounts at the cell surface, and then N-CAM is reexpressed. In mode II, one or the other CAM disappears from epithelia expressing both CAMs. As a result of the primary processes of development, collectives of cells linked by N-CAM and undergoing modulation mode I are brought into the proximity of collectives of cells linked by L-CAM plus N-CAM or by L-CAM undergoing modulation mode II. Such adjoining cell collectives or CAM couples were found at all sites of embryonic induction examined. Images PMID:3863135

  4. GABA regulates synaptic integration of newly generated neurons in the adult brain

    NASA Astrophysics Data System (ADS)

    Ge, Shaoyu; Goh, Eyleen L. K.; Sailor, Kurt A.; Kitabatake, Yasuji; Ming, Guo-Li; Song, Hongjun

    2006-02-01

    Adult neurogenesis, the birth and integration of new neurons from adult neural stem cells, is a striking form of structural plasticity and highlights the regenerative capacity of the adult mammalian brain. Accumulating evidence suggests that neuronal activity regulates adult neurogenesis and that new neurons contribute to specific brain functions. The mechanism that regulates the integration of newly generated neurons into the pre-existing functional circuitry in the adult brain is unknown. Here we show that newborn granule cells in the dentate gyrus of the adult hippocampus are tonically activated by ambient GABA (γ-aminobutyric acid) before being sequentially innervated by GABA- and glutamate-mediated synaptic inputs. GABA, the major inhibitory neurotransmitter in the adult brain, initially exerts an excitatory action on newborn neurons owing to their high cytoplasmic chloride ion content. Conversion of GABA-induced depolarization (excitation) into hyperpolarization (inhibition) in newborn neurons leads to marked defects in their synapse formation and dendritic development in vivo. Our study identifies an essential role for GABA in the synaptic integration of newly generated neurons in the adult brain, and suggests an unexpected mechanism for activity-dependent regulation of adult neurogenesis, in which newborn neurons may sense neuronal network activity through tonic and phasic GABA activation.

  5. Tolerance to allopregnanolone with focus on the GABA-A receptor

    PubMed Central

    Turkmen, Sahruh; Backstrom, Torbjorn; Wahlstrom, Goran; Andreen, Lotta; Johansson, Inga-Maj

    2011-01-01

    Many studies have suggested a relationship between stress, sex steroids, and negative mental and mood changes in humans. The progesterone metabolite allopregnanolone is a potent endogenous ligand of the γ-amino butyric acid –A (GABA-A) receptor, and the most discussed neuroactive steroid. Variations in the levels of neuroactive steroids that influence the activity of the GABA-A receptor cause a vulnerability to mental and emotional pathology. There are physiological conditions in which allopregnanolone production increases acutely (e.g. stress) or chronically (e.g. menstrual cycle, pregnancy), thus exposing the GABA-A receptor to high and continuous allopregnanolone concentrations. In such conditions, tolerance to allopregnanolone may develop. We have shown that both acute and chronic tolerances can develop to the effects of allopregnanolone. Following the development of acute allopregnanolone tolerance, there is a decrease in the abundance of the GABA-A receptor α4 subunit and the expression of the α4 subunit mRNA in the ventral-posteriomedial nucleus of the thalamus. Little is known about the mechanism behind allopregnanolone tolerance and its effects on assembly of the GABA-A receptor composition. The exact mechanism of the allopregnanolone tolerance phenomena remains unclear. The purpose of this review is to summarize certain aspects of current knowledge concerning allopregnanolone tolerance and changes in the GABA-A receptors. PMID:20883478

  6. Implementing bacterial acid resistance into cell-free protein synthesis for buffer-free expression and screening of enzymes.

    PubMed

    Kim, Ho-Cheol; Kim, Kwang-Soo; Kang, Taek-Jin; Choi, Jong Hyun; Song, Jae Jun; Choi, Yun Hee; Kim, Byung-Gee; Kim, Dong-Myung

    2015-12-01

    Cell-free protein synthesis utilizes translational machinery isolated from the cells for in vitro expression of template genes. Because it produces proteins without gene cloning and cell cultivation steps, cell-free protein synthesis can be used as a versatile platform for high-throughput expression of enzyme libraries. Furthermore, the open nature of cell-free protein synthesis allows direct integration of enzyme synthesis with subsequent screening steps. However, the presence of high concentration of chemical buffers in the conventional reaction mixture makes it difficult to streamline cell-free protein synthesis with pH-based assay of the synthesized enzymes. In this study, we have implemented an enzyme-assisted bacterial acid resistance mechanism into an Escherichia coli (E.coli) extract-based cell-free protein synthesis system in place of chemical buffers. When deployed in the reaction mixture for cell-free synthesis of enzymes, through proton-consuming conversion of glutamate into γ-aminobutyric acid (GABA), an engineered glutamate decarboxylase (GADβ) was able to maintain the pH of reaction mixture during enzyme synthesis. Because the reaction mixture becomes free of buffering capacity upon the depletion of glutamate, synthesized enzyme could be directly assayed without purification steps. The designed method was successfully applied to the screening of mutant library of sialyltransferase genes to identify mutants with improved enzymatic activity.

  7. GABA(B) receptor isoforms GBR1a and GBR1b, appear to be associated with pre- and post-synaptic elements respectively in rat and human cerebellum.

    PubMed

    Billinton, A; Upton, N; Bowery, N G

    1999-03-01

    1. Metabotropic gamma-aminobutyric acid (GABA) receptors, GABA(B), are coupled through G-proteins to K+ and Ca2+ channels in neuronal membranes. Cloning of the GABAB receptor has not uncovered receptor subtypes, but demonstrated two isoforms, designated GBR1a and GBR1b, which differ in their N terminal regions. In the rodent cerebellum GABA(B) receptors are localized to a greater extent in the molecular layer, and are reported to exist on granule cell parallel fibre terminals and Purkinje cell (PC) dendrites, which may represent pre- and post-synaptic receptors. 2. The objective of this study was to localize the mRNA splice variants, GBR1a and GBR1b for GABA(B) receptors in rat cerebellum, for comparison with the localization in human cerebellum using in situ hybridization. 3. Receptor autoradiography was performed utilizing [3H]-CGP62349 to localize GABA(B) receptors in rat and human cerebellum. Radioactively labelled oligonucleotide probes were used to localize GBR1a and GBR1b, and by dipping slides in photographic emulsion, silver grain images were obtained for quantification at the cellular level. 4. Binding of 0.5 nM [3H]-CGP62349 demonstrated significantly higher binding to GABA(B) receptors in the molecular layer than the granule cell (GC) layer of rat cerebellum (molecular layer binding 200+/-11% of GC layer; P<0.0001). GBR1a mRNA expression was found to be predominantly in the GC layer (PC layer grains 6+/-6% of GC layer grains; P<0.05), and GBR1b expression predominantly in PCs (PC layer grains 818+/-14% of GC layer grains; P<0.0001). 5. The differential distribution of GBR1a and GBR1b mRNA splice variants for GABA(B) receptors suggests a possible association of GBR1a and GBR1b with pre- and post-synaptic elements respectively.

  8. Striatal cholinergic interneurons Drive GABA release from dopamine terminals.

    PubMed

    Nelson, Alexandra B; Hammack, Nora; Yang, Cindy F; Shah, Nirao M; Seal, Rebecca P; Kreitzer, Anatol C

    2014-04-01

    Striatal cholinergic interneurons are implicated in motor control, associative plasticity, and reward-dependent learning. Synchronous activation of cholinergic interneurons triggers large inhibitory synaptic currents in dorsal striatal projection neurons, providing one potential substrate for control of striatal output, but the mechanism for these GABAergic currents is not fully understood. Using optogenetics and whole-cell recordings in brain slices, we find that a large component of these inhibitory responses derive from action-potential-independent disynaptic neurotransmission mediated by nicotinic receptors. Cholinergically driven IPSCs were not affected by ablation of striatal fast-spiking interneurons but were greatly reduced after acute treatment with vesicular monoamine transport inhibitors or selective destruction of dopamine terminals with 6-hydroxydopamine, indicating that GABA release originated from dopamine terminals. These results delineate a mechanism in which striatal cholinergic interneurons can co-opt dopamine terminals to drive GABA release and rapidly inhibit striatal output neurons.

  9. Cyan fluorescent protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina

    PubMed Central

    Vila, Alejandro; Huynh, Uyen-Chi N.; Brecha, Nicholas C.

    2008-01-01

    Purpose To characterize cyan fluorescent protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. Methods CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD67), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. Results CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 μm in diameter, and they had a density of 2636±347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD67, GAT-1, and ChAT indicated that the small, weakly fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. Conclusions The total number and density of CFP-fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells. PMID:18728756

  10. Selank Administration Affects the Expression of Some Genes Involved in GABAergic Neurotransmission

    PubMed Central

    Volkova, Anastasiya; Shadrina, Maria; Kolomin, Timur; Andreeva, Lyudmila; Limborska, Svetlana; Myasoedov, Nikolay; Slominsky, Petr

    2016-01-01

    Clinical studies have shown the similarity of the spectrum of physiological effects of Selank and classical benzodiazepines, such as diazepam and phenazepam. These data suggest that there is a similar basis of their mechanism of action. To test this hypothesis we studied the effect of Selank and GABA on the expression of genes involved in neurotransmission. We analyzed the expression of 84 genes involved in neurotransmission (e.g., major subunit of the GABA receptor, transporters, ion channels, dopamine, and serotonin receptors) in the frontal cortex of rats 1 and 3 h after the administration of Selank or GABA (300 μg/kg) using real-time PCR method. We found significant changes in the expression of 45 genes 1 h after the administration of the compounds. Three hours after Selank or GABA administration, 22 genes changed their expression. We found positive correlation between the changes in genes expression within 1 h after administration of Selank or GABA. Our results showed that Selank caused a number of alterations in the expression of genes involved in neurotransmission. The data obtained indicate that Selank is characterized by its complex effects on nerve cells, and one of its possible molecular mechanisms is associated with allosteric modulation of the GABAergic system. PMID:26924987

  11. Dental enamel cells express functional SOCE channels.

    PubMed

    Nurbaeva, Meerim K; Eckstein, Miriam; Concepcion, Axel R; Smith, Charles E; Srikanth, Sonal; Paine, Michael L; Gwack, Yousang; Hubbard, Michael J; Feske, Stefan; Lacruz, Rodrigo S

    2015-10-30

    Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) dynamics in enamel cells are unclear. Store-operated Ca(2+) entry (SOCE) channels are important Ca(2+) influx mechanisms in many cells. SOCE involves release of Ca(2+) from intracellular pools followed by Ca(2+) entry. The best-characterized SOCE channels are the Ca(2+) release-activated Ca(2+) (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca(2+) uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca(2+) release mechanism. Passive depletion of ER Ca(2+) stores with thapsigargin resulted in a significant raise in [Ca(2+)]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca(2+) entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca(2+) uptake in enamel formation.

  12. Dental enamel cells express functional SOCE channels

    PubMed Central

    Nurbaeva, Meerim K.; Eckstein, Miriam; Concepcion, Axel R.; Smith, Charles E.; Srikanth, Sonal; Paine, Michael L.; Gwack, Yousang; Hubbard, Michael J.; Feske, Stefan; Lacruz, Rodrigo S.

    2015-01-01

    Dental enamel formation requires large quantities of Ca2+ yet the mechanisms mediating Ca2+ dynamics in enamel cells are unclear. Store-operated Ca2+ entry (SOCE) channels are important Ca2+ influx mechanisms in many cells. SOCE involves release of Ca2+ from intracellular pools followed by Ca2+ entry. The best-characterized SOCE channels are the Ca2+ release-activated Ca2+ (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca2+ uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca2+ release mechanism. Passive depletion of ER Ca2+ stores with thapsigargin resulted in a significant raise in [Ca2+]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca2+ entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca2+ uptake in enamel formation. PMID:26515404

  13. Cell cycle gene expression under clinorotation

    NASA Astrophysics Data System (ADS)

    Artemenko, Olga

    2016-07-01

    Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

  14. Conditional deletion of Mecp2 in parvalbumin-expressing GABAergic cells results in the absence of critical period plasticity.

    PubMed

    He, Ling-jie; Liu, Nan; Cheng, Tian-lin; Chen, Xiao-jing; Li, Yi-ding; Shu, You-sheng; Qiu, Zi-long; Zhang, Xiao-hui

    2014-10-09

    Mutations in the X-linked gene encoding the transcriptional modulator methyl-CpG-binding protein 2 (MeCP2) impair postnatal development of the brain. Here we use neuronal-type specific gene deletion in mice to show that conditional Mecp2 deletion in GABAergic parvalbumin-expressing (PV) cells (PV-Mecp2(-/y)) does not cause most Rett-syndrome-like behaviours, but completely abolishes experience-dependent critical period plasticity of primary visual cortex (V1) that develops normal visual functions. However, selective loss of Mecp2 in GABAergic somatostatin-expressing cells or glutamatergic pyramidal cells does not affect the critical period plasticity. MeCP2-deficient PV cells exhibit high intrinsic excitability, selectively reduced efficacy of recurrent excitatory synapses in V1 layer 4 circuits, and decreased evoked visual responses in vivo. Enhancing cortical gamma-aminobutyric acid (GABA) inhibition with diazepam infusion can restore critical period plasticity in both young and adult PV-Mecp2(-/y) mice. Thus, MeCP2 expression in inhibitory PV cells during the critical period is essential for local circuit functions underlying experience-dependent cortical plasticity.

  15. The Arabidopsis pop2-1 mutant reveals the involvement of GABA transaminase in salt stress tolerance

    PubMed Central

    2010-01-01

    Background GABA (γ-aminobutyric acid) is a non protein amino acid that has been reported to accumulate in a number of plant species when subjected to high salinity and many other environmental constraints. However, no experimental data are to date available on the molecular function of GABA and the involvement of its metabolism in salt stress tolerance in higher plants. Here, we investigated the regulation of GABA metabolism in Arabidopsis thaliana at the metabolite, enzymatic activity and gene transcription levels upon NaCl stress. Results We identified the GABA transaminase (GABA-T), the first step of GABA catabolism, as the most responsive to NaCl. We further performed a functional analysis of the corresponding gene POP2 and demonstrated that the previously isolated loss-of-function pop2-1 mutant was oversensitive to ionic stress but not to osmotic stress suggesting a specific role in salt tolerance. NaCl oversensitivity was not associated with overaccumulation of Na+ and Cl- but mutant showed a slight decrease in K+. To bring insights into POP2 function, a promoter-reporter gene strategy was used and showed that POP2 was mainly expressed in roots under control conditions and was induced in primary root apex and aerial parts of plants in response to NaCl. Additionally, GC-MS- and UPLC-based metabolite profiling revealed major changes in roots of pop2-1 mutant upon NaCl stress including accumulation of amino acids and decrease in carbohydrates content. Conclusions GABA metabolism was overall up-regulated in response to NaCl in Arabidopsis. Particularly, GABA-T was found to play a pivotal function and impairment of this step was responsible for a decrease in salt tolerance indicating that GABA catabolism was a determinant of Arabidopsis salt tolerance. GABA-T would act in salt responses in linking N and C metabolisms in roots. PMID:20122158

  16. Auto-modulation of neuroactive steroids on GABA A receptors: a novel pharmacological effect.

    PubMed

    Wegner, Florian; Rassler, Cornelia; Allgaier, Clemens; Strecker, Karl; Wohlfarth, Kai

    2007-02-01

    GABA(A) receptor function is modulated by various important drugs including neuroactive steroids that act on allosteric modulatory sites and can directly activate GABA(A) receptor channels at high concentrations. We used whole cell patch-clamp recordings and rapid applications of the neuroactive steroid alphaxalone to investigate repetitive steroid effects. Alphaxalone potentiation of submaximal GABA-evoked currents was enhanced significantly by repetitive coapplications at all investigated recombinant isoforms (alpha1beta3delta, alpha1beta3gamma2L, alpha6beta3delta, alpha6beta3gamma2L) and at GABA(A) receptors of differentiated human NT2 neurons. A similar increase of current amplitudes was induced by repetitive applications of a high steroid concentration without GABA. We refer to these reversible effects as auto-modulation because repeated interactions of steroids enhanced their own pharmacological impact at the receptor sites in a time and concentration dependent manner without affecting GABA controls. Pronounced auto-modulatory actions were also measured using the neurosteroid 5alpha-THDOC in contrast to indiplon, THIP, and pentobarbital indicating a steroid specificity. Protein kinase A inhibition significantly reduced alphaxalone auto-modulation at alpha1beta3gamma2L, alpha6beta3gamma2L, and alpha6beta3delta subtypes while it enhanced potentiation at alpha1beta3delta isoforms suggesting a crucial influence of receptor subunit composition and phosphorylation for steroid actions. Especially at extrasynaptic GABA(A) receptor sites containing the delta subunit steroid auto-modulation may have a critical role in enhancing potentiation of GABA-induced currents.

  17. Glutamate and GABA activate different receptors and Cl(-) conductances in crab peptide-secretory neurons.

    PubMed

    Duan, S; Cooke, I M

    2000-01-01

    Responses to rapid application of glutamic acid (Glu) and gamma-aminobutyric acid (GABA), 0.01-3 mM, were recorded by whole-cell patch clamp of cultured crab (Cardisoma carnifex) X-organ neurons. Responses peaked within 200 ms. Both Glu and GABA currents had reversal potentials that followed the Nernst Cl(-) potential when [Cl(-)](i) was varied. A Boltzmann fit to the normalized, averaged dose-response curve for Glu indicated an EC(50) of 0.15 mM and a Hill coefficient of 1.05. Rapid (t(1/2) approximately 1 s) desensitization occurred during Glu but not GABA application that required >2 min for recovery. Desensitization was unaffected by concanavalin A or cyclothiazide. N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, quisqualate, and kainate (to 1 mM) were ineffective, nor were Glu responses influenced by glycine (1 microM) or Mg(2+) (0-26 mM). Glu effects were imitated by ibotenic acid (0.1 mM). The following support the conclusion that Glu and GABA act on different receptors: 1) responses sum; 2) desensitization to Glu or ibotenic acid did not diminish GABA responses; 3) the Cl(-)-channel blockers picrotoxin and niflumic acid (0.5 mM) inhibited Glu responses by approximately 90 and 80% but GABA responses by approximately 50 and 20%; and 4) polyvinylpyrrolydone-25 (2 mM in normal crab saline) eliminated Glu responses but left GABA responses unaltered. Thus crab secretory neurons have separate receptors responsive to Glu and to GABA, both probably ionotropic, and mediating Cl(-) conductance increases. In its responses and pharmacology, this crustacean Glu receptor resembles Cl(-)-permeable Glu receptors previously described in invertebrates and differs from cation-permeable Glu receptors of vertebrates and invertebrates.

  18. Resting GABA and glutamate concentrations do not predict visual gamma frequency or amplitude

    PubMed Central

    Cousijn, Helena; Haegens, Saskia; Wallis, George; Near, Jamie; Stokes, Mark G.; Harrison, Paul J.; Nobre, Anna C.

    2014-01-01

    Gamma band oscillations arise in neuronal networks of interconnected GABAergic interneurons and excitatory pyramidal cells. A previous study found a correlation between visual gamma peak frequency, as measured with magnetoencephalography, and resting GABA levels, as measured with magnetic resonance spectroscopy (MRS), in 12 healthy volunteers. If true, this would allow studies in clinical populations testing modulation of this relationship, but this finding has not been replicated. We addressed this important question by measuring gamma oscillations and GABA, as well as glutamate, in 50 healthy volunteers. Visual gamma activity was evoked using an established gratings paradigm, and we applied a beamformer spatial filtering technique to extract source-reconstructed gamma peak frequency and amplitude from the occipital lobe. We determined gamma peak frequency and amplitude from the location with maximal activation and from the location of the MRS voxel to assess the relationship of GABA with gamma. Gamma peak frequency was estimated from the highest value of the raw spectra and by a Gaussian fit to the spectra. MRS data were acquired from occipital cortex. We did not replicate the previously found correlation between gamma peak frequency and GABA concentration. Calculation of a Bayes factor provided strong evidence in favor of the null hypothesis. We also did not find a correlation between gamma activity and glutamate or between gamma and the ratio of GABA/glutamate. Our results suggest that cortical gamma oscillations do not have a consistent, demonstrable relationship to excitatory/inhibitory network activity as proxied by MRS measurements of GABA and glutamate. PMID:24927588

  19. Resting GABA and glutamate concentrations do not predict visual gamma frequency or amplitude.

    PubMed

    Cousijn, Helena; Haegens, Saskia; Wallis, George; Near, Jamie; Stokes, Mark G; Harrison, Paul J; Nobre, Anna C

    2014-06-24

    Gamma band oscillations arise in neuronal networks of interconnected GABAergic interneurons and excitatory pyramidal cells. A previous study found a correlation between visual gamma peak frequency, as measured with magnetoencephalography, and resting GABA levels, as measured with magnetic resonance spectroscopy (MRS), in 12 healthy volunteers. If true, this would allow studies in clinical populations testing modulation of this relationship, but this finding has not been replicated. We addressed this important question by measuring gamma oscillations and GABA, as well as glutamate, in 50 healthy volunteers. Visual gamma activity was evoked using an established gratings paradigm, and we applied a beamformer spatial filtering technique to extract source-reconstructed gamma peak frequency and amplitude from the occipital lobe. We determined gamma peak frequency and amplitude from the location with maximal activation and from the location of the MRS voxel to assess the relationship of GABA with gamma. Gamma peak frequency was estimated from the highest value of the raw spectra and by a Gaussian fit to the spectra. MRS data were acquired from occipital cortex. We did not replicate the previously found correlation between gamma peak frequency and GABA concentration. Calculation of a Bayes factor provided strong evidence in favor of the null hypothesis. We also did not find a correlation between gamma activity and glutamate or between gamma and the ratio of GABA/glutamate. Our results suggest that cortical gamma oscillations do not have a consistent, demonstrable relationship to excitatory/inhibitory network activity as proxied by MRS measurements of GABA and glutamate. PMID:24927588

  20. Kinetic studies on the inhibition of GABA-T by gamma-vinyl GABA and taurine.

    PubMed

    Sulaiman, Saba A J; Suliman, Fakhr Eldin O; Barghouthi, Samira

    2003-08-01

    Gamma-aminobutyric acid transaminase (GABA-T, EC 2.6.1.19) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the degradation of gamma-aminobutyric acid. The kinetics of this reaction are studied in vitro, both in the absence, and in the presence of two inhibitors: gamma-vinyl GABA (4-aminohex-5-enoic acid), and a natural product, taurine (ethylamine-2-sulfonic acid). A kinetic model that describes the transamination process is proposed. GABA-T from Pseudomonas fluorescens is inhibited by gamma-vinyl GABA and taurine at concentrations of 51.0 and 78.5 mM. Both inhibitors show competitive inhibition behavior when GABA is the substrate and the inhibition constant (Ki) values for gamma-vinyl GABA and taurine were found to be 26 +/- 3 mM and 68 +/- 7 mM respectively. The transamination process of alpha-ketoglutarate was not affected by the presence of gamma-vinyl GABA, whereas, taurine was a noncompetitive inhibitor of GABA-T when alpha-ketoglutarate was the substrate. The inhibition dissociation constant (Kii) for this system was found to be 96 +/- 10 mM. The Michaelis-Menten constant (Km) in the absence of inhibition, was found to be 0.79 +/- 0.11 mM, and 0.47 +/- 0.10 mM for GABA and alpha-ketoglutarate respectively.

  1. Optogenetics for gene expression in mammalian cells.

    PubMed

    Müller, Konrad; Naumann, Sebastian; Weber, Wilfried; Zurbriggen, Matias D

    2015-02-01

    Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system.

  2. GABA and glycine in the developing brain.

    PubMed

    Ito, Susumu

    2016-09-01

    GABA and glycine are major inhibitory neurotransmitters in the CNS and act on receptors coupled to chloride channels. During early developmental periods, both GABA and glycine depolarize membrane potentials due to the relatively high intracellular Cl(-) concentration. Therefore, they can act as excitatory neurotransmitters. GABA and glycine are involved in spontaneous neural network activities in the immature CNS such as giant depolarizing potentials (GDPs) in neonatal hippocampal neurons, which are generated by the synchronous activity of GABAergic interneurons and glutamatergic principal neurons. GDPs and GDP-like activities in the developing brains are thought to be important for the activity-dependent functiogenesis through Ca(2+) influx and/or other intracellular signaling pathways activated by depolarization or stimulation of metabotropic receptors. However, if GABA and glycine do not shift from excitatory to inhibitory neurotransmitters at the birth and in maturation, it may result in neural disorders including autism spectrum disorders. PMID:26951057

  3. Cleavage of the vesicular GABA transporter under excitotoxic conditions is followed by accumulation of the truncated transporter in nonsynaptic sites.

    PubMed

    Gomes, João R; Lobo, Andrea C; Melo, Carlos V; Inácio, Ana R; Takano, Jiro; Iwata, Nobuhisa; Saido, Takaomi C; de Almeida, Luís P; Wieloch, Tadeusz; Duarte, Carlos B

    2011-03-23

    GABA is the major inhibitory neurotransmitter in the CNS and changes in GABAergic neurotransmission affect the overall activity of neuronal networks. The uptake of GABA into synaptic vesicles is mediated by the vesicular GABA transporter (VGAT), and changes in the expression of the transporter directly regulate neurotransmitter release. In this work we investigated the changes in VGAT protein levels during ischemia and in excitotoxic conditions, which may affect the demise process. We found that VGAT is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, giving rise to a stable truncated cleavage product (tVGAT). VGAT cleavage was also observed after transient middle cerebral artery occlusion in mice, a cerebral ischemia model, and following intrahippocampal injection of kainate, but no effect was observed in transgenic mice overexpressing calpastatin, a calpain inhibitor. Incubation of isolated cerebrocortical synaptic vesicles with recombinant calpain also induced the cleavage of VGAT and formation of stable tVGAT. Immunoblot experiments using antibodies targeting different regions of VGAT and N-terminal sequencing analysis showed that calpain cleaves the transporter in the N-terminal region, at amino acids 52 and 60. Immunocytochemistry of GABAergic striatal neurons expressing GFP fusion proteins with the full-length VGAT or tVGAT showed that cleavage of the transporter induces a loss of synaptic delivery, leading to a homogeneous distribution of the protein along neurites. Our results show that excitotoxicity downregulates full-length VGAT, with a concomitant generation of tVGAT, which is likely to affect GABAergic neurotransmission and may influence cell death during ischemia.

  4. Distribution and ultrastructure of neurons in opossum piriform cortex displaying immunoreactivity to GABA and GAD and high-affinity tritiated GABA uptake

    SciTech Connect

    Haberly, L.B.; Hansen, D.J.; Feig, S.L.; Presto, S.

    1987-12-08

    GABAergic neurons have been identified in the piriform cortex of the opossum at light and electron microscopic levels by immunocytochemical localization of GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase and by autoradiographic visualization of high-affinity /sup 3/H-GABA uptake. Four major neuron populations have been distinguished on the basis of soma size, shape, and segregation at specific depths and locations: large horizontal cells in layer Ia of the anterior piriform cortex, small globular cells with thin dendrites concentrated in layers Ib and II of the posterior piriform cortex, and multipolar and fusiform cells concentrated in the deep part of layer III in anterior and posterior parts of the piriform cortex and the subjacent endopiriform nucleus. All four populations were well visualized with both antisera, but the large layer Ia horizontal cells displayed only very light /sup 3/H-GABA uptake, thus suggesting a lack of local axon collaterals or lack of high-affinity GABA uptake sites. The large, ultrastructurally distinctive somata of layer Ia horizontal cells receive a very small number of symmetrical synapses; the thin, axonlike dendrites of small globular cells are exclusively postsynaptic and receive large numbers of both symmetrical and asymmetrical synapses, in contrast to somata which receive a small number of both types; and the deep multipolar and fusiform cells receive a highly variable number of symmetrical and asymmetrical synapses on somata and proximal dendrites. Labeled puncta of axon terminal dimensions were found in large numbers in the neuropil surrounding pyramidal cell somata in layer II and in the endopiriform nucleus. Moderately large numbers of labeled puncta were found in layer I at the depth of pyramidal cell apical dendrites with greater numbers in layer Ia at the depth of distal apical segments than in layer Ib.

  5. Regulation of α3-containing GABAA receptors in guinea-pig adrenal medullary cells by adrenal steroids.

    PubMed

    Inoue, M; Harada, K; Nakamura, J; Matsuoka, H

    2013-12-01

    GABA is thought to function as a paracrine factor in adrenal medullary (AM) cells. Thus, we electrophysiologically and immunologically examined the properties of GABAA receptors (GABAARs) in guinea-pig AM cells. Bath application of GABA produced an inward current at -60 mV in a dose-dependent manner with an EC50 of 32.3 μM. This GABA-induced current was enhanced by allopregnanolone at concentrations of 0.01 μM and more. A prior exposure to allopregnanolone resulted in a decrease in an EC50 for GABA in activating GABAARs. The GABA-induced current was suppressed by Zn(2+) in a dose-dependent manner with an IC50 of 18 μM, whereas it was enhanced by 100 μM La(3+). The benzodiazepine analog diazepam was three times more potent than zolpidem in enhancing the GABA current, and it was also augmented by L-838,417, which has no action on α1-containing GABAARs. The GABAAR α3, but not α1, and γ2 subunits were immunologically detected at the cell periphery. The expression of α3 subunits in PC12 cells was enhanced by glucocorticoid activity. The results indicated that GABAARs in guinea-pig AM cells mainly comprise α3, β, and γ2 subunits and are enhanced by allopreganalone and glucocorticoids may play a major role in the expression of α3 subunits. PMID:24012744

  6. Mutations in γ-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence.

    PubMed

    Park, Duck Hwan; Mirabella, Rossana; Bronstein, Philip A; Preston, Gail M; Haring, Michel A; Lim, Chun Keun; Collmer, Alan; Schuurink, Robert C

    2010-10-01

    Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid γ-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome harbors three genes annotated as gabT GABA transaminases. A DC3000 mutant lacking all three gabT genes was constructed and found to be unable to utilize GABA as a sole carbon and nitrogen source. In complete minimal media supplemented with GABA, the mutant grew less well than wild-type DC3000 and showed strongly reduced expression of hrpL and avrPto, which encode an alternative sigma factor and effector, respectively, associated with the type III secretion system. The growth of the gabT triple mutant was weakly reduced in Arabidopsis ecotype Landberg erecta (Ler) and strongly reduced in the Ler pop2-1 GABA transaminase-deficient mutant that accumulates higher levels of GABA. Much of the ability to grow on GABA-amended minimal media or in Arabidopsis pop2-1 leaves could be restored to the gabT triple mutant by expression in trans of just gabT2. The ability of DC3000 to elicit the hypersensitive response (HR) in tobacco leaves is dependent upon deployment of the type III secretion system, and the gabT triple mutant was less able than wild-type DC3000 to elicit this HR when bacteria were infiltrated along with GABA at levels of 1 mm or more. GABA may have multiple effects on P. syringae-plant interactions, with elevated levels increasing disease resistance.

  7. The regulation of transmitter expression in postembryonic lineages in the moth Manduca sexta. I. Transmitter identification and developmental acquisition of expression.

    PubMed

    Witten, J L; Truman, J W

    1991-07-01

    The majority of the neurons in the adult nervous system of Manduca sexta are born postembryonically, during larval life. Stereotypic arrays of identifiable neuroblasts generate their clonal families or lineages commencing at the end of the second larval instar through pupal day 2, when the neuroblasts die (Booker and Truman, 1987a). We have used immunohistochemical techniques to follow the neurochemical differentiation of GABA and a peptide similar to molluscan small cardioactive peptide B (SCPB) in identified lineages. We report here the distribution and developmental acquisition of the expression of these putative transmitters. There are 24 postembryonic lineages in the second thoracic ganglion of the larvae (Booker and Truman, 1987a). Immunoreactivity against GABA and SCPB is seen only in a subset of these 24 clonal families. GABA immunoreactivity is confined to the progeny of the E, K, M, N, T, and X neuroblasts and is expressed by most or all of the neurons in these lineages. The SCPB-like immunoreactivity is found in a subset of the neurons in only two clonal groups, the K and M groups, and is colocalized with GABA. These results show that, though heterogeneity in transmitter type exists (GABA, GABA/SCPB), members of a given lineage share at least some features (GABA) in common. The onset of transmitter expression was followed in detail for the K- and M-lineage neurons. During the larval stages, the postembryonic lineage cells are developmentally arrested in a partially differentiated state (Booker and Truman, 1987a) and do not express transmitter immunoreactivity at this time. Their maturation resumes with the onset of metamorphosis.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. mRNA and Protein Levels for GABA[subscript A][alpha]4, [alpha]5, [beta]1 and GABA[subscript B]R1 Receptors are Altered in Brains from Subjects with Autism

    ERIC Educational Resources Information Center

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rooney, Robert J.; Patel, Diven H.; Thuras, Paul D.

    2010-01-01

    We have shown altered expression of gamma-aminobutyric acid A (GABA[subscript A]) and gamma-aminobutyric acid B (GABA[subscript B]) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3…

  9. Striatal GABA receptor alterations in hypoxic neonatal rats: role of glucose, oxygen and epinephrine treatment.

    PubMed

    Anju, T R; Binoy, J; Anitha, M; Paulose, C S

    2012-03-01

    Hypoxia in neonates disrupts the oxygen flow to the brain, essentially starving the brain and preventing it from performing vital biochemical processes important for central nervous system development. Hypoxia results in a permanent brain damage by gene and receptor level alterations mediated through neurotransmitters. The present study evaluated GABA, GABAA, GABAB receptor functions and gene expression changes in glutamate decarboxylase in the corpus striatum of hypoxic neonatal rats and the treatment groups with glucose, oxygen and epinephrine. Since GABA is the principal neurotransmitter involved in hypoxic ventilatory decline, the alterations in its level under hypoxic stress points to an important aspect of respiratory control. Following hypoxic stress, a significant decrease in total GABA, GABAA and GABAB receptors function and GAD expression was observed in the striatum, which accounts for the ventilator decline. Hypoxic rats treated with glucose alone and with oxygen showed a reversal of the receptor alterations and changes in GAD to near control. Being a source of immediate energy, glucose can reduce the ATP-depletion-induced changes in GABA and oxygenation helps in overcoming reduction in oxygen supply. Treatment with oxygen alone and epinephrine was not effective in reversing the altered receptor functions. Thus, our study point to the functional role of GABA receptors in mediating ventilatory response to hypoxia and the neuroprotective role of glucose treatment. This has immense significance in the proper management of neonatal hypoxia for a better intellect in the later stages of life.

  10. The inhibitory role of γ-aminobutyric acid (GABA) on immunomodulation of Pacific oyster Crassostrea gigas.

    PubMed

    Li, Meijia; Qiu, Limei; Wang, Lingling; Wang, Weilin; Xin, Lusheng; Li, Yiqun; Liu, Zhaoqun; Song, Linsheng

    2016-05-01

    γ-aminobutyric acid (GABA) is an inhibitory neurotransmitter to suppress the immune-mediated pro-inflammatory reactions, and it has been used in the treatment of many inflammation-related diseases in vertebrates, while its immunomodulatory role in invertebrates has never been reported. In the present study, GABA was found to exist in the hemolymph of Pacific oyster Crassostrea gigas, and its concentration decreased slightly from 8.00 ± 0.37 μmol L(-1) at normal condition to 7.73 ± 0.15 μmol L(-1) at 6 h after LPS stimulation, and then increased to 9.34 ± 0.15 μmol L(-1), 8.86 ± 0.68 μmol L(-1) at 12 h and 48 h, respectively. After LPS stimulation, the mRNA expressions of pro-inflammatory cytokines (CgIL-17 and CgTNF) and immune effectors (CgSOD and CgBPI), and the protein expression of NOS increased significantly, and these increased trends were remarkably inhibited by GABA stimulation. At the same time, the phagocytosis rate and apoptosis rate of immunocytes also increased obviously after LPS stimulation, whereas the increase was repressed with the addition of GABA. The results collectively demonstrated that GABA was an indispensable inhibitory agent for both humoral and cellular immune response, which mainly functioned at the late phase of immune response to avoid the excess immune reactions and maintain the immune homeostasis.

  11. Synaptic GABA release prevents GABA transporter type-1 reversal during excessive network activity

    PubMed Central

    Savtchenko, Leonid; Megalogeni, Maria; Rusakov, Dmitri A.; Walker, Matthew C.; Pavlov, Ivan

    2015-01-01

    GABA transporters control extracellular GABA, which regulates the key aspects of neuronal and network behaviour. A prevailing view is that modest neuronal depolarization results in GABA transporter type-1 (GAT-1) reversal causing non-vesicular GABA release into the extracellular space during intense network activity. This has important implications for GABA uptake-targeting therapies. Here we combined a realistic kinetic model of GAT-1 with experimental measurements of tonic GABAA receptor currents in ex vivo hippocampal slices to examine GAT-1 operation under varying network conditions. Our simulations predict that synaptic GABA release during network activity robustly prevents GAT-1 reversal. We test this in the 0 Mg2+ model of epileptiform discharges using slices from healthy and chronically epileptic rats and find that epileptiform activity is associated with increased synaptic GABA release and is not accompanied by GAT-1 reversal. We conclude that sustained efflux of GABA through GAT-1 is unlikely to occur during physiological or pathological network activity. PMID:25798861

  12. Modulation by general anaesthetics of rat GABAA receptors comprised of α1β3 and β3 subunits expressed in human embryonic kidney 293 cells

    PubMed Central

    Davies, Paul A; Kirkness, Ewen F; Hales, Tim G

    1997-01-01

    Radioligand binding and patch-clamp techniques were used to study the actions of γ-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5α-pregnan-3α-ol-20-one on rat α1 and β3 GABAA receptor subunits, expressed either alone or in combination.Membranes from HEK293 cells after transfection with α1 cDNA did not bind significant levels of [35S]-tert-butyl bicyclophosphorothionate ([35S]-TBPS) (<0.03 pmol mg−1 protein). GABA (100 μM) applied to whole-cells transfected with α1 cDNA and clamped at −60 mV, also failed to activate discernible currents.The membranes of cells expressing β3 cDNAs bound [35S]-TBPS (∼1 pmol mg−1 protein). However, the binding was not influenced by GABA (10 nM–100 μM). Neither GABA (100 μM) nor picrotoxin (10 μM) affected currents recorded from cells expressing β3 cDNA, suggesting that β3 subunits do not form functional GABAA receptors or spontaneously active ion channels.GABA (10 nM–100 μM) modulated [35S]-TBPS binding to the membranes of cells transfected with both α1 and β3 cDNAs. GABA (0.1 μM–1 mM) also dose-dependently activated inward currents with an EC50 of 9 μM recorded from cells transfected with α1 and β3 cDNAs, clamped at −60 mV.Propofol (10 nM–100 μM), pentobarbitone (10 nM–100 μM) and 5α-pregnan-3α-ol-20-one (1 nM–30 μM) modulated [35S]-TBPS binding to the membranes of cells expressing either α1β3 or β3 receptors. Propofol (100 μM), pentobarbitone (1 mM) and 5α-pregnan-3α-ol-20-one (10 μM) also activated currents recorded from cells expressing α1β3 receptors.Propofol (1 μM–1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing β3 homomers. In contrast, application of 5α-pregnan-3α-ol-20-one (10 μM) failed to activate detectable currents.Propofol (100 μM)-activated currents recorded from cells expressing either α1β3 or β3

  13. GABA-cAMP response element-binding protein signaling regulates maturation and survival of newly generated neurons in the adult hippocampus.

    PubMed

    Jagasia, Ravi; Steib, Kathrin; Englberger, Elisabeth; Herold, Sabine; Faus-Kessler, Theresa; Saxe, Michael; Gage, Fred H; Song, Hongjun; Lie, D Chichung

    2009-06-24

    Survival and integration of new neurons in the hippocampal circuit are rate-limiting steps in adult hippocampal neurogenesis. Neuronal network activity is a major regulator of these processes, yet little is known about the respective downstream signaling pathways. Here, we investigate the role of cAMP response element-binding protein (CREB) signaling in adult hippocampal neurogenesis. CREB is activated in new granule neurons during a distinct developmental period. Loss of CREB function in a cell-autonomous manner impairs dendritic development, decreases the expression of the neurogenic transcription factor NeuroD and of the neuronal microtubule-associated protein, doublecortin (DCX), and compromises the survival of newborn neurons. In addition, GABA-mediated excitation regulates CREB activation at early developmental stages. Importantly, developmental defects after loss of GABA-mediated excitation can be compensated by enhanced CREB signaling. These results indicate that CREB signaling is a central pathway in adult hippocampal neurogenesis, regulating the development and survival of new hippocampal neurons downstream of GABA-mediated excitation.

  14. Expression of bacterial genes in plant cells.

    PubMed Central

    Fraley, R T; Rogers, S G; Horsch, R B; Sanders, P R; Flick, J S; Adams, S P; Bittner, M L; Brand, L A; Fink, C L; Fry, J S; Galluppi, G R; Goldberg, S B; Hoffmann, N L; Woo, S C

    1983-01-01

    Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed. Images PMID:6308651

  15. Impairment of GABA release in the hippocampus at the time of the first spontaneous seizure in the pilocarpine model of temporal lobe epilepsy.

    PubMed

    Soukupová, Marie; Binaschi, Anna; Falcicchia, Chiara; Zucchini, Silvia; Roncon, Paolo; Palma, Eleonora; Magri, Eros; Grandi, Enrico; Simonato, Michele

    2014-07-01

    The alterations in GABA release have not yet been systematically measured along the natural course of temporal lobe epilepsy. In this work, we analyzed GABA extracellular concentrations (using in vivo microdialysis under basal and high K(+)-evoked conditions) and loss of two GABA interneuron populations (parvalbumin and somatostatin neurons) in the ventral hippocampus at different time-points after pilocarpine-induced status epilepticus in the rat, i.e. during development and progression of epilepsy. We found that (i) during the latent period between the epileptogenic insult, status epilepticus, and the first spontaneous seizure, basal GABA outflow was reduced to about one third of control values while the number of parvalbumin-positive cells was reduced by about 50% and that of somatostatin-positive cells by about 25%; nonetheless, high K(+) stimulation increased extracellular GABA in a proportionally greater manner during latency than under control conditions; (ii) at the time of the first spontaneous seizure (i.e., when the diagnosis of epilepsy is made in humans) this increased responsiveness to stimulation disappeared, i.e. there was no longer any compensation for GABA cell loss; (iii) thereafter, this dysfunction remained constant until a late phase of the disease. These data suggest that a GABAergic hyper-responsiveness can compensate for GABA cell loss and protect from occurrence of seizures during latency, whereas impaired extracellular GABA levels can favor the occurrence of spontaneous recurrent seizures and the maintenance of an epileptic state.

  16. Molecular, pharmacological and functional properties of GABAA receptors in anterior pituitary cells

    PubMed Central

    Zemkova, Hana W; Bjelobaba, Ivana; Tomic, Melanija; Zemkova, Hana; Stojilkovic, Stanko S

    2008-01-01

    Anterior pituitary cells express γ-aminobutyric acid (GABA)-A receptor-channels, but their structure, distribution within the secretory cell types, and nature of action have not been clarified. Here we addressed these questions using cultured anterior pituitary cells from postpubertal female rats and immortalized αT3-1 and GH3 cells. Our results show that mRNAs for all GABAA receptor subunits are expressed in pituitary cells and that α1/β1 subunit proteins are present in all secretory cells. In voltage-clamped gramicidin-perforated cells, GABA induced dose-dependent increases in current amplitude that were inhibited by bicuculline and picrotoxin and facilitated by diazepam and zolpidem in a concentration-dependent manner. In intact cells, GABA and the GABAA receptor agonist muscimol caused a rapid and transient increase in intracellular calcium, whereas the GABAB receptor agonist baclofen was ineffective, suggesting that chloride-mediated depolarization activates voltage-gated calcium channels. Consistent with this finding, RT-PCR analysis indicated high expression of NKCC1, but not KCC2 cation/chloride transporter mRNAs in pituitary cells. Furthermore, the GABAA channel reversal potential for chloride ions was positive to the baseline membrane potential in most cells and the activation of ion channels by GABA resulted in depolarization of cells and modulation of spontaneous electrical activity. These results indicate that secretory pituitary cells express functional GABAA receptor-channels that are depolarizing. PMID:18450776

  17. Functional plasticity of GAT-3 in avian Müller cells is regulated by neurons via a glutamatergic input.

    PubMed

    Schitine, Clarissa S; Mendez-Flores, Orquidia G; Santos, Luis E; Ornelas, Isis; Calaza, Karin C; Pérez-Toledo, Karla; López-Bayghen, Esther; Ortega, Arturo; Gardino, Patrícia F; de Mello, Fernando G; Reis, Ricardo A M

    2015-03-01

    GABA (γ-amino butyric acid) is the major inhibitory transmitter in the central nervous system and its action is terminated by specific transporters (GAT), found in neurons and glial cells. We have previously described that GAT-3 is responsible for GABA uptake activity in cultured avian Müller cells and that it operates in a Na(+) and Cl(-) dependent manner. Here we show that glutamate decreases [(3)H] GABA uptake in purified cultured glial cells up to 50%, without causing cell death. This effect is mediated by ionotropic glutamatergic receptors. Glutamate inhibition on GABA uptake is not reverted by inhibitors of protein kinase C or modified by agents that modulate cyclic AMP/PKA. Biotinylation experiments demonstrate that this reduction in GABA uptake correlates with a decrease in GAT-3 plasma membrane levels. Interestingly, both GAT-1 and GAT-3 mRNA levels are also decreased by glutamate. Conditioned media (CM) prepared from retinal neurons could also decrease GABA influx, and glutamate receptor antagonists (MK-801 + CNQX) were able to prevent this effect. However, glutamate levels in CM were not different from those found in fresh media, indicating that a glutamatergic co-agonist or modulator could be regulating GABA uptake by Müller cells in this scenario. In the whole avian retina, GAT-3 is present from embryonic day 5 (E5) increasing up to the end of embryonic development and post-hatch period exclusively in neuronal layers. However, this pattern may change in pathological conditions, which drive GAT-3 expression in Müller cells. Our data suggest that in purified cultures and upon extensive neuronal lesion in vivo, shown as a Brn3a reduced neuronal cells and an GFAP increased gliosis, Müller glia may change its capacity to take up GABA due to GAT-3 up regulation and suggests a regulatory interplay mediated by glutamate between neurons and glial cells in this process.

  18. In the developing rat hippocampus a tonic GABAA-mediated conductance selectively enhances the glutamatergic drive of principal cells

    PubMed Central

    Marchionni, Ivan; Omrani, Azar; Cherubini, Enrico

    2007-01-01

    In the adult hippocampus, two different forms of GABAA receptor-mediated inhibition have been identified: phasic and tonic. The first is due to the activation of GABAA receptors facing the presynaptic releasing sites, whereas the second is due to the activation of receptors localized away from the synapses. Because of their high affinity and low desensitization rate, extrasynaptic receptors are persistently able to sense low concentrations of GABA. Here we show that, early in postnatal life, between postnatal day (P) 2 and P6, CA1 and CA3 pyramidal cells but not stratum radiatum interneurons, express a tonic GABAA-mediated conductance. Block of the neuronal GABA transporter GAT-1 slightly enhanced the persistent GABA conductance in principal cells but not in GABAergic interneurons. However, in adulthood, a tonic GABAA-mediated conductance could be revealed in stratum radiatum interneurons, indicating that the ability of these cells to sense ambient GABA levels is developmentally regulated. Pharmacological analysis of the tonic conductance in principal cells demonstrated the involvement of β2/β3, α5 and γ2 GABAA receptor subunits. Removal of the tonic depolarizing action of GABA with picrotoxin, reduced the excitability and the glutamatergic drive of principal cells but did not modify the excitability of stratum radiatum interneurons. The increased cell excitability and synaptic activity following the activation of extrasynaptic GABAA receptors by ambient GABA would facilitate the induction of giant depolarizing potentials. PMID:17317750

  19. Gene expression profiles in irradiated cancer cells

    NASA Astrophysics Data System (ADS)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C.

    2013-07-01

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  20. Gene expression profiles in irradiated cancer cells

    SciTech Connect

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C.

    2013-07-26

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  1. Early life stress is a risk factor for excessive alcohol drinking and impulsivity in adults and is mediated via a CRF/GABA(A) mechanism.

    PubMed

    Gondré-Lewis, Marjorie C; Warnock, Kaitlin T; Wang, Hong; June, Harry L; Bell, Kimberly A; Rabe, Holger; Tiruveedhula, Veera Venkata Naga Phani Babu; Cook, James; Lüddens, Hartmut; Aurelian, Laure; June, Harry L

    2016-01-01

    Childhood stress and trauma are associated with substance use disorders in adulthood, but the neurological changes that confer increased vulnerability are largely unknown. In this study, maternal separation (MS) stress, restricted to the pre-weaning period, was used as a model to study mechanisms of protracted effects of childhood stress/traumatic experiences on binge drinking and impulsivity. Using an operant self-administration model of binge drinking and a delay discounting assay to measure impulsive-like behavior, we report that early life stress due to MS facilitated acquisition of binge drinking and impulsivity during adulthood in rats. Previous studies have shown heightened levels of corticotropin releasing factor (CRF) after MS, and here, we add that MS increased expression levels of GABA(A) α2 subunit in central stress circuits. To investigate the precise role of these circuits in regulating impulsivity and binge drinking, the CRF1 receptor antagonist antalarmin and the novel GABA(A) α2 subunit ligand 3-PBC were infused into the central amygdala (CeA) and medial prefrontal cortex (mPFC). Antalarmin and 3-PBC at each site markedly reduced impulsivity and produced profound reductions on binge-motivated alcohol drinking, without altering responding for sucrose. Furthermore, whole-cell patch-clamp studies showed that low concentrations of 3-PBC directly reversed the effect of relatively high concentrations of ethanol on α2β3γ2 GABA(A) receptors, by a benzodiazepine site-independent mechanism. Together, our data provide strong evidence that maternal separation, i.e. early life stress, is a risk factor for binge drinking, and is linked to impulsivity, another key risk factor for excessive alcohol drinking. We further show that pharmacological manipulation of CRF and GABA receptor signaling is effective to reverse binge drinking and impulsive-like behavior in MS rats. These results provide novel insights into the role of the brain stress systems in the

  2. Early life stress is a risk factor for excessive alcohol drinking and impulsivity in adults and is mediated via a CRF/GABA(A) mechanism.

    PubMed

    Gondré-Lewis, Marjorie C; Warnock, Kaitlin T; Wang, Hong; June, Harry L; Bell, Kimberly A; Rabe, Holger; Tiruveedhula, Veera Venkata Naga Phani Babu; Cook, James; Lüddens, Hartmut; Aurelian, Laure; June, Harry L

    2016-01-01

    Childhood stress and trauma are associated with substance use disorders in adulthood, but the neurological changes that confer increased vulnerability are largely unknown. In this study, maternal separation (MS) stress, restricted to the pre-weaning period, was used as a model to study mechanisms of protracted effects of childhood stress/traumatic experiences on binge drinking and impulsivity. Using an operant self-administration model of binge drinking and a delay discounting assay to measure impulsive-like behavior, we report that early life stress due to MS facilitated acquisition of binge drinking and impulsivity during adulthood in rats. Previous studies have shown heightened levels of corticotropin releasing factor (CRF) after MS, and here, we add that MS increased expression levels of GABA(A) α2 subunit in central stress circuits. To investigate the precise role of these circuits in regulating impulsivity and binge drinking, the CRF1 receptor antagonist antalarmin and the novel GABA(A) α2 subunit ligand 3-PBC were infused into the central amygdala (CeA) and medial prefrontal cortex (mPFC). Antalarmin and 3-PBC at each site markedly reduced impulsivity and produced profound reductions on binge-motivated alcohol drinking, without altering responding for sucrose. Furthermore, whole-cell patch-clamp studies showed that low concentrations of 3-PBC directly reversed the effect of relatively high concentrations of ethanol on α2β3γ2 GABA(A) receptors, by a benzodiazepine site-independent mechanism. Together, our data provide strong evidence that maternal separation, i.e. early life stress, is a risk factor for binge drinking, and is linked to impulsivity, another key risk factor for excessive alcohol drinking. We further show that pharmacological manipulation of CRF and GABA receptor signaling is effective to reverse binge drinking and impulsive-like behavior in MS rats. These results provide novel insights into the role of the brain stress systems in the

  3. Presynaptic GluN2D receptors detect glutamate spillover and regulate cerebellar GABA release.

    PubMed

    Dubois, Christophe J; Lachamp, Philippe M; Sun, Lu; Mishina, Masayoshi; Liu, Siqiong June

    2016-01-01

    Glutamate directly activates N-methyl-d-aspartate (NMDA) receptors on presynaptic inhibitory interneurons and enhances GABA release, altering the excitatory-inhibitory balance within a neuronal circuit. However, which class of NMDA receptors is involved in the detection of glutamate spillover is not known. GluN2D subunit-containing NMDA receptors are ideal candidates as they exhibit a high affinity for glutamate. We now show that cerebellar stellate cells express both GluN2B and GluN2D NMDA receptor subunits. Genetic deletion of GluN2D subunits prevented a physiologically relevant, stimulation-induced, lasting increase in GABA release from stellate cells [long-term potentiation of inhibitory transmission (I-LTP)]. NMDA receptors are tetramers composed of two GluN1 subunits associated to either two identical subunits (di-heteromeric receptors) or to two different subunits (tri-heteromeric receptors). To determine whether tri-heteromeric GluN2B/2D NMDA receptors mediate I-LTP, we tested the prediction that deletion of GluN2D converts tri-heteromeric GluN2B/2D to di-heteromeric GluN2B NMDA receptors. We find that prolonged stimulation rescued I-LTP in GluN2D knockout mice, and this was abolished by GluN2B receptor blockers that failed to prevent I-LTP in wild-type mice. Therefore, NMDA receptors that contain both GluN2D and GluN2B mediate the induction of I-LTP. Because these receptors are not present in the soma and dendrites, presynaptic tri-heteromeric GluN2B/2D NMDA receptors in inhibitory interneurons are likely to mediate the cross talk between excitatory and inhibitory transmission.

  4. Interleukin-15 receptor is essential to facilitate GABA transmission and hippocampal dependent memory

    PubMed Central

    He, Yi; Wu, Xiaojun; Hsuchou, Hung; Kastin, Abba J.; Khan, Reas S.; Pistell, Paul J.; Wang, Wei-Hsung; Feng, Jiming; Li, Zengbiao; Guo, Xiaochuan; Pan, Weihong

    2010-01-01

    Interleukin-15 (IL15) is a cytokine produced by normal brain, but the functions of the IL15 system in normal adults are not yet clear. The hypothesis that the hippocampal IL15 system is essential for memory consolidation was tested by use of IL15Rα knockout mice in behavioral, biochemical, immunohistological, and electron microscopic analyses. The knockout mice showed deficits in memory, determined by the Stone T-maze and fear conditioning. In their hippocampi, the concentration of γ-aminobutyric acid (GABA) was significantly lower. There were region specific changes of the GABA synthesizing enzyme glutamic acid decarboxidase (GAD), with increased GAD-67 immunopositive interneurons in the stratum oriens of the CA1 region of the hippocampus, accompanied by non-significant reduction of GAD-67 synapses in the CA3 region. Western blotting showed an increase of GAD-65, but not GAD-67, in the hippocampal homogenate. The ultrastructure of the hippocampus remained intact in the knockout mice. To further test the hypothesis that IL15 directly modulates GABA turnover by reuptake mechanisms, the dose-response relationship of IL15 on 3H-GABA uptake was determined in two neuronal cell lines. The effective and non-toxic dose was further used in the synaptosomal uptake studies. IL15 decreased the uptake of 3H-GABA in synaptosomes from the forebrain of wildtype mice. Consistent with this, IL15Rα knockout mice had increased synaptosomal uptake of 3H-GABA. Overall, the results show novel functions of a unique cytokine in normal hippocampal activity by regulating GABA transmission. PMID:20357123

  5. Adult human bone marrow stromal spheres express neuronal traits in vitro and in a rat model of Parkinson's disease

    PubMed Central

    Suon, Sokreine; Yang, Ming; Iacovitti, Lorraine

    2007-01-01

    Adult human bone marrow stromal cells (hMSCs) grown in suspension culture gave rise to spheres of neural progenitor (NP) cells, capable of expressing both dopaminergic (DA) and GABAergic (GABA) traits. After transplantation into the Parkinsonian rat, human NPs and neurons were present at 2 weeks. Although no DA neurons appeared to survive transplantation, there were abundant GABA neurons present in the graft. By 4 weeks, however, all cells had died. Finding ways to prolong survival and promote the appropriate neurotransmitter phenotype is essential if hMSCs are to be clinically useful. PMID:16828720

  6. Inhibition of GABA synthesis in the prefrontal cortex increases locomotor activity but does not affect attention in the 5-choice serial reaction time task.

    PubMed

    Asinof, Samuel K; Paine, Tracie A

    2013-02-01

    Attention deficits are a core cognitive symptom of schizophrenia; the neuropathology underlying these deficits is not known. Attention is regulated, at least in part, by the prefrontal cortex (PFC), a brain area in which pathology of γ-aminobutyric acid (GABA) neurons has been consistently observed in post-mortem analysis of the brains of people with schizophrenia. Specifically, expression of the 67-kD isoform of the GABA synthesis enzyme glutamic acid decarboxylase (GAD67) is reduced in parvalbumin-containing fast-spiking GABA interneurons. Thus it is hypothesized that reduced cortical GABA synthesis and release may contribute to the attention deficits in schizophrenia. Here the effect of reducing cortical GABA synthesis with l-allylglycine (LAG) on attention was tested using three different versions of the 5-choice serial reaction time task (5CSRTT). Because 5CSRTT performance can be affected by locomotor activity, we also measured this behavior in an open field. Finally, the expression of Fos protein was used as an indirect measure of reduced GABA synthesis. Intra-cortical LAG (10 μg/0.5 μl/side) infusions increased Fos expression and resulted in hyperactivity in the open field. Intra-cortical LAG infusions did not affect attention in any version of the 5CSRTT. These results suggest that a general decrease in GABA synthesis is not sufficient to cause attention deficits. It remains to be tested whether a selective decrease in GABA synthesis in parvalbumin-containing GABA neurons could cause attention deficits. Decreased cortical GABA synthesis did increase locomotor activity; this may reflect the positive symptoms of schizophrenia.

  7. Glutamate and GABA imbalance following traumatic brain injury.

    PubMed

    Guerriero, Réjean M; Giza, Christopher C; Rotenberg, Alexander

    2015-05-01

    Traumatic brain injury (TBI) leads to multiple short- and long-term changes in neuronal circuits that ultimately conclude with an imbalance of cortical excitation and inhibition. Changes in neurotransmitter concentrations, receptor populations, and specific cell survival are important contributing factors. Many of these changes occur gradually, which may explain the vulnerability of the brain to multiple mild impacts, alterations in neuroplasticity, and delays in the presentation of posttraumatic epilepsy. In this review, we provide an overview of normal glutamate and GABA homeostasis and describe acute, subacute, and chronic changes that follow injury. We conclude by highlighting opportunities for therapeutic interventions in this paradigm. PMID:25796572

  8. Glutamate and GABA imbalance following traumatic brain injury

    PubMed Central

    Guerriero, Réjean M.; Giza, Christopher C.; Rotenberg, Alexander

    2015-01-01

    Traumatic brain injury (TBI) leads to multiple short and long term changes in neuronal circuits that ultimately conclude with an imbalance of cortical excitation and inhibition. Changes in neurotransmitter concentrations, receptor populations and specific cell survival are important contributing factors. Many of these changes occur gradually, which may explain the vulnerability of the brain to multiple mild impacts, alterations in neuroplasticity, and delays in the presentation of post-traumatic epilepsy. In this review we provide an overview of normal glutamate and GABA homeostasis, and describe acute, subacute and chronic changes that follow injury. We conclude by highlighting opportunities for therapeutic interventions in this paradigm. PMID:25796572

  9. Gene Expression by Mouse Inner Ear Hair Cells during Development

    PubMed Central

    Scheffer, Déborah I.; Shen, Jun

    2015-01-01

    Hair cells of the inner ear are essential for hearing and balance. As a consequence, pathogenic variants in genes specifically expressed in hair cells often cause hereditary deafness. Hair cells are few in number and not easily isolated from the adjacent supporting cells, so the biochemistry and molecular biology of hair cells can be difficult to study. To study gene expression in hair cells, we developed a protocol for hair cell isolation by FACS. With nearly pure hair cells and surrounding cells, from cochlea and utricle and from E16 to P7, we performed a comprehensive cell type-specific RNA-Seq study of gene expression during mouse inner ear development. Expression profiling revealed new hair cell genes with distinct expression patterns: some are specific for vestibular hair cells, others for cochlear hair cells, and some are expressed just before or after maturation of mechanosensitivity. We found that many of the known hereditary deafness genes are much more highly expressed in hair cells than surrounding cells, suggesting that genes preferentially expressed in hair cells are good candidates for unknown deafness genes. PMID:25904789

  10. T cells stimulate catabolic gene expression by the stromal cells from giant cell tumor of bone

    SciTech Connect

    Cowan, Robert W.; Ghert, Michelle; Singh, Gurmit

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Two T cell lines stimulate PTHrP, RANKL, MMP13 gene expression in GCT cell cultures. Black-Right-Pointing-Pointer CD40 expressed by stromal cells; CD40L detected in whole tumor but not cultures. Black-Right-Pointing-Pointer Effect of CD40L treatment on GCT cells increased PTHrP and MMP13 gene expression. Black-Right-Pointing-Pointer PTHrP treatment increased MMP13 expression, while inhibition decreased expression. Black-Right-Pointing-Pointer T cells may stimulate GCT stromal cells and promote the osteolysis of the tumor. -- Abstract: The factors that promote the localized bone resorption by giant cell tumor of bone (GCT) are not fully understood. We investigated whether T cells could contribute to bone resorption by stimulating expression of genes for parathyroid hormone-related protein (PTHrP), matrix metalloproteinase (MMP)-13, and the receptor activator of nuclear-factor {kappa}B ligand (RANKL). Two cell lines, Jurkat clone E6-1 and D1.1, were co-cultured with isolated GCT stromal cells. Real-time PCR analyses demonstrated a significant increase of all three genes following 48 h incubation, and PTHrP and MMP-13 gene expression was also increased at 24 h. Further, we examined the expression of CD40 ligand (CD40L), a protein expressed by activated T cells, and its receptor, CD40, in GCT. Immunohistochemistry results revealed expression of the CD40 receptor in both the stromal cells and giant cells of the tumor. RNA collected from whole GCT tissues showed expression of CD40LG, which was absent in cultured stromal cells, and suggests that CD40L is expressed within GCT. Stimulation of GCT stromal cells with CD40L significantly increased expression of the PTHrP and MMP-13 genes. Moreover, we show that inhibition of PTHrP with neutralizing antibodies significantly decreased MMP13 expression by the stromal cells compared to IgG-matched controls, whereas stimulation with PTHrP (1-34) increased MMP-13 gene expression. These

  11. Imbalanced expression of glutamate-glutamine cycle enzymes induced by human T-cell lymphotropic virus type 1 Tax protein in cultivated astrocytes.

    PubMed Central

    Akaoka, H; Hardin-Pouzet, H; Bernard, A; Verrier, B; Belin, M F; Giraudon, P

    1996-01-01

    Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent involved in the disease HTLV-1-associated myelopathy, or tropical spastic paraparesis (HAM/TSP). The pathogenesis of HAM/TSP is poorly understood, but it is probable that viral infection has an indirect, deleterious effect on neural function. In this regard, dysfunction in astrocytes may be severely detrimental, as they supply neurons with metabolic precursors, control the extracellular levels of ion and excitatory neurotransmitters, and are electrically coupled with oligodendrocytes. In a model in vitro, we demonstrate that HTLV-1 induces an imbalance in the expression of two astrocyte enzymes, at both the transcriptional and translational levels. In both human astrocyte precursors and rat glial cells, the levels of expression of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were increased and decreased, respectively, after coculture with HTLV-1 T cells. The enhancement of GS expression may result from the action of the protein Tax, which is demonstrated to transactivate the GS gene promoter, while the decreased expression of GDH seems to reflect some compensatory mechanism in response to GS induction. GS and GDH are involved in the conversion of glutamate into glutamine or alpha-ketoglutarate, which then acts as a precursor for glutamatergic and gamma-aminobutyric acid (GABA)-ergic neurons. Metabolism in astrocytes altered by Tax protein may lead to deleterious effects if it modifies the extracellular levels of glutamine, glutamate, and GABA and thus modulates neuronal excitability and osmotic equilibrium in the central nervous system of HTLV-1-infected patients. PMID:8971000

  12. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes.

    PubMed

    Kang, Hoin; Park, Jong Im; Roh, Sangho

    2016-01-01

    In vivo-matured mouse oocytes were enucleated, and a single murine embryonic fibroblast (control or reprogrammed by introducing extracts from murine testis tissue, which showed expression of male germ cell-specific genes) was injected into the cytoplasm of the oocytes. The rate of blastocyst development and expression levels of Oct-4, Eomes and Cdx-2 were not significantly different in both experimental groups. However, the expression levels of Nanog, Sox9 and Glut-1 were significantly increased when reprogrammed cells were used as donor nuclei. Increased expression of Nanog can be supportive of complete reprogramming of somatic cell nuclear transfer murine embryos. The present study suggested that donor cells expressing male germ cell-specific genes can be reconstructed and can develop into embryos with normal high expression of developmentally essential genes. PMID:26369430

  13. Rapid, activity-independent turnover of vesicular transmitter content at a mixed glycine/GABA synapse.

    PubMed

    Apostolides, Pierre F; Trussell, Laurence O

    2013-03-13

    The release of neurotransmitter via the fusion of transmitter-filled, presynaptic vesicles is the primary means by which neurons relay information. However, little is known regarding the molecular mechanisms that supply neurotransmitter destined for vesicle filling, the endogenous transmitter concentrations inside presynaptic nerve terminals, or the dynamics of vesicle refilling after exocytosis. We addressed these issues by recording from synaptically coupled pairs of glycine/GABA coreleasing interneurons (cartwheel cells) of the mouse dorsal cochlear nucleus. We find that the plasma membrane transporter GlyT2 and the intracellular enzyme glutamate decarboxylase supply the majority of glycine and GABA, respectively. Pharmacological block of GlyT2 or glutamate decarboxylase led to rapid and complete rundown of transmission, whereas increasing GABA synthesis via intracellular glutamate uncaging dramatically potentiated GABA release within 1 min. These effects were surprisingly independent of exocytosis, indicating that prefilled vesicles re-equilibrated upon acute changes in cytosolic transmitter. Titration of cytosolic transmitter with postsynaptic responses indicated that endogenous, nonvesicular glycine/GABA levels in nerve terminals are 5-7 mm, and that vesicular transport mechanisms are not saturated under basal conditions. Thus, cytosolic transmitter levels dynamically set the strength of inhibitory synapses in a release-independent manner. PMID:23486948

  14. Glutamate and GABA in Vestibulo-Sympathetic Pathway Neurons

    PubMed Central

    Holstein, Gay R.; Friedrich, Victor L. Jr.; Martinelli, Giorgio P.

    2016-01-01

    The vestibulo-sympathetic reflex (VSR) actively modulates blood pressure during changes in posture. This reflex allows humans to stand up and quadrupeds to rear or climb without a precipitous decline in cerebral perfusion. The VSR pathway conveys signals from the vestibular end organs to the caudal vestibular nuclei. These cells, in turn, project to pre-sympathetic neurons in the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively). The present study assessed glutamate- and GABA-related immunofluorescence associated with central vestibular neurons of the VSR pathway in rats. Retrograde FluoroGold tract tracing was used to label vestibular neurons with projections to RVLM or CVLM, and sinusoidal galvanic vestibular stimulation (GVS) was employed to activate these pathways. Central vestibular neurons of the VSR were identified by co-localization of FluoroGold and cFos protein, which accumulates in some vestibular neurons following galvanic stimulation. Triple-label immunofluorescence was used to co-localize glutamate- or GABA- labeling in the identified VSR pathway neurons. Most activated projection neurons displayed intense glutamate immunofluorescence, suggestive of glutamatergic neurotransmission. To support this, anterograde tracer was injected into the caudal vestibular nuclei. Vestibular axons and terminals in RVLM and CVLM co-localized the anterograde tracer and vesicular glutamate transporter-2 signals. Other retrogradely-labeled cFos-positive neurons displayed intense GABA immunofluorescence. VSR pathway neurons of both phenotypes were present in the caudal medial and spinal vestibular nuclei, and projected to both RVLM and CVLM. As a group, however, triple-labeled vestibular cells with intense glutamate immunofluorescence were located more rostrally in the vestibular nuclei than the GABAergic neurons. Only the GABAergic VSR pathway neurons showed a target preference, projecting predominantly to CVLM. These data provide the first

  15. Glutamate and GABA in Vestibulo-Sympathetic Pathway Neurons.

    PubMed

    Holstein, Gay R; Friedrich, Victor L; Martinelli, Giorgio P

    2016-01-01

    The vestibulo-sympathetic reflex (VSR) actively modulates blood pressure during changes in posture. This reflex allows humans to stand up and quadrupeds to rear or climb without a precipitous decline in cerebral perfusion. The VSR pathway conveys signals from the vestibular end organs to the caudal vestibular nuclei. These cells, in turn, project to pre-sympathetic neurons in the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively). The present study assessed glutamate- and GABA-related immunofluorescence associated with central vestibular neurons of the VSR pathway in rats. Retrograde FluoroGold tract tracing was used to label vestibular neurons with projections to RVLM or CVLM, and sinusoidal galvanic vestibular stimulation (GVS) was employed to activate these pathways. Central vestibular neurons of the VSR were identified by co-localization of FluoroGold and cFos protein, which accumulates in some vestibular neurons following galvanic stimulation. Triple-label immunofluorescence was used to co-localize glutamate- or GABA- labeling in the identified VSR pathway neurons. Most activated projection neurons displayed intense glutamate immunofluorescence, suggestive of glutamatergic neurotransmission. To support this, anterograde tracer was injected into the caudal vestibular nuclei. Vestibular axons and terminals in RVLM and CVLM co-localized the anterograde tracer and vesicular glutamate transporter-2 signals. Other retrogradely-labeled cFos-positive neurons displayed intense GABA immunofluorescence. VSR pathway neurons of both phenotypes were present in the caudal medial and spinal vestibular nuclei, and projected to both RVLM and CVLM. As a group, however, triple-labeled vestibular cells with intense glutamate immunofluorescence were located more rostrally in the vestibular nuclei than the GABAergic neurons. Only the GABAergic VSR pathway neurons showed a target preference, projecting predominantly to CVLM. These data provide the first

  16. Effects of ABA and CaCl₂ on GABA accumulation in fava bean germinating under hypoxia-NaCl stress.

    PubMed

    Yang, Runqiang; Hui, Qianru; Gu, Zhenxin

    2016-01-01

    Effects of exogenous abscisic acid (ABA) and CaCl2 on γ-aminobutyric acid (GABA) accumulation of germinated fava bean under hypoxia-NaCl stress were investigated. Exogenous ABA resulted in the enhancement of glutamate decarboxylase (GAD) and diamine oxidase (DAO) activity as well as GABA content in cotyledon and shoot. CaCl2 increased both enzyme activities in shoot and GABA content in cotyledon and shoot. ABA downregulated GAD expression in cotyledon and radicle, while upregulated that in shoot; it also upregulated DAO expression in each organ. CaCl2 upregulated GAD expression in cotyledon, while downregulated that in radicle. However, it upregulated DAO expression in shoot, downregulated that in radicle. ABA inhibitor fluridon and ethylenediaminetetraacetic acid inhibited GAD and DAO activities significantly so that inhibited GABA accumulation through reducing ABA biosynthesis and chelating Ca(2+), respectively. However, they upregulated GAD and DAO expression in varying degrees. These results indicate that ABA and Ca(2+) participate in GABA biosynthesis in fava bean during germination under hypoxia-NaCl stress.

  17. Effects of ABA and CaCl₂ on GABA accumulation in fava bean germinating under hypoxia-NaCl stress.

    PubMed

    Yang, Runqiang; Hui, Qianru; Gu, Zhenxin

    2016-01-01

    Effects of exogenous abscisic acid (ABA) and CaCl2 on γ-aminobutyric acid (GABA) accumulation of germinated fava bean under hypoxia-NaCl stress were investigated. Exogenous ABA resulted in the enhancement of glutamate decarboxylase (GAD) and diamine oxidase (DAO) activity as well as GABA content in cotyledon and shoot. CaCl2 increased both enzyme activities in shoot and GABA content in cotyledon and shoot. ABA downregulated GAD expression in cotyledon and radicle, while upregulated that in shoot; it also upregulated DAO expression in each organ. CaCl2 upregulated GAD expression in cotyledon, while downregulated that in radicle. However, it upregulated DAO expression in shoot, downregulated that in radicle. ABA inhibitor fluridon and ethylenediaminetetraacetic acid inhibited GAD and DAO activities significantly so that inhibited GABA accumulation through reducing ABA biosynthesis and chelating Ca(2+), respectively. However, they upregulated GAD and DAO expression in varying degrees. These results indicate that ABA and Ca(2+) participate in GABA biosynthesis in fava bean during germination under hypoxia-NaCl stress. PMID:26644273

  18. Activation of toll like receptor 4 attenuates GABA synthesis and postsynaptic GABA receptor activities in the spinal dorsal horn via releasing interleukin-1 beta.

    PubMed

    Yan, Xisheng; Jiang, Enshe; Weng, Han-Rong

    2015-01-09

    Toll like receptor 4 (TLR4) is an innate immune pattern recognition receptor, expressed predominantly on microglia in the CNS. Activation of spinal TLR4 plays a critical role in the genesis of pathological pain induced by nerve injury, bone cancer, and tissue inflammation. Currently, it remains unknown how synaptic activities in the spinal dorsal horn are regulated by TLR4 receptors. Through recording GABAergic currents in neurons and glial glutamate transporter currents in astrocytes in rodent spinal slices, we determined whether and how TLR4 modulates GABAergic synaptic activities in the superficial spinal dorsal horn. We found that activation of TLR4 by lipopolysaccharide (LPS) reduces GABAergic synaptic activities through both presynaptic and postsynaptic mechanisms. Specifically, LPS causes the release of IL-1β from microglia. IL-1β in turn suppresses GABA receptor activities at the postsynaptic site through activating protein kinase C (PKC) in neurons. GABA synthesis at the presynaptic site is reduced upon activation of TLR4. Glial glutamate transporter activities are suppressed by IL-1β and PKC activation induced by LPS. The suppression of glial glutamate transporter activities leads to a deficiency of glutamine supply, which results in an attenuation of the glutamate-glutamine cycle-dependent GABA synthesis. These findings shed light on understanding synaptic plasticity induced by activation of TLR4 under neuroinflammation and identify GABA receptors, glial glutamate transporters, IL-1β and PKC as therapeutic targets to abrogate abnormal neuronal activities following activation of TLR4 in pathological pain conditions.

  19. Detection of Reduced GABA Synthesis Following Inhibition of GABA Transaminase Using in Vivo Magnetic Resonance Signal of [13C]GABA C1

    PubMed Central

    Yang, Jehoon; Johnson, Christopher; Shen, Jun

    2009-01-01

    Previous in vivo magnetic resonance spectroscopy (MRS) studies of gamma-aminobutyric acid (GABA) synthesis have relied on 13C label incorporation into GABA C2 from [1-13C] or [1,6-13C2]glucose. In this study, the [13C]GABA C1 signal at 182.3 ppm in the carboxylic/amide spectral region of localized in vivo 13C spectra was detected. GABA-transaminase of rat brain was inhibited by administration of gabaculine after pre-labeling of GABA C1 and its metabolic precursors with exogenous [2,5-13C2]glucose. A subsequent isotope chase experiment was performed by infusing unlabeled glucose, which revealed a markedly slow change in the labeling of GABA C1 accompanying the blockade of the GABA shunt. This slow labeling of GABA at elevated GABA concentration was attributed to the relatively small intercompartmental GABA-glutamine cycling flux that constitutes the main route of 13C label loss during the isotope chase. Because this study showed that using low RF power broadband stochastic proton decoupling is feasible at very high field strength, it has important implications for the development of carboxylic/amide 13C MRS methods to study brain metabolism and neurotransmission in human subjects at high magnetic fields. PMID:19540876

  20. Genes in the GABA Pathway Increase in the Lateral Thalamus of Sprague-Dawley Rats During the Proestrus/Estrus Phase.

    PubMed

    Umorin, Mikhail; Stinson, Crystal; Bellinger, Larry L; Kramer, Phillip R

    2016-05-01

    Pain can vary over the estrous cycle as a result of changes in estradiol concentration but the mechanism causing this variation is unclear. Because the thalamus is important in pain control, gene expression in the lateral thalamus (ventral posteromedial, ventral posterolateral, reticular thalamic nuclei) was screened at different phases of the estrous cycle. Gene expression changes in Sprague-Dawley rats were further analyzed by real-time PCR and ELISA and plasma estradiol levels were measured by RIAs at different phases of the estrous cycle. Our results indicated that both the RNA and protein expression of glutamate decarboxylase 1 and 2 (GAD1, GAD2), GABA(A) receptor-associated protein like 1 (GABARAPL1), and vesicular GABA transporter (VGAT) significantly increased in the lateral thalamus when plasma estradiol levels were elevated. Estradiol levels were elevated during the proestrus and estrus phases of the estrous cycle. Estrogen receptor α (ERα) was observed to be co-localized in thalamic cells and thalamic infusion of an ERα antagonist significantly reduced GAD1 and VGAT transcript. GAD1, GAD2, GABARAPL1, and VGAT have been shown to effect neuronal responses suggesting that attenuation of pain during the estrous cycle can be dependent, in part, through estradiol induced changes in thalamic gene expression. PMID:26388520

  1. Zinc-induced collapse of augmented inhibition by GABA in a temporal lobe epilepsy model.

    PubMed

    Buhl, E H; Otis, T S; Mody, I

    1996-01-19

    In the kindling model of temporal lobe epilepsy, several physiological indicators of inhibition by gamma-aminobutyric acid (GABA) in the hippocampal dentate gyrus are consistent with an augmented, rather than a diminished, inhibition. In brain slices obtained from epileptic (kindled) rats, the excitatory drive onto inhibitory interneurons was increased and was paralleled by a reduction in the presynaptic autoinhibition of GABA release. This augmented inhibition was sensitive to zinc most likely after a molecular reorganization of GABAA receptor subunits. Consequently, during seizures, inhibition by GABA may be diminished by the zinc released from aberrantly sprouted mossy fiber terminals of granule cells, which are found in many experimental models of epilepsy and in human temporal lobe epilepsy.

  2. GABA-mediated repulsive coupling between circadian clock neurons in the SCN encodes seasonal time

    PubMed Central

    Myung, Jihwan; Hong, Sungho; DeWoskin, Daniel; De Schutter, Erik; Forger, Daniel B.; Takumi, Toru

    2015-01-01

    The mammalian suprachiasmatic nucleus (SCN) forms not only the master circadian clock but also a seasonal clock. This neural network of ∼10,000 circadian oscillators encodes season-dependent day-length changes through a largely unknown mechanism. We show that region-intrinsic changes in the SCN fine-tune the degree of network synchrony and reorganize the phase relationship among circadian oscillators to represent day length. We measure oscillations of the clock gene Bmal1, at single-cell and regional levels in cultured SCN explanted from animals raised under short or long days. Coupling estimation using the Kuramoto framework reveals that the network has couplings that can be both phase-attractive (synchronizing) and -repulsive (desynchronizing). The phase gap between the dorsal and ventral regions increases and the overall period of the SCN shortens with longer day length. We find that one of the underlying physiological mechanisms is the modulation of the intracellular chloride concentration, which can adjust the strength and polarity of the ionotropic GABAA-mediated synaptic input. We show that increasing day-length changes the pattern of chloride transporter expression, yielding more excitatory GABA synaptic input, and that blocking GABAA signaling or the chloride transporter disrupts the unique phase and period organization induced by the day length. We test the consequences of this tunable GABA coupling in the context of excitation–inhibition balance through detailed realistic modeling. These results indicate that the network encoding of seasonal time is controlled by modulation of intracellular chloride, which determines the phase relationship among and period difference between the dorsal and ventral SCN. PMID:26130804

  3. GABA-mediated repulsive coupling between circadian clock neurons in the SCN encodes seasonal time.

    PubMed

    Myung, Jihwan; Hong, Sungho; DeWoskin, Daniel; De Schutter, Erik; Forger, Daniel B; Takumi, Toru

    2015-07-21

    The mammalian suprachiasmatic nucleus (SCN) forms not only the master circadian clock but also a seasonal clock. This neural network of ∼10,000 circadian oscillators encodes season-dependent day-length changes through a largely unknown mechanism. We show that region-intrinsic changes in the SCN fine-tune the degree of network synchrony and reorganize the phase relationship among circadian oscillators to represent day length. We measure oscillations of the clock gene Bmal1, at single-cell and regional levels in cultured SCN explanted from animals raised under short or long days. Coupling estimation using the Kuramoto framework reveals that the network has couplings that can be both phase-attractive (synchronizing) and -repulsive (desynchronizing). The phase gap between the dorsal and ventral regions increases and the overall period of the SCN shortens with longer day length. We find that one of the underlying physiological mechanisms is the modulation of the intracellular chloride concentration, which can adjust the strength and polarity of the ionotropic GABAA-mediated synaptic input. We show that increasing day-length changes the pattern of chloride transporter expression, yielding more excitatory GABA synaptic input, and that blocking GABAA signaling or the chloride transporter disrupts the unique phase and period organization induced by the day length. We test the consequences of this tunable GABA coupling in the context of excitation-inhibition balance through detailed realistic modeling. These results indicate that the network encoding of seasonal time is controlled by modulation of intracellular chloride, which determines the phase relationship among and period difference between the dorsal and ventral SCN.

  4. Probing cell-free gene expression noise in femtoliter volumes.

    PubMed

    Karig, David K; Jung, Seung-Yong; Srijanto, Bernadeta; Collier, C Patrick; Simpson, Michael L

    2013-09-20

    Cell-free systems offer a simplified and flexible context that enables important biological reactions while removing complicating factors such as fitness, division, and mutation that are associated with living cells. However, cell-free expression in unconfined spaces is missing important elements of expression in living cells. In particular, the small volume of living cells can give rise to significant stochastic effects, which are negligible in bulk cell-free reactions. Here, we confine cell-free gene expression reactions to cell-relevant 20 fL volumes (between the volumes of Escherichia coli and Saccharomyces cerevisiae ), in polydimethylsiloxane (PDMS) containers. We demonstrate that expression efficiency varies widely among different containers, likely due to non-Poisson distribution of expression machinery at the observed scale. Previously, this phenomenon has been observed only in liposomes. In addition, we analyze gene expression noise. This analysis is facilitated by our use of cell-free systems, which allow the mapping of the measured noise properties to intrinsic noise models. In contrast, previous live cell noise analysis efforts have been complicated by multiple noise sources. Noise analysis reveals signatures of translational bursting, while noise dynamics suggest that overall cell-free expression is limited by a diminishing translation rate. In addition to offering a unique approach to understanding noise in gene circuits, our work contributes to a deeper understanding of the biophysical properties of cell-free expression systems, thus aiding efforts to harness cell-free systems for synthetic biology applications.

  5. Expression of bone morphogenetic proteins of human neoplastic epithelial cells.

    PubMed

    Hatakeyama, S; Gao, Y H; Ohara-Nemoto, Y; Kataoka, H; Satoh, M

    1997-07-01

    Bone morphogenetic proteins (BMPs) are crucial factors of osteogenesis. We investigated the expressions of BMP subtypes in human salivary adenocarcinoma cell line (HSG-S8), tongue squamous cell (HSC-4) and gingival squamous cell (Ca9-22) carcinoma cell lines, gastric poorly differentiated adenocarcinoma cell (MNK45) and signet ring cell (KATOIII) carcinoma cell lines, rectal adenocarcinoma (RCM-1, RCM-2, and RCM-3), and thyroid (8505C) and bladder (T24) carcinoma cell lines by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR disclosed that BMP-1 was expressed in all cell lines examined, and BMP-2 was amplified in almost all cells except MKN45. Two squamous cell carcinomas, HSC-4 and Ca9-22, and KATOIII expressed only BMP-1 and BMP-2. MKN45 did not express BMP-2, but expressed BMP-7 and weakly BMP-4 and BMP-5. In addition to the expression BMP-7, and HSG-S8 expressed BMP-6. These findings indicated that the neoplastic epithelial cells possessed a rather great potency to express BMP mRNAs. On the other hand, among these carcinoma cells, HSG-S8 solely induced bone in nude mouse tumors, and HSC-4 and KATOIII contained many calcified masses in tumors while the rest did not induce either. PMID:9247707

  6. Plasmalemmal and Vesicular γ-Aminobutyric Acid Transporter Expression in the Developing Mouse Retina

    PubMed Central

    GUO, CHENYING; STELLA, SALVATORE L.; HIRANO, ARLENE A.; BRECHA, NICHOLAS C.

    2009-01-01

    Plasmalemmal and vesicular γ-aminobutyric acid (GABA) transporters influence neurotransmission by regulating high-affinity GABA uptake and GABA release into the synaptic cleft and extracellular space. Postnatal expression of the plasmalemmal GABA transporter-1 (GAT-1), GAT-3, and the vesicular GABA/glycine transporter (VGAT) were evaluated in the developing mouse retina by using immunohistochemistry with affinity-purified antibodies. Weak transporter immunoreactivity was observed in the inner retina at postnatal day 0 (P0). GAT-1 immunostaining at P0 and at older ages was in amacrine and displaced amacrine cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), respectively, and in their processes in the inner plexiform layer (IPL). At P10, weak GAT-1 immunostaining was in Müller cell processes. GAT-3 immunostaining at P0 and older ages was in amacrine cells and their processes, as well as in Müller cells and their processes that extended radially across the retina. At P10, Müller cell somata were observed in the middle of the INL. VGAT immunostaining was present at P0 and older ages in amacrine cells in the INL as well as processes in the IPL. At P5, weak VGAT immunostaining was also observed in horizontal cell somata and processes. By P15, the GAT and VGAT immunostaining patterns appear similar to the adult immunostaining patterns; they reached adult levels by about P20. These findings demonstrate that GABA uptake and release are initially established in the inner retina during the first postnatal week and that these systems subsequently mature in the outer retina during the second postnatal week. PMID:18975268

  7. Effect of two GABA-ergic drugs on the cognitive functions of rapid eye movement in sleep-deprived and recovered rats

    PubMed Central

    Bao, Lidao; Si, Lengge; Wang, Yuehong; Wuyun, Gerile; Bo, Agula

    2016-01-01

    Rapid eye movement (REM) sleep is closely associated with nervous functions. The present study aimed to evaluate the effects of gabazine and tiagabine on the cognitive functions (CF) of REM sleep-deprived and sleep recovered rats. Rats were divided into REM sleep deprivation, blank control (CC) and environmental groups. The REM sleep deprivation group was further divided into non-operation (nonOP), sham-operated (Sham), gabazine (SR) and tiagabine groups. Each group was evaluated over five time points: Sleep deprived for 1 day (SD 1 day), SD 3 day, SD 5 day, sleep recovery 6 h (RS 6 h) and RS 12 h. A rat model of REM sleep deprivation was established by a modified multi-platform water method, with CF assessed by Morris water maze. Hypothalamic γ-aminobutyric acid (GABA) and glutamic acid contents were measured via high performance liquid chromatography. The number and morphology of hypocretin (Hcrt) neurons and Fos in the hypothalamus, and GABAARα1-induced integral optical density were detected by immunofluorescence. Compared to the CC group, the nonOP and Sham group rats CF were significantly diminished, Fos-positive and Fos-Hcrt double positive cells were significantly increased, and GABA content and GABAARα1 expression levels were significantly elevated (P<0.05). The tiagabine and CC groups exhibited similar results at three time points. The CF of rats in the SR group were diminished and the number of Fos-positive and Fos-Hcrt double positive cells were significantly increased (P<0.05) at RS 6 h and RS l2 h. GABA content and GABAARα1 expression levels were significantly increased in the SR group at all time points (P<0.05), whereas only GABAARα1 expression levels were significantly increased in the tiagabine group at SD 5 day (P<0.05). The results of the present study indicated that REM sleep deprivation diminished CF, increased the number of Hcrt neurons, GABA content and GABAARα1 expression. Furthermore, all alterations were positively correlated with

  8. The transmembrane sodium gradient influences ambient GABA concentration by altering the equilibrium of GABA transporters.

    PubMed

    Wu, Yuanming; Wang, Wengang; Richerson, George B

    2006-11-01

    Tonic inhibition is widely believed to be caused solely by "spillover" of GABA that escapes the synaptic cleft and activates extrasynaptic GABA(A) receptors. However, an exclusively vesicular source is not consistent with the observation that tonic inhibition can still occur after blocking vesicular release. Here, we made patch-clamp recordings from neurons in rat hippocampal cultures and measured the tonic current that was blocked by bicuculline or gabazine. During perforated patch recordings, the tonic GABA current was decreased by the GAT1 antagonist SKF-89976a. Zero calcium solution did not change the amount of tonic current, despite a large reduction in vesicular GABA release. Perturbations that would be expected to alter the transmembrane sodium gradient influenced the tonic current. For example, in zero calcium Ringer, TTX (which can decrease cytosolic [Na(+)]) reduced tonic current, whereas veratridine (which can increase cytosolic [Na(+)]) increased tonic current. Likewise, removal of extracellular sodium led to a large increase in tonic current. The increases in tonic current induced by veratridine and sodium removal were completely blocked by SKF89976a. When these experiments were repeated in hippocampal slices, similar results were obtained except that a GAT1- and GAT3-independent nonvesicular source(s) of GABA was found to contribute to the tonic current. We conclude that multiple sources can contribute to ambient GABA, including spillover and GAT1 reversal. The source of GABA release may be conceptually less important in determining the amount of tonic inhibition than the factors that control the equilibrium of GABA transporters.

  9. Opioid-Induced GABA potentiation after chronic morphine attenuates the rewarding effects of opioids in the ventral tegmental area.

    PubMed

    Madhavan, Anuradha; Bonci, Antonello; Whistler, Jennifer L

    2010-10-20

    GABA transmission in the ventral tegmental area (VTA) is critical for fine tuning the activity of dopamine neurons in response to opioids. However, the precise mechanism by which GABA input shapes opioid reward is poorly understood. We observed a reduction of conditioned place preference for low doses of the opioid [d-Ala2, N-MePhe4, Gly5-ol]-enkephalin (DAMGO) and a switch in the functional effects of μ-opioid receptor modulation of GABA postsynaptic currents in the mouse VTA 1 d after chronic morphine treatment. Specifically, whereas in naive mice DAMGO inhibits GABA postsynaptic currents, GABAergic currents are potentiated by DAMGO after chronic morphine treatment. Importantly, pretreatment with the cAMP signaling inhibitor (R)-adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium both restored DAMGO reward and reversed the DAMGO-mediated potentiation, thereby reestablishing the inhibitory effects of opioids on GABA currents. Thus, a paradoxical bidirectionality in μ-receptor-mediated control of GABA transmission following chronic morphine treatment is a critical mechanism that determines the expression of opioid reward in the VTA.

  10. Immunocytochemical evidence for SNARE protein-dependent transmitter release from guinea pig horizontal cells

    PubMed Central

    Lee, Helen; Brecha, Nicholas C.

    2013-01-01

    Horizontal cells are lateral interneurons that participate in visual processing in the outer retina but the cellular mechanisms underlying transmitter release from these cells are not fully understood. In non-mammalian horizontal cells, GABA release has been shown to occur by a non-vesicular mechanism. However, recent evidence in mammalian horizontal cells favors a vesicular mechanism as they lack plasmalemmal GABA transporters and some soluble NSF attachment protein receptor (SNARE) core proteins have been identified in rodent horizontal cells. Moreover, immunoreactivity for GABA and the molecular machinery to synthesize GABA have been found in guinea pig horizontal cells, suggesting that if components of the SNARE complex are expressed they could contribute to the vesicular release of GABA. In this study we investigated whether these vesicular and synaptic proteins are expressed by guinea pig horizontal cells using immunohistochemistry with well-characterized antibodies to evaluate their cellular distribution. Components of synaptic vesicles including vesicular GABA transporter, synapsin I and synaptic vesicle protein 2A were localized to horizontal cell processes and endings, along with the SNARE core complex proteins, syntaxin-1a, syntaxin-4 and synaptosomal-associated protein 25 (SNAP-25). Complexin I/II, a cytosolic protein that stabilizes the activated SNARE fusion core, strongly immunostained horizontal cell soma and processes. In addition, the vesicular Ca2+-sensor, synaptotagmin-2, which is essential for Ca2+-mediated vesicular release, was also localized to horizontal cell processes and somata. These morphological findings from guinea pig horizontal cells suggest that mammalian horizontal cells have the capacity to utilize a regulated Ca2+-dependent vesicular pathway to release neurotransmitter, and that this mechanism may be shared among many mammalian species. PMID:20384779

  11. Menthol shares general anesthetic activity and sites of action on the GABA(A) receptor with the intravenous agent, propofol.

    PubMed

    Watt, Erin E; Betts, Brooke A; Kotey, Francesca O; Humbert, Danielle J; Griffith, Theanne N; Kelly, Elizabeth W; Veneskey, Kelley C; Gill, Nikhila; Rowan, Kathleen C; Jenkins, Andrew; Hall, Adam C

    2008-08-20

    Menthol and related compounds were investigated for modulation of recombinant human gamma-aminobutyric acid type A (GABA(A), alpha(1)beta(2)gamma(2s)) receptor currents expressed in Xenopus oocytes. Sub-maximal (EC(20)) GABA currents were typically enhanced by co-applications of 3-300 microM (+)-menthol (e.g. by approximately 2-fold at 50 microM) > isopulegol > isomenthol> alpha-terpineol > cyclohexanol. We studied menthol's actions on GABA(A) receptors compared to sedatives (benzodiazepines) and intravenous anesthetics (barbiturates, steroids, etomidate and propofol). Flumazenil (a benzodiazepine antagonist) did not inhibit menthol enhancements while currents directly activated by 50 microM propofol were significantly inhibited (by 26+/-3%) by 50 microM (+)-menthol. GABA(A) receptors containing beta(2) subunits with either a point mutation in a methionine residue to a tryptophan at the 286 position (in transmembrane domain 3, TM-3) or a tyrosine to a tryptophan at the 444 position (TM-4) are insensitive to modulation by propofol. Enhancements of GABA EC(20) currents by menthol were equally abolished in GABA(A) alpha(1)beta(2)(M286W)gamma(2s) and alpha(1)beta(2)(Y444W)gamma(2s) receptors while positive modulations by benzodiazepines, barbiturates and steroids were unaffected. Menthol may therefore exert its actions on GABA(A) receptors via sites distinct from benzodiazepines, steroids and barbiturates, and via sites important for modulation by propofol. Finally, using an in vivo tadpole assay, addition of (+)-menthol resulted in a loss of righting reflex with an EC(50) of 23.5+/-4.7 microM (approximately10-fold less potent anesthesia than propofol). Thus, menthol and analogs share general anesthetic action with propofol, possibly via action at similar sites on the GABA(A) receptor. PMID:18593637

  12. Hemoglobin enhances tissue factor expression on human malignant cells.

    PubMed

    Siddiqui, F A; Amirkhosravi, A; Amaya, M; Meyer, T; Biggerstaff, J; Desai, H; Francis, J L

    2001-04-01

    Tissue Factor (TF) is a transmembrane glycoprotein that complexes with factor VII/activated factor VII to initiate blood coagulation. TF may be expressed on the surface of various cells including monocytes and endothelial cells. Over-expression of TF in human tumor cell lines promotes metastasis. We recently showed that hemoglobin (Hb) forms a specific complex with TF purified from human malignant melanoma cells and enhances its procoagulant activity (PCA). To further study this interaction, we examined the effect of Hb on the expression of TF on human malignant (TF+) cells and KG1 myeloid leukemia (TF-) cells. Human melanoma A375 and J82 bladder carcinoma cells, which express TF at moderate and relatively high levels, respectively, were incubated with varying concentrations (0-1.5 mg/ml) of Hb. After washing, cells were analyzed for Hb binding and TF expression using flow cytometry and confocal microscopy. Hb bound to the cells in a concentration-dependent manner, and increased both TF expression and PCA. The human A375 malignant melanoma cells incubated with Hb (1 mg/ml) expressed up to six times more TF antigen than cells without Hb. This increase in TF expression and PCA of intact cells incubated with Hb was significantly inhibited by cycloheximide at a concentration of 10 microg/ml (P < 0.01). An increase in total cellular TF antigen content was demonstrated by specific immunoassay. In contrast, Hb (5 mg/ml) did not induce TF expression and PCA on KG1 cells as determined by flow cytometry and TF (FXAA) activity. We conclude that Hb specifically binds to TF-bearing malignant cells and increases their PCA. This effect seems to be at least partly due to de novo synthesis of TF and increased surface expression. However, the exact mechanism by which Hb binds and upregulates TF expression remains to be determined.

  13. Hemoglobin enhances tissue factor expression on human malignant cells.

    PubMed

    Siddiqui, F A; Amirkhosravi, A; Amaya, M; Meyer, T; Biggerstaff, J; Desai, H; Francis, J L

    2001-04-01

    Tissue Factor (TF) is a transmembrane glycoprotein that complexes with factor VII/activated factor VII to initiate blood coagulation. TF may be expressed on the surface of various cells including monocytes and endothelial cells. Over-expression of TF in human tumor cell lines promotes metastasis. We recently showed that hemoglobin (Hb) forms a specific complex with TF purified from human malignant melanoma cells and enhances its procoagulant activity (PCA). To further study this interaction, we examined the effect of Hb on the expression of TF on human malignant (TF+) cells and KG1 myeloid leukemia (TF-) cells. Human melanoma A375 and J82 bladder carcinoma cells, which express TF at moderate and relatively high levels, respectively, were incubated with varying concentrations (0-1.5 mg/ml) of Hb. After washing, cells were analyzed for Hb binding and TF expression using flow cytometry and confocal microscopy. Hb bound to the cells in a concentration-dependent manner, and increased both TF expression and PCA. The human A375 malignant melanoma cells incubated with Hb (1 mg/ml) expressed up to six times more TF antigen than cells without Hb. This increase in TF expression and PCA of intact cells incubated with Hb was significantly inhibited by cycloheximide at a concentration of 10 microg/ml (P < 0.01). An increase in total cellular TF antigen content was demonstrated by specific immunoassay. In contrast, Hb (5 mg/ml) did not induce TF expression and PCA on KG1 cells as determined by flow cytometry and TF (FXAA) activity. We conclude that Hb specifically binds to TF-bearing malignant cells and increases their PCA. This effect seems to be at least partly due to de novo synthesis of TF and increased surface expression. However, the exact mechanism by which Hb binds and upregulates TF expression remains to be determined. PMID:11414630

  14. Electrophysiological characteristics of inhibitory neurons of the prepositus hypoglossi nucleus as analyzed in Venus-expressing transgenic rats.

    PubMed

    Shino, M; Kaneko, R; Yanagawa, Y; Kawaguchi, Y; Saito, Y

    2011-12-01

    The identification and characterization of excitatory and inhibitory neurons are significant steps in understanding neural network functions. In this study, we investigated the intrinsic electrophysiological properties of neurons in the prepositus hypoglossi nucleus (PHN), a brainstem structure that is involved in gaze holding, using whole-cell recordings in brainstem slices from vesicular GABA transporter (VGAT)-Venus transgenic rats, in which inhibitory neurons express the fluorescent protein Venus. To characterize the intrinsic properties of these neurons, we recorded afterhyperpolarization (AHP) profiles and firing patterns from Venus-expressing [Venus⁺] and Venus-non-expressing [Venus⁻] PHN neurons. Although both types of neurons showed a wide variety of AHP profiles and firing patterns, oscillatory firing was specific to Venus⁺ neurons, while a firing pattern showing only a few spikes was specific to Venus⁻ neurons. In addition, AHPs without a slow component and delayed spike generation were preferentially displayed by Venus⁺ neurons, whereas a firing pattern with constant interspike intervals was preferentially displayed by Venus⁻ neurons. We evaluated the mRNAs expression of glutamate decarboxylase (GAD65, GAD67) and glycine transporter 2 (GlyT2) to determine whether the recorded Venus⁺ neurons were GABAergic or glycinergic. Of the 67 Venus⁺ neurons tested, GlyT2 expression alone was detected in only one neuron. Approximately 40% (28/67) expressed GAD65 and/or GAD67 (GABAergic neuron), and the remainder (38/67) expressed both GAD(s) and GlyT2 (GABA&GLY neuron). These results suggest that most inhibitory PHN neurons use either GABA or both GABA and glycine as neurotransmitters. Although the overall distribution of firing patterns in GABAergic neurons was similar to that of GABA&GLY neurons, only GABA&GLY neurons exhibited a firing pattern with a long first interspike interval. These differential electrophysiological properties will be useful

  15. Characteristics of GABA release induced by free radicals in mouse hippocampal slices.

    PubMed

    Saransaari, Pirjo; Oja, Simo S

    2008-03-01

    The release of the inhibitory neurotransmitter GABA is generally enhanced under potentially cell-damaging conditions. The properties and regulation of preloaded [3H]GABA release from mouse hippocampal slices were now studied in free radical-containing medium in a superfusion system. Free radical production was induced by 0.01% of H2O2 in the medium. H2O2 markedly potentiated GABA release, which was further enhanced about 1.5-fold by K+ stimulation (50 mM). In Ca2+-free media this stimulation was not altered, indicating that the release was mostly Ca2+-independent. Moreover, omission of Na+ increased the release, suggesting that it is mediated by Na+-dependent transporters operating outwards, a conception confirmed by the enhancement with GABA homoexchange. Inhibition of the release with the ion channel inhibitors diisothiocyanostilbene-2,2'-disulphonate and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate indicates that Cl(-) channels also participate in the process. This release was not modified by the adenosine receptor (A1 and A2a) agonists and ionotropic glutamate receptor agonists kainate, N-methy-D: -aspartate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, whereas the agonists of metabotropic glutamate receptors of group I [(S)-3,5-dihydroxyphenylglycine] and of group II [(2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate] enhanced it by receptor-mediated mechanisms, the effects being abolished by their respective antagonists. The group III agonist L+-2-amino-4-phosphonobutyrate reduced the evoked GABA release, but this was not affected by the antagonist. Furthermore, the release was reduced by activation of protein kinase C by 4 beta-phorbol 12-myristate 13-acetate and by inhibition of tyrosine kinase by genistein and of phoshoplipase by quinacrine. On the other hand, increasing cGMP levels with the phosphodiesterase inhibitor zaprinast, selective for PDE5, 6 and 9, and NO production with the NO-generating compounds hydroxylamine, sodium nitroprusside

  16. Impact of exogenous GABA treatments on endogenous GABA metabolism in anthurium cut flowers in response to postharvest chilling temperature.

    PubMed

    Aghdam, Morteza Soleimani; Naderi, Roohangiz; Jannatizadeh, Abbasali; Babalar, Mesbah; Sarcheshmeh, Mohammad Ali Askari; Faradonbe, Mojtaba Zamani

    2016-09-01

    Anthurium flowers are susceptible to chilling injury, and the optimum storage temperature is 12.5-20 °C. The γ-aminobutyric acid (GABA) shunt pathway may alleviate chilling stress in horticultural commodities by providing energy (ATP), reducing molecules (NADH), and minimizing accumulation of reactive oxygen species (ROS). In this experiment, the impact of a preharvest spray treatment with 1 mM GABA and postharvest treatment of 5 mM GABA stem-end dipping on GABA shunt pathway activity of anthurium cut flowers (cv. Sirion) in response to cold storage (4 °C for 21 days) was investigated. GABA treatments resulted in lower glutamate decarboxylase (GAD) and higher GABA transaminase (GABA-T) activities in flowers during cold storage, which was associated with lower GABA content and coincided with higher ATP content. GABA treatments also enhanced accumulation of endogenous glycine betaine (GB) in flowers during cold storage, as well as higher spathe relative water content (RWC). These findings suggest that GABA treatments may alleviate chilling injury of anthurium cut flowers by enhancing GABA shunt pathway activity leading to provide sufficient ATP and promoting endogenous GB accumulation. PMID:27135813

  17. Impact of exogenous GABA treatments on endogenous GABA metabolism in anthurium cut flowers in response to postharvest chilling temperature.

    PubMed

    Aghdam, Morteza Soleimani; Naderi, Roohangiz; Jannatizadeh, Abbasali; Babalar, Mesbah; Sarcheshmeh, Mohammad Ali Askari; Faradonbe, Mojtaba Zamani

    2016-09-01

    Anthurium flowers are susceptible to chilling injury, and the optimum storage temperature is 12.5-20 °C. The γ-aminobutyric acid (GABA) shunt pathway may alleviate chilling stress in horticultural commodities by providing energy (ATP), reducing molecules (NADH), and minimizing accumulation of reactive oxygen species (ROS). In this experiment, the impact of a preharvest spray treatment with 1 mM GABA and postharvest treatment of 5 mM GABA stem-end dipping on GABA shunt pathway activity of anthurium cut flowers (cv. Sirion) in response to cold storage (4 °C for 21 days) was investigated. GABA treatments resulted in lower glutamate decarboxylase (GAD) and higher GABA transaminase (GABA-T) activities in flowers during cold storage, which was associated with lower GABA content and coincided with higher ATP content. GABA treatments also enhanced accumulation of endogenous glycine betaine (GB) in flowers during cold storage, as well as higher spathe relative water content (RWC). These findings suggest that GABA treatments may alleviate chilling injury of anthurium cut flowers by enhancing GABA shunt pathway activity leading to provide sufficient ATP and promoting endogenous GB accumulation.

  18. Neuroligin-2 accelerates GABAergic synapse maturation in cerebellar granule cells.

    PubMed

    Fu, Zhanyan; Vicini, Stefano

    2009-09-01

    Neuroligins (NLGs) are postsynaptic cell adhesion molecules that are thought to function in synaptogenesis. To investigate the role of NLGs on synaptic transmission once the synapse is formed, we transfected neuroligin-2 (NLG-2) in cultured mouse cerebellar granule cells (CGCs), and recorded GABA(A) (gamma-aminobutyric acid) receptor mediated miniature postsynaptic currents (mIPSCs). NLG-2 transfected cells had mIPSCs with faster decay than matching GFP expressing controls at young culture ages (days in vitro, DIV7-8). Down-regulation of NLG-2 by the isoform specific shRNA-NLG-2 resulted in an opposite effect. We and others have shown that the switch of alpha subunits of GABA(A)Rs from alpha2/3 to alpha1 underlies developmental speeding of the IPSC decay in various CNS regions, including the cerebellum. To assess whether the reduced decay time of mIPSCs by NLG-2 is due to the recruitment of more alpha1 containing GABA(A)Rs at the synapses, we examined the prolongation of current decay by the Zolpidem, which has been shown to preferentially enhance the activity of alpha1 subunit-containing GABA channel. The application of Zolpidem resulted in a significantly greater prolongation kinetics of synaptic currents in NLG-2 over-expressing cells than control cells, suggesting that NLG-2 over-expression accelerates synapse maturation by promoting incorporation of the alpha1 subunit-containing GABA(A)Rs at postsynaptic sites in immature cells. In addition, the effect of NLG-2 on the speeding of decay time course of synaptic currents was abolished when we used CGC cultures from alpha1-/- mice. Lastly, to exclude the possibility that the fast decay of mIPSCs induced by NLG-2 could be also due to the impacts of NLG-2 on the GABA transient in synaptic cleft, we measured the sensitivity of mIPSCs to the fast-off competitive antagonists TPMPA. We found that TPMPA similarly inhibits mIPSCs in control and NLG-2 over-expressing CGCs both at young age (DIV8) and old age (DIV14) of

  19. Synthesis, biological evaluation and structure-activity relationship of new GABA uptake inhibitors, derivatives of 4-aminobutanamides.

    PubMed

    Kowalczyk, Paula; Sałat, Kinga; Höfner, Georg C; Mucha, Marta; Rapacz, Anna; Podkowa, Adrian; Filipek, Barbara; Wanner, Klaus T; Kulig, Katarzyna

    2014-08-18

    Six series of 2-substituted 4-aminobutanamide derivatives were synthesized and evaluated for their ability to inhibit GABA transport proteins mGAT1-4 stably expressed in HEK-293 cell lines. The pIC50 values determined were in the range 4.23-5.23. Two compounds (15b and 15c) were selected for further in vitro studies. These compounds were also subjected to preliminary behavioral studies to evaluate their anticonvulsant, antidepressant-like, and antinociceptive activities in mice. Their influence on motor coordination was also assessed. We report that, among a spectrum of in vivo activities, both 15b and 15c displayed significant activity against pentylenetetrazole (PTZ)-induced seizures.

  20. Calreticulin modulates cell adhesiveness via regulation of vinculin expression

    PubMed Central

    1996-01-01

    Calreticulin is an ubiquitous and highly conserved high capacity Ca(2+)- binding protein that plays a major role in Ca2+ storage within the lumen of the ER. Here, using L fibroblast cell lines expressing different levels of calreticulin, we show that calreticulin plays a role in the control of cell adhesiveness via regulation of expression of vinculin, a cytoskeletal protein essential for cell-substratum and cell-cell attachments. Both vinculin protein and mRNA levels are increased in cells overexpressing calreticulin and are downregulated in cells expressing reduced level of calreticulin. Abundance of actin, talin, alpha 5 and beta 1 integrins, pp125 focal adhesion kinase, and alpha-catenin is not affected by the differential calreticulin expression. Overexpression of calreticulin increases both cell- substratum and cell-cell adhesiveness of L fibroblasts that, most surprisingly, establish vinculin-rich cell-cell junctions. Upregulation of calreticulin also affects adhesion-dependent phenomena such as cell motility (which decreases) and cell spreading (which increases). Downregulation of calreticulin brings about inverse effects. Cell adhesiveness is Ca2+ regulated. The level of calreticulin expression, however, has no effect on either the resting cytoplasmic Ca2+ concentration or the magnitude of FGF-induced Ca2+ transients. Calreticulin, however, participates in Ca2+ homeostasis as its level of expression affects cell viability at low concentrations of extracellular Ca2+. Consequently, we infer that it is not the Ca2+ storage function of calreticulin that affects cell adhesiveness. Neither endogenous calreticulin nor overexpressed green fluorescent protein-calreticulin construct can be detected outside of the ER. Since all of the adhesion-related effects of differential calreticulin expression can be explained by its regulation of vinculin expression, we conclude that it is the ER-resident calreticulin that affects cellular adhesiveness. PMID:8991101

  1. Cumulus Cells Gene Expression Profiling in Terms of Oocyte Maturity in Controlled Ovarian Hyperstimulation Using GnRH Agonist or GnRH Antagonist

    PubMed Central

    Devjak, Rok; Fon Tacer, Klementina; Juvan, Peter; Virant Klun, Irma; Rozman, Damjana; Vrtačnik Bokal, Eda

    2012-01-01

    In in vitro fertilization (IVF) cycles controlled ovarian hyperstimulation (COH) is established by gonadotropins in combination with gonadotropin-releasing hormone (GnRH) agonists or antagonists, to prevent premature luteinizing hormone (LH) surge. The aim of our study was to improve the understanding of gene expression profile of cumulus cells (CC) in terms of ovarian stimulation protocol and oocyte maturity. We applied Affymetrix gene expression profiling in CC of oocytes at different maturation stages using either GnRH agonists or GnRH antagonists. Two analyses were performed: the first involved CC of immature metaphase I (MI) and mature metaphase II (MII) oocytes where 359 genes were differentially expressed, and the second involved the two GnRH analogues where no differentially expressed genes were observed at the entire transcriptome level. A further analysis of 359 differentially genes was performed, focusing on anti-Müllerian hormone receptor 2 (AMHR2), follicle stimulating hormone receptor (FSHR), vascular endothelial growth factor C (VEGFC) and serine protease inhibitor E2 (SERPINE2). Among other differentially expressed genes we observed a marked number of new genes connected to cell adhesion and neurotransmitters such as dopamine, glycine and γ-Aminobutyric acid (GABA). No differential expression in CC between the two GnRH analogues supports the findings of clinical studies where no significant difference in live birth rates between both GnRH analogues has been proven. PMID:23082142

  2. Probing cell-free gene expression noise in femtoliter volumes

    SciTech Connect

    Karig, David K; Jung, Seung-Yong; Srijanto, Bernadeta R; Collier, Pat; Simpson, Michael L

    2013-01-01

    Cell-free systems offer a simplified and flexible context that enables important biological reactions while removing complicating factors such as fitness, division, and mutation that are associated with living cells. However, cell-free expression in unconfined spaces is missing important elements of expression in living cells. In particular, the small volume of living cells can give rise to significant stochastic effects, which are negligible in bulk cell-free reactions. Here, we confine cell-free gene expression reactions to cell relevant 20 fL volumes (between the volumes of E. coli and S. cerevisiae), in polydimethylsiloxane (PDMS) containers. We demonstrate that expression efficiency varies widely at this volume, and we analyze gene expression noise. Noise analysis reveals signatures of translational bursting while noise dynamics suggest that overall cell-free expression is limited by a diminishing translation rate. In addition to offering a unique approach to understanding noise in gene circuits, our work contributes to a deeper understanding of the biophysical properties of cell-free expression systems, thus aiding efforts to harness cell-free systems for synthetic biology applications.

  3. Expression of HOX C homeobox genes in lymphoid cells.

    PubMed

    Lawrence, H J; Stage, K M; Mathews, C H; Detmer, K; Scibienski, R; MacKenzie, M; Migliaccio, E; Boncinelli, E; Largman, C

    1993-08-01

    The class I homeobox genes located in four clusters in mammalian genomes (HOX A, HOX B, HOX C, and HOX D) appear to play a major role in fetal development. Previous surveys of homeobox gene expression in human leukemic cell lines have shown that certain HOX A genes are expressed only in myeloid cell lines, whereas HOX B gene expression is largely restricted to cells with erythroid potential. We now report a survey of the expression patterns of 9 homeobox genes from the HOX C locus in a panel of 24 human and 7 murine leukemic cell lines. The most striking observation is the lymphoid-specific pattern of expression of HOX C4, located at the 3' end of the locus. A major transcript of 1.9 kilobases is observed in both T-cell and B-cell lines. HOX C4 expression is also detected in normal human marrow and peripheral blood lymphocytes, but not in mature granulocytes or monocytes. HOX C8 is also expressed in human lymphoid cells but is expressed in other blood cell types as well. However, the HOX C8 transcript pattern is lineage specific. These data, in conjunction with earlier findings, suggest that homeobox gene expression influences lineage determination during hematopoiesis.

  4. Freedom of expression: cell-type-specific gene profiling.

    PubMed

    Otsuki, Leo; Cheetham, Seth W; Brand, Andrea H

    2014-01-01

    Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single-cell expression profiling.

  5. Teratogenesis of polychlorocycloalkane insecticides in chicken embryos resulting from their interactions at the convulsant recognition sites of the GABA (pro)receptor complex

    SciTech Connect

    Seifert, J. )

    1989-05-01

    There is an increasing concern for the effects of toxicants expressed during embryonic development. This paper considers the teratogenicity of polychlorocycloalkane insecticides. They are neurotoxicants which act at the convulsant recognition site(s) of the GABA receptor-chloride ionophore and antagonize GABA-mediated inhibitory neurotransmission. This study uses the chicken embryo as a model organism. It focuses on malformations related to interactions of polychlorocycloalkane insecticides with the convulsant recognition sites in the brain. Information provided by this study is important for investigations on the teratogenicity of insecticidal GABA antagonists in mammals.

  6. Presynaptic Control of Corticostriatal Synapses by Endogenous GABA

    PubMed Central

    Logie, Christopher; Bagetta, Vincenza

    2013-01-01

    Corticostriatal terminals have presynaptic GABAB receptors that limit glutamate release, but how these receptors are activated by endogenous GABA released by different types of striatal neurons is still unknown. To address this issue, we used single and paired whole-cell recordings combined with stimulation of corticostriatal fibers in rats and mice. In the presence of opioid, GABAA, and NK1 receptor antagonists, antidromic stimulation of a population of striatal projection neurons caused suppression of subsequently evoked EPSPs in projection neurons. These effects were larger at intervals of 500 ms than 1 or 2 s, and were fully blocked by the selective GABAB receptor antagonist CGP 52432. Bursts of spikes in individual projection neurons were not able to inhibit evoked EPSPs. Similarly, spikes in fast spiking interneurons and low-threshold spike interneurons failed to elicit detectable effects mediated by GABAB receptors. Conversely, spikes in individual neurogliaform interneurons suppressed evoked EPSPs, and these effects were blocked by CGP 52432. These results provide the first demonstration of how GABAB receptors are activated by endogenous GABA released by striatal neuronal types. PMID:24068811

  7. Zolpidem modulates GABA(A) receptor function in subthalamic nucleus.

    PubMed

    Chen, Lei; Xie, Jun-Xia; Fung, Kam-Shuen; Yung, Wing-Ho

    2007-05-01

    The subthalamic nucleus occupies a position in the indirect pathway of basal ganglia circuit, which plays an important role in the movement regulation. Zolpidem is an imidazopyridine agonist with a high affinity on the benzodiazepine site of GABA(A) receptors containing alpha 1 subunit. Recently, zolpidem has been reported to be useful in treating subgroups of parkinsonian patients. A high density of zolpidem binding sites has been shown in rat subthalamic nucleus. To further investigate the modulation of zolpidem on GABA(A) receptor-mediated inhibitory synaptic current in subthalamic nucleus, whole-cell patch clamp recordings were used in the present study. Zolpidem at 100nM significantly prolonged the decay time and rise time of miniature inhibitory postsynaptic currents, with no effect on the amplitude and frequency. The benzodiazepine antagonist flumazenil could completely block the potentiation induced by zolpidem, confirming the specificity on the benzodiazepine site. At a high concentration of 1 microM, zolpidem significantly increased the decay time, rise time, amplitude and frequency of miniature inhibitory postsynaptic currents. In the behaving rats, unilateral microinjection of zolpidem into subthalamic nucleus induced a significant contralateral rotation. The present findings on the effect of zolpidem in subthalamic nucleus provide a rationale for further investigations into its potential in the treatment of Parkinson's disease. PMID:17337310

  8. Nestin-expressing cells in the human hippocampus.

    PubMed

    Kruglyakova, E P; Khovryakov, A V; Shikhanov, N P; Maccann, G M; Vael', Ikhsan; Kruglyakov, P P; Sosunov, A A

    2005-11-01

    Nestin, a protein of the intermediate filament family, is typical of undifferentiated neural stem and progenitor cells. The present report describes studies of nestin expression in the hippocampus of patients with epilepsy and identifies five types of nestin-immunopositive cells differing in terms of their morphological phenotype and immunological characteristics. These were cells with the phenotype of radial glial cells, bipolar cells, small dendritic cells, cells of the subependymal zone, and astrocyte-like cells. Two types of cell - radial gliocytes of the dentate fascia and NG2-immunopositive bipolar cells - can be regarded as neural precursors of different levels of commitment.

  9. Characteristics and EGFP expression of goat mammary gland epithelial cells.

    PubMed

    Zheng, Y-M; He, X-Y; Zhang, Y

    2010-12-01

    The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing. PMID:20113446

  10. Characteristics and EGFP expression of goat mammary gland epithelial cells.

    PubMed

    Zheng, Y-M; He, X-Y; Zhang, Y

    2010-12-01

    The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing.

  11. P2Y1 receptor inhibits GABA transport through a calcium signalling-dependent mechanism in rat cortical astrocytes.

    PubMed

    Jacob, Pedro F; Vaz, Sandra H; Ribeiro, Joaquim A; Sebastião, Ana M

    2014-08-01

    Astrocytes express a variety of purinergic (P2) receptors, involved in astrocytic communication through fast increases in [Ca(2+) ]i . Of these, the metabotropic ATP receptors (P2Y) regulate cytoplasmic Ca(2+) levels through the PLC-PKC pathway. GABA transporters are a substrate for a number of Ca(2+) -related kinases, raising the possibility that calcium signalling in astrocytes impact the control of extracellular levels of the major inhibitory transmitter in the brain. To access this possibility we tested the influence of P2Y receptors upon GABA transport into astrocytes. Mature primary cortical astroglial-enriched cultures expressed functional P2Y receptors, as evaluated through Ca(2+) imaging, being P2Y1 the predominant P2Y receptor subtype. ATP (100 μM, for 1 min) caused an inhibition of GABA transport through either GAT-1 or GAT-3 transporters, decreasing the Vmax kinetic constant. ATP-induced inhibition of GATs activity was still evident in the presence of adenosine deaminase, precluding an adenosine-mediated effect. This, was mimicked by a specific agonist for the P2Y1,12,13 receptor (2-MeSADP). The effect of 2-MeSADP on GABA transport was blocked by the P2 (PPADS) and P2Y1 selective (MRS2179) receptor antagonists, as well as by the PLC inhibitor (U73122). 2-MeSADP failed to inhibit GABA transport in astrocytes where intracellular calcium had been chelated (BAPTA-AM) or where calcium stores were depleted (α-cyclopiazonic acid, CPA). In conclusion, P2Y1 receptors in astrocytes inhibit GABA transport through a mechanism dependent of P2Y1 -mediated calcium signalling, suggesting that astrocytic calcium signalling, which occurs as a consequence of neuronal firing, may operate a negative feedback loop to enhance extracellular levels of GABA. PMID:24733747

  12. Robust Inference of Cell-to-Cell Expression Variations from Single- and K-Cell Profiling

    PubMed Central

    Narayanan, Manikandan; Martins, Andrew J.; Tsang, John S.

    2016-01-01

    Quantifying heterogeneity in gene expression among single cells can reveal information inaccessible to cell-population averaged measurements. However, the expression level of many genes in single cells fall below the detection limit of even the most sensitive technologies currently available. One proposed approach to overcome this challenge is to measure random pools of k cells (e.g., 10) to increase sensitivity, followed by computational “deconvolution” of cellular heterogeneity parameters (CHPs), such as the biological variance of single-cell expression levels. Existing approaches infer CHPs using either single-cell or k-cell data alone, and typically within a single population of cells. However, integrating both single- and k-cell data may reap additional benefits, and quantifying differences in CHPs across cell populations or conditions could reveal novel biological information. Here we present a Bayesian approach that can utilize single-cell, k-cell, or both simultaneously to infer CHPs within a single condition or their differences across two conditions. Using simulated as well as experimentally generated single- and k-cell data, we found situations where each data type would offer advantages, but using both together can improve precision and better reconcile CHP information contained in single- and k-cell data. We illustrate the utility of our approach by applying it to jointly generated single- and k-cell data to reveal CHP differences in several key inflammatory genes between resting and inflammatory cytokine-activated human macrophages, delineating differences in the distribution of ‘ON’ versus ‘OFF’ cells and in continuous variation of expression level among cells. Our approach thus offers a practical and robust framework to assess and compare cellular heterogeneity within and across biological conditions using modern multiplexed technologies. PMID:27438699

  13. Advantages and applications of CAR-expressing natural killer cells

    PubMed Central

    Glienke, Wolfgang; Esser, Ruth; Priesner, Christoph; Suerth, Julia D.; Schambach, Axel; Wels, Winfried S.; Grez, Manuel; Kloess, Stephan; Arseniev, Lubomir; Koehl, Ulrike

    2015-01-01

    In contrast to donor T cells, natural killer (NK) cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR) expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy. PMID:25729364

  14. Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

    PubMed Central

    Brown, Laura E.; Fuchs, Celine; Nicholson, Martin W.; Stephenson, F. Anne; Thomson, Alex M.; Jovanovic, Jasmina N.

    2014-01-01

    Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed

  15. Transferrin receptor expression by stimulated cells in mixed lymphocyte culture.

    PubMed Central

    Salmon, M; Bacon, P A; Symmons, D P; Walton, K W

    1985-01-01

    Transferrin receptor (TRFr) expression by cells in mixed lymphocyte culture increases steadily for the first 5 days, but then reaches a plateau. By the sixth day in culture, about 20% of viable cells express TRFr in two-way mixed lymphocyte reactions. This subpopulation of TRFr-positive cells represents the proliferating population; it is heterogeneous, containing T-cell blasts and smaller cells which are a mixture of T and non-T cells. A small group of non-T cells have phenotypic similarity to natural killer (NK) cells. T cells appear to divide earlier in the course of the response than non-T cells. The biphasic nature of this response and the slower non-T reactivity may be due to a secondary stimulation of non-T cells by factors released from activated T cells (such as interleukin-2). PMID:2982734

  16. The regulation of gene expression in hair cells.

    PubMed

    Ryan, Allen F; Ikeda, Ryoukichi; Masuda, Masatsugu

    2015-11-01

    No genes have been discovered for which expression is limited only to inner ear hair cells. This is hardly surprising, since the number of mammalian genes is estimated to be 20-25,000, and each gene typically performs many tasks in various locations. Many genes are expressed in inner ear sensory cells and not in other cells of the labyrinth. However, these genes are also expressed in other locations, often in other sensory or neuronal cell types. How gene transcription is directed specifically to hair cells is unclear. Key transcription factors that act during development can specify cell phenotypes, and the hair cell is no exception. The transcription factor ATOH1 is well known for its ability to transform nonsensory cells of the developing inner ear into hair cells. And yet, ATOH1 also specifies different sensory cells at other locations, neuronal phenotypes in the brain, and epithelial cells in the gut. How it specifies hair cells in the inner ear, but alternate cell types in other locations, is not known. Studies of regulatory DNA and transcription factors are revealing mechanisms that direct gene expression to hair cells, and that determine the hair cell identity. The purpose of this review is to summarize what is known about such gene regulation in this key auditory and vestibular cell type.

  17. Chemokine receptor expression by inflammatory T cells in EAE.

    PubMed

    Mony, Jyothi Thyagabhavan; Khorooshi, Reza; Owens, Trevor

    2014-01-01

    Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20 receptor CCR6 has been reported to be selectively expressed by CD4(+) T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4(+) T cells produced IFNγ in flow cytometric cytokine assays, whereas less than 1% produced IL-17. About 1% of CD4(+) T cells produced both cytokines. CCR6 was expressed by Th1, Th1+17 and by Th17 cells, but not by CD8(+) T cells. CD8(+) T cells expressed CXCR3, which was also expressed by CD4(+) T cells, with no correlation to cytokine profile. Messenger RNA for IFNγ, IL-17A, and the Th1 and Th17-associated transcription factors T-bet and RORγt was detected in both CCR6(+) and CXCR3(+) CD4(+) T cells. IFNγ, but not IL-17A mRNA expression was detected in CD8(+) T cells in CNS. CCR6 and CD4 were co-localized in spinal cord infiltrates by double immunofluorescence. Consistent with flow cytometry data some but not all CD4(+) T cells expressed CCR6 within infiltrates. CD4-negative CCR6(+) cells included macrophage/microglial cells. Thus we have for the first time directly studied CD4(+) and CD8(+) T cells in the CNS of mice with peak EAE, and determined IFNγ and IL17 expression by cells expressing CCR6 and CXCR3. We show that neither CCR6 or CXCR3 align with CD4 T cell subsets, and Th1 or mixed Th1+17 predominate in EAE.

  18. Geometry of the Gene Expression Space of Individual Cells.

    PubMed

    Korem, Yael; Szekely, Pablo; Hart, Yuval; Sheftel, Hila; Hausser, Jean; Mayo, Avi; Rothenberg, Michael E; Kalisky, Tomer; Alon, Uri

    2015-07-01

    There is a revolution in the ability to analyze gene expression of single cells in a tissue. To understand this data we must comprehend how cells are distributed in a high-dimensional gene expression space. One open question is whether cell types form discrete clusters or whether gene expression forms a continuum of states. If such a continuum exists, what is its geometry? Recent theory on evolutionary trade-offs suggests that cells that need to perform multiple tasks are arranged in a polygon or polyhedron (line, triangle, tetrahedron and so on, generally called polytopes) in gene expression space, whose vertices are the expression profiles optimal for each task. Here, we analyze single-cell data from human and mouse tissues profiled using a variety of single-cell technologies. We fit the data to shapes with different numbers of vertices, compute their statistical significance, and infer their tasks. We find cases in which single cells fill out a continuum of expression states within a polyhedron. This occurs in intestinal progenitor cells, which fill out a tetrahedron in gene expression space. The four vertices of this tetrahedron are each enriched with genes for a specific task related to stemness and early differentiation. A polyhedral continuum of states is also found in spleen dendritic cells, known to perform multiple immune tasks: cells fill out a tetrahedron whose vertices correspond to key tasks related to maturation, pathogen sensing and communication with lymphocytes. A mixture of continuum-like distributions and discrete clusters is found in other cell types, including bone marrow and differentiated intestinal crypt cells. This approach can be used to understand the geometry and biological tasks of a wide range of single-cell datasets. The present results suggest that the concept of cell type may be expanded. In addition to discreet clusters in gene-expression space, we suggest a new possibility: a continuum of states within a polyhedron, in which the

  19. Geometry of the Gene Expression Space of Individual Cells

    PubMed Central

    Korem, Yael; Szekely, Pablo; Hart, Yuval; Sheftel, Hila; Hausser, Jean; Mayo, Avi; Rothenberg, Michael E.; Kalisky, Tomer; Alon, Uri

    2015-01-01

    There is a revolution in the ability to analyze gene expression of single cells in a tissue. To understand this data we must comprehend how cells are distributed in a high-dimensional gene expression space. One open question is whether cell types form discrete clusters or whether gene expression forms a continuum of states. If such a continuum exists, what is its geometry? Recent theory on evolutionary trade-offs suggests that cells that need to perform multiple tasks are arranged in a polygon or polyhedron (line, triangle, tetrahedron and so on, generally called polytopes) in gene expression space, whose vertices are the expression profiles optimal for each task. Here, we analyze single-cell data from human and mouse tissues profiled using a variety of single-cell technologies. We fit the data to shapes with different numbers of vertices, compute their statistical significance, and infer their tasks. We find cases in which single cells fill out a continuum of expression states within a polyhedron. This occurs in intestinal progenitor cells, which fill out a tetrahedron in gene expression space. The four vertices of this tetrahedron are each enriched with genes for a specific task related to stemness and early differentiation. A polyhedral continuum of states is also found in spleen dendritic cells, known to perform multiple immune tasks: cells fill out a tetrahedron whose vertices correspond to key tasks related to maturation, pathogen sensing and communication with lymphocytes. A mixture of continuum-like distributions and discrete clusters is found in other cell types, including bone marrow and differentiated intestinal crypt cells. This approach can be used to understand the geometry and biological tasks of a wide range of single-cell datasets. The present results suggest that the concept of cell type may be expanded. In addition to discreet clusters in gene-expression space, we suggest a new possibility: a continuum of states within a polyhedron, in which the

  20. Characteristics and EGFP expression of porcine mammary gland epithelial cells.

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

    The aims of this study were to establish a porcine mammary gland epithelial (PMGE) cell line, and to determine if these PMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of PMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating pig. The passage sixteen PMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in PMGE cells was tested by immunofluorescence. Βeta-Casein gene mRNA was tested for PMGE cells by RT-PCR. The results showed that PMGE cells could form dome-like structure which looked like nipple, and the cells contained different cell types. The expression of Cell keratins demonstrated the property of epithelial cells, and the PMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the PMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected porcine mammary gland epithelial (ET-PMGE) cell line. PMID:20400167

  1. Characteristics and EGFP expression of porcine mammary gland epithelial cells.

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

    The aims of this study were to establish a porcine mammary gland epithelial (PMGE) cell line, and to determine if these PMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of PMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating pig. The passage sixteen PMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in PMGE cells was tested by immunofluorescence. Βeta-Casein gene mRNA was tested for PMGE cells by RT-PCR. The results showed that PMGE cells could form dome-like structure which looked like nipple, and the cells contained different cell types. The expression of Cell keratins demonstrated the property of epithelial cells, and the PMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the PMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected porcine mammary gland epithelial (ET-PMGE) cell line.

  2. The pharmacology of spontaneously open alpha 1 beta 3 epsilon GABA A receptor-ionophores.

    PubMed

    Maksay, Gábor; Thompson, Sally A; Wafford, Keith A

    2003-06-01

    Human alpha(1)beta(3) epsilon GABA(A) receptors were expressed in Xenopus oocytes and examined using the conventional two-electrode voltage-clamp technique and compared to alpha(1)beta(3)gamma(2) receptors. The effects of several GABA(A) agonists were studied, and the allosteric modulation of the channel by a number of GABAergic modulators investigated. The presence of the epsilon subunit increased the potency and efficacy of direct activation by partial GABA(A) agonists (piperidine-4-sulphonic acid and thio-4-PIOL), pentobarbital and neuro-steroids. Direct activation by 3-hydroxylated neurosteroids was restricted to 3alpha epimers, while chirality at C5 was indifferent. The 3beta-sulfate esters of pregnenolone and dehydroepiandrosterone inhibited the spontaneous currents with efficacies higher, while bicuculline methiodide and SR 95531 did so lower than picrotoxin and TBPS. Furosemide, fipronil, triphenylcyanoborate and Zn(2+) blocked the spontaneous currents of alpha(1)beta(3) epsilon receptors with different efficacies. Flunitrazepam and 4'-chlorodiazepam inhibited the spontaneous currents with micromolar potencies. In conclusion, spontaneously active alpha(1)beta(3) epsilon GABA(A) receptors can be potentiated and blocked by GABAergic agents within a broad range of efficacy.

  3. Etoposide sensitizes neuroblastoma cells expressing caspase 8 to TRAIL.

    PubMed

    Kim, Hye Ryung; Lee, Myoung Woo; Kim, Dae Seong; Jo, Ha Yeong; Lee, Soo Hyun; Chueh, Hee Won; Jung, Hye Lim; Yoo, Keon Hee; Sung, Ki Woong; Koo, Hong Hoe

    2012-01-01

    TRAIL [TNF (tumour necrosis factor)-related apoptosis-inducing ligand] is a promising agent for clinical use since it kills a wide range of tumour cells without affecting normal cells. We provide evidence that pretreatment with etoposide significantly enhanced TRAIL-mediated apoptosis via up-regulation of DR5 (death receptor 5 or TRAIL-R2) expression in the caspase 8 expressing neuroblastoma cell line, SK-N-MC. In addition, sequential treatment with etoposide and TRAIL increased caspases 8, 9 and 3 activation, Mcl-1 cleavage and Bid truncation, which suggests that the ability of etoposide and TRAIL to induce apoptosis is mediated through activation of an intrinsic signalling pathway. Although TRAIL-R2 expression increased in IMR-32 cells in response to etoposide treatment, cell death was not increased by concurrent treatment with TRAIL compared with etoposide alone, because the cells lacked caspase 8 expression. Restoration of caspase 8 expression by exposure to IFNγ (interferon γ) sensitizes IMR-32 cells to TRAIL. Moreover, pretreatment with etoposide increased TRAIL-induced apoptosis in caspase 8 restored IMR-32 cells through activation of a caspase cascade that included caspases 8, 9 and 3. These results indicate that the etoposide-mediated sensitization of neuroblastoma cells to TRAIL is associated with an increase in TRAIL-R2 expression and requires caspase 8 expression. These observations support the potential use of a combination of etoposide and TRAIL in future clinical trials. PMID:23124518

  4. Identifying gene expression modules that define human cell fates.

    PubMed

    Germanguz, I; Listgarten, J; Cinkornpumin, J; Solomon, A; Gaeta, X; Lowry, W E

    2016-05-01

    Using a compendium of cell-state-specific gene expression data, we identified genes that uniquely define cell states, including those thought to represent various developmental stages. Our analysis sheds light on human cell fate through the identification of core genes that are altered over several developmental milestones, and across regional specification. Here we present cell-type specific gene expression data for 17 distinct cell states and demonstrate that these modules of genes can in fact define cell fate. Lastly, we introduce a web-based database to disseminate the results.

  5. GABA shapes the dynamics of bistable perception.

    PubMed

    van Loon, Anouk M; Knapen, Tomas; Scholte, H Steven; St John-Saaltink, Elexa; Donner, Tobias H; Lamme, Victor A F

    2013-05-01

    Sometimes, perception fluctuates spontaneously between two distinct interpretations of a constant sensory input. These bistable perceptual phenomena provide a unique window into the neural mechanisms that create the contents of conscious perception. Models of bistable perception posit that mutual inhibition between stimulus-selective neural populations in visual cortex plays a key role in these spontaneous perceptual fluctuations. However, a direct link between neural inhibition and bistable perception has not yet been established experimentally. Here, we link perceptual dynamics in three distinct bistable visual illusions (binocular rivalry, motion-induced blindness, and structure from motion) to measurements of gamma-aminobutyric acid (GABA) concentrations in human visual cortex (as measured with magnetic resonance spectroscopy) and to pharmacological stimulation of the GABAA receptor by means of lorazepam. As predicted by a model of neural interactions underlying bistability, both higher GABA concentrations in visual cortex and lorazepam administration induced slower perceptual dynamics, as reflected in a reduced number of perceptual switches and a lengthening of percept durations. Thus, we show that GABA, the main inhibitory neurotransmitter, shapes the dynamics of bistable perception. These results pave the way for future studies into the competitive neural interactions across the visual cortical hierarchy that elicit conscious perception. PMID:23602476

  6. Differential expression and function of CD27 in chronic lymphocytic leukemia cells expressing ZAP-70.

    PubMed

    Lafarge, Sandrine T; Hou, Sen; Pauls, Samantha D; Johnston, James B; Gibson, Spencer B; Marshall, Aaron J

    2015-07-01

    Chronic lymphocytic leukemia is a malignancy driven by abberant B cell signaling and survival. Leukemic B cells accumulate in the peripheral blood and the lymphoid organs where contact with stromal cells and T cells provide critical survival signals. Clinical severity of CLL is associated with several prognostic markers including expression of the kinase ZAP-70. ZAP-70 expression enhances signaling via the B cell antigen receptor and is associated with increased cell adhesion and migration capacity. Here we report that ZAP-70-positive CLL patients display significantly higher expression of the TNF superfamily receptor and memory marker CD27 than do ZAP-70 negative patients. CD27 expression by CLL was acutely elevated upon BCR cross-linking, or upon ectopic expression of ZAP-70. CD27 expression correlated with functional capacity to adhere to stromal cells and antibody blockade of CD27 impaired CLL binding to stroma. These results provide the first evidence for differential expression of CD27 among CLL prognostic groups, suggest a role for ZAP-70 dependent signaling in CD27 induction and implicate CD27 in cell-cell interactions with the lymphoid tissue microenvironment.

  7. [Autoantibodies to glutamate and GABA in opiate addiction].

    PubMed

    Vetrile, L A; Fomina, V G; Nevidimova, T I; Vetlugina, T P; Batukhtina, E I; Savochkina, D N; Zakharova, I A; Davydova, T V

    2015-01-01

    Blood serum from 129 patients with opium addiction at different stages of the disease and 63 donors (control group) was examined for the presence of autoantibodies to the exciting and inhibitory amino acids glutamate and GABA. It was shown enhanced production of autoantibodies to glutamate and GABA. Dependence of the level and frequency of detec- tion of autoantibodies to glutamate and GABA on the stage of the disease was revealed.

  8. [Autoantibodies to glutamate and GABA in opiate addiction].

    PubMed

    Vetrile, L A; Fomina, V G; Nevidimova, T I; Vetlugina, T P; Batukhtina, E I; Savochkina, D N; Zakharova, I A; Davydova, T V

    2015-01-01

    Blood serum from 129 patients with opium addiction at different stages of the disease and 63 donors (control group) was examined for the presence of autoantibodies to the exciting and inhibitory amino acids glutamate and GABA. It was shown enhanced production of autoantibodies to glutamate and GABA. Dependence of the level and frequency of detec- tion of autoantibodies to glutamate and GABA on the stage of the disease was revealed. PMID:26852594

  9. Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data

    PubMed Central

    Moignard, Victoria; Göttgens, Berthold; Adryan, Boris

    2016-01-01

    Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete pipeline for the analysis of single cell qPCR data that uses the mathematics behind bursty expression to develop more accurate and robust algorithms for analyzing the origin of heterogeneity in experimental samples, specifically an algorithm for clustering cells by their bursting behavior (Simulated Annealing for Bursty Expression Clustering, SABEC) and a statistical tool for comparing the kinetic parameters of bursty expression across populations of cells (Estimation of Parameter changes in Kinetics, EPiK). We applied these methods to hematopoiesis, including a new single cell dataset in which transcription factors (TFs) involved in the earliest branchpoint of blood differentiation were individually up- and down-regulated. We could identify two unique sub-populations within a seemingly homogenous group of hematopoietic stem cells. In addition, we could predict regulatory mechanisms controlling the expression levels of eighteen key hematopoietic transcription factors throughout differentiation. Detailed information about gene regulatory mechanisms can therefore be obtained simply from high throughput single cell gene expression data, which should be widely applicable given the rapid expansion of single cell genomics. PMID:27551778

  10. Expression, regulation, and function of B cell-expressed CD154 in germinal centers.

    PubMed

    Grammer, A C; McFarland, R D; Heaney, J; Darnell, B F; Lipsky, P E

    1999-10-15

    Activated B cells and T cells express CD154/CD40 ligand in vitro. The in vivo expression and function of B cell CD154 remain unclear and therefore were examined. Tonsillar B and T cells expressed CD154 at a similar density both in situ and immediately ex vivo, whereas a significantly higher percentage of the former expressed CD154. CD154-expressing B cells were most frequent in the CD38positiveIgD+ pre-germinal center (GC)/GC founder, CD38positive GC and CD38-IgD- memory populations, and were also found in the CD38-IgD+ naive and CD38brightIgD+ plasmablast subsets, but not in the CD38brightIgD- plasma cell subset. B cell expression of CD154 was induced by engaging surface Ig or CD40 by signals that predominantly involved activation of AP-1/NF-AT and NF-kappaB, respectively. The functional importance of CD154-mediated homotypic B cell interactions in vivo was indicated by the finding that mAb to CD154 inhibited differentiation of CD38positiveIgD- GC B cells to CD38-IgD- memory cells. In addition, mAb to CD154 inhibited proliferation induced by engaging sIg or CD40, indicating the role of up-regulation of this molecule in facilitating B cell responsiveness. Of note, CD154 itself not only functioned as a ligand but also as a direct signaling molecule as anti-CD154-conjugated Sepharose beads costimulated B cell responses induced by engaging surface Ig. These results indicate that CD154 is expressed by human B cells in vivo and plays an important role in mediating B cell responses.

  11. Fibronectin induces MMP2 expression in human prostate cancer cells.

    PubMed

    Moroz, Andrei; Delella, Flávia K; Lacorte, Lívia M; Deffune, Elenice; Felisbino, Sérgio L

    2013-01-25

    High-grade prostate cancers express high levels of matrix metalloproteinases (MMPs), major enzymes involved in tumor invasion and metastasis. However, the tumor cell lines commonly employed for prostate cancer research express only small amounts of MMPs when cultivated as monolayer cultures, in common culture media. The present study was conducted to ascertain whether culture conditions that include fibronectin can alter MMP2 and MMP9 expression by the human prostatic epithelial cell lines RWPE-1, LNCaP and PC-3. These cells were individually seeded at 2×10(4) cells/cm(2), cultivated until they reached 80% confluence, and then exposed for 4h to fibronectin, after which the conditioned medium was analyzed by gelatin zymography. Untreated cells were given common medium. Only RWPE-1 cells express detectable amounts of MMP9 when cultivated in common medium, whereas the addition of fibronectin induced high expression levels of pro and active forms of MMP2 in all tested cell lines. Our findings demonstrate that normal and tumor prostate cell lines express MMP2 activity when in contact with extracellular matrix components or blood plasma proteins such as fibronectin. Future studies of transcriptomes and proteomes in prostate cancer research using these cell lines should not neglect these important conclusions.

  12. LKB1 expression reverses the tumorigenicity of L02 cells.

    PubMed

    Liang, Xiaoyan; Xu, Ge; Gao, Qing; Tao, Xiaohong

    2016-08-01

    The tumor-suppressor liver kinase B1 (LKB1), a highly conserved and ubiquitously expressed protein kinase, plays a critical role in tumorigenesis. In the present study, we revealed that human hepatic L02 cells had severely impaired endogenous LKB1 expression as gauged by western blot, northern blot and RT-PCR analyses. Stable ectopic expression of LKB1 in L02 cells resulted in decreased cell growth, hypophosphorylation of Rb, and marked attenuation of colony formation on soft agar. Inoculation of L02 cells into immunocompromised mice resulted in the development of subcutaneous tumors, which could be completely abrogated by ectopic LKB1 expression. The tumors that formed in the mouse model recapitulated the histopathological features of hepatocellular carcinoma under the microscope. Our results jointly suggest that severely compromised endogenous LKB1 expression in the L02 cell line may confer to L02 cells tumor-initiating capacities in vivo and in vitro, and ectopic LKB1 expression antagonizes the tumorigenic properties of L02 cells. Our findings imply that caution may be needed to interpret the results obtained on the widely used human hepatic L02 cell line. The L02 cell line may be a new model to define the cellular mechanisms of liver transformation, and to unravel the molecular mechanisms underlying the growth suppressive effect of LKB1. PMID:27349837

  13. LKB1 expression reverses the tumorigenicity of L02 cells.

    PubMed

    Liang, Xiaoyan; Xu, Ge; Gao, Qing; Tao, Xiaohong

    2016-08-01

    The tumor-suppressor liver kinase B1 (LKB1), a highly conserved and ubiquitously expressed protein kinase, plays a critical role in tumorigenesis. In the present study, we revealed that human hepatic L02 cells had severely impaired endogenous LKB1 expression as gauged by western blot, northern blot and RT-PCR analyses. Stable ectopic expression of LKB1 in L02 cells resulted in decreased cell growth, hypophosphorylation of Rb, and marked attenuation of colony formation on soft agar. Inoculation of L02 cells into immunocompromised mice resulted in the development of subcutaneous tumors, which could be completely abrogated by ectopic LKB1 expression. The tumors that formed in the mouse model recapitulated the histopathological features of hepatocellular carcinoma under the microscope. Our results jointly suggest that severely compromised endogenous LKB1 expression in the L02 cell line may confer to L02 cells tumor-initiating capacities in vivo and in vitro, and ectopic LKB1 expression antagonizes the tumorigenic properties of L02 cells. Our findings imply that caution may be needed to interpret the results obtained on the widely used human hepatic L02 cell line. The L02 cell line may be a new model to define the cellular mechanisms of liver transformation, and to unravel the molecular mechanisms underlying the growth suppressive effect of LKB1.

  14. Tachykinin-related peptide and GABA-mediated presynaptic inhibition of crayfish photoreceptors.

    PubMed

    Glantz, R M; Miller, C S; Nässel, D R

    2000-03-01

    Off-axis illumination elicits lateral inhibition at the primary visual synapse in crustacea and insects. The evidence suggests that the inhibitory action is presynaptic (i.e., on the photoreceptor terminal) and that the amacrine neurons of the lamina ganglionaris (the first synaptic layer) may be part of the inhibitory pathway. The neurotransmitters and the synaptic mechanisms are unknown. We show by immunocytochemistry that GABA and a tachykinin-related peptide (TRP) are localized in the amacrine neurons of the crayfish lamina ganglionaris. Indirect evidence suggests that GABA and TRP may be colocalized in these neurons. The extensive processes of the amacrine neurons occupy lamina layers containing the terminals of photoreceptors. Application of exogenous GABA and TRP to photoreceptor terminals produces a short-latency, dose-dependent hyperpolarization with a decay time constant on the order of a few seconds. TRP also exhibits actions that evolve over several minutes. These include a reduction of the receptor potential (and the light-elicited current) by approximately 40% and potentiation of the action of GABA by approximately 100%. The mechanisms of TRP action in crayfish are not known, but a plausible pathway is a TRP-dependent elevation of intracellular Ca(2+) that reduces photoreceptor sensitivity in arthropods. Although the mechanisms are not established, the results indicate that in crayfish photoreceptors TRP displays actions on two time scales and can exert profound modulatory control over cell function.

  15. Friedreich ataxia: failure of GABA-ergic and glycinergic synaptic transmission in the dentate nucleus.

    PubMed

    Koeppen, Arnulf H; Ramirez, R Liane; Becker, Alyssa B; Feustel, Paul J; Mazurkiewicz, Joseph E

    2015-02-01

    Atrophy of large neurons in the dentate nucleus (DN) is an important pathologic correlate of neurologic disability in patients with Friedreich ataxia (FA). Thinning of the DN was quantified in 29 autopsy cases of FA and 2 carriers by measuring the thickness of the gray matter ribbon on stains with anti-glutamic acid decarboxylase, the rate-limiting enzyme in the biosynthesis of γ-amino-butyric acid (GABA). The DN was thinner than normal in all cases of FA, and atrophy correlated inversely with disease duration but not with age at onset or length of the homozygous guanine-adenine-adenine trinucleotide expansions. In 13 of the FA cases, frozen DN tissue was available for assay of frataxin. Dentate nucleus atrophy was more severe when frataxin was very low. Immunohistochemical staining for glutamic acid decarboxylase revealed grumose reaction and preservation of small GABA-ergic neurons in the DN of FA patients. Residual small DN neurons and varicose axons also contained the glycine transporter 2, identifying them as glycinergic. Immunohistochemistry also confirmed severe loss of GABA-A and glycine receptors in the DN with comparable depletion of the receptor-anchoring protein gephyrin. Thus, loss of gephyrin and failure to position GABA-A and glycine receptors correctly may reduce trophic support of large DN neurons and contribute to their atrophy. By contrast, Purkinje cells may escape retrograde atrophy in FA by issuing new axonal sprouts to small surviving DN neurons where they form reparative grumose clusters.

  16. Phenotypic expression of marrow cells when grown on various substrata.

    PubMed

    Fried, A; Shamay, A; Wientroub, S; Benayahu, D

    1996-05-01

    Our aim was to study the role of various extracellular matrices (ECM) on growth and differentiation of marrow stromal cells in vitro. Morphology changes, gene expression, and enzymatic activities were monitored in stromal osteoblastic MBA-15 and adipocytic 14F1.1 cells. These stromal cells were plated on dishes precoated with different substrata, such as matrigel (basement membrane), collagen type I, and endothelial ECM, and compared with cells plated on protein-free dishes. Striking morphological differences were observed when the cells grew on these different substrata. Changes in cell shape and growth also led to differential mRNA expression and enzymatic activities. When MBA-15 cells were plated on collagen, there was a decrease in mRNA for alkaline phosphatase (ALK-P), osteopontin (OP), and osteonectin (ON), and an increase in mRNA for procollagen (I). A differential effect was noted on 14F1.1 cells, the mRNA for ALK-P increased, the expressions of OP and ON lowered, and no expression for procollagen (I) was monitored. MBA-15 cells cultured on matrigel had decreased mRNA for ALK-P and OP, while they had increased ON mRNA expression and remained unchanged for procollagen I. No change in mRNA expression by 14F1.1 cells was monitored when cultured on matrigel. Functional enzymatic activities of ALK-P markedly decreased in MBA-15 cells cultured on various substrata, and increased or were unchanged in 14F1.1 cells. An additional enzyme, neutral endopeptidase (CD10/NEP), altered differentially in both cell types; this enzymatic activity increased or was unchanged when cells were cultured on these matrices. The results indicate a specific role for different ECM on various stromal cell types and their function. PMID:9173088

  17. The inhibitory neurotransmitter GABA evokes long‐lasting Ca2+ oscillations in cortical astrocytes

    PubMed Central

    Mariotti, Letizia; Losi, Gabriele; Sessolo, Michele; Marcon, Iacopo

    2015-01-01

    Studies over the last decade provided evidence that in a dynamic interaction with neurons glial cell astrocytes contribut to fundamental phenomena in the brain. Most of the knowledge on this derives, however, from studies monitoring the astrocyte Ca2+ response to glutamate. Whether astrocytes can similarly respond to other neurotransmitters, including the inhibitory neurotransmitter GABA, is relatively unexplored. By using confocal and two photon laser‐scanning microscopy the astrocyte response to GABA in the mouse somatosensory and temporal cortex was studied. In slices from developing (P15‐20) and adult (P30‐60) mice, it was found that in a subpopulation of astrocytes GABA evoked somatic Ca2+ oscillations. This response was mediated by GABAB receptors and involved both Gi/o protein and inositol 1,4,5‐trisphosphate (IP3) signalling pathways. In vivo experiments from young adult mice, revealed that also cortical astrocytes in the living brain exibit GABAB receptor‐mediated Ca2+ elevations. At all astrocytic processes tested, local GABA or Baclofen brief applications induced long‐lasting Ca2+ oscillations, suggesting that all astrocytes have the potential to respond to GABA. Finally, in patch‐clamp recordings it was found that Ca2+ oscillations induced by Baclofen evoked astrocytic glutamate release and slow inward currents (SICs) in pyramidal cells from wild type but not IP3R2−/− mice, in which astrocytic GABAB receptor‐mediated Ca2+ elevations are impaired. These data suggest that cortical astrocytes in the mouse brain can sense the activity of GABAergic interneurons and through their specific recruitment contribut to the distinct role played on the cortical network by the different subsets of GABAergic interneurons. GLIA 2016;64:363–373 PMID:26496414

  18. BAFF suppresses IL-15 expression in B cells.

    PubMed

    Ma, Ning; Xing, Chen; Xiao, He; He, Youdi; Han, Gencheng; Chen, Guojiang; Hou, Chunmei; Marrero, Bernadette; Wang, Yujuan; Zhang, Shengquan; Shen, Beifen; Li, Yan; Wang, Renxi

    2014-05-01

    Clinical trials have shown that BAFF inhibitors do not reduce memory B cell levels but can reduce the number of mature B cells. It remains uncertain whether BAFF affects memory-maintaining cytokines such as IL-15. We found that BAFF suppressed IL-15 expression in B cells from lupus-like or experimental allergic encephalomyelitis mice. When BAFF was blocked with atacicept-IgG, IL-15 expression was upregulated in lupus-like or experimental allergic encephalomyelitis mice. Finally, we showed that BAFF suppressed IL-15 expression in transitional 2 B cells by reducing Foxo1 expression and inducing Foxo1 phosphorylation. This study suggests that BAFF suppresses IL-15 expression in autoimmune diseases, and this opens up the possible opportunity for the clinical application of BAFF- and IL-15-specific therapeutic agents.

  19. Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs cycle in rat pancreatic islets.

    PubMed

    Hernández-Fisac, Inés; Fernández-Pascual, Sergio; Ortsäter, Henrik; Pizarro-Delgado, Javier; Martín del Río, Rafael; Bergsten, Peter; Tamarit-Rodriguez, Jorge

    2006-11-15

    OMP (oxo-4-methylpentanoic acid) stimulates by itself a biphasic secretion of insulin whereas L-leucine requires the presence of L-glutamine. L-Glutamine is predominantly converted into GABA (gamma-aminobutyric acid) in rat islets and L-leucine seems to promote its metabolism in the 'GABA shunt' [Fernández-Pascual, Mukala-Nsengu-Tshibangu, Martín del Río and Tamarit-Rodríguez (2004) Biochem. J. 379, 721-729]. In the present study, we have investigated how 10 mM OMP affects L-glutamine metabolism to uncover possible differences with L-leucine that might help to elucidate whether they share a common mechanism of stimulation of insulin secretion. In contrast with L-leucine, OMP alone stimulated a biphasic insulin secretion in rat perifused islets and decreased the islet content of GABA without modifying its extracellular release irrespective of the concentration of L-glutamine in the medium. GABA was transaminated to L-leucine whose intracellular concentration did not change because it was efficiently transported out of the islet cells. The L-[U-14C]-Glutamine (at 0.5 and 10.0 mM) conversion to 14CO2 was enhanced by 10 mM OMP within 30% and 70% respectively. Gabaculine (250 microM), a GABA transaminase inhibitor, suppressed OMP-induced oxygen consumption but not L-leucine- or glucose-stimulated respiration. It also suppressed the OMP-induced decrease in islet GABA content and the OMP-induced increase in insulin release. These results support the view that OMP promotes islet metabolism in the 'GABA shunt' generating 2-oxo-glutarate, in the branched-chain alpha-amino acid transaminase reaction, which would in turn trigger GABA deamination by GABA transaminase. OMP, but not L-leucine, suppressed islet semialdehyde succinic acid reductase activity and this might shift the metabolic flux of the 'GABA shunt' from gamma-hydroxybutyrate to succinic acid production.

  20. Cyclin Dl expression in B-cell non Hodgkin lymphoma.

    PubMed

    Aref, Salah; Mossad, Y; El-Khodary, T; Awad, M; El-Shahat, E

    2006-10-01

    Disorders of the cell cycle regulatory machinery play a key role in the pathogenesis of cancer. Over-expression of cyclin D1 protein has been reported in several solid tumors and certain lymphoid malignancies, but little is known about the effect of its expression on clinical behavior and outcome in B-cell Non-Hodgkin lymphoma (NHL). In this study, we investigated the expression of cyclin Dl in group of patients with NHL and correlated the results with the clinical and laboratory data. The degree of expression of cyclin Dl protein was evaluated by flow cytometry in a group of NHL patients (n = 46) and in normal control group (n = 10). Cyclin Dl over expression was detected in 10 out of 46 (21.7%) patients; they were 5/5-mantle cell lymphoma (MCL) (100%) and 5/28 large B-cell lymphoma (17.8%). All other NHL subtypes showed normal cyclin D1 expression. The clinical signs (hepatomegaly, splenomegaly and B-symptoms, clinical staging) and laboratory data (hemoglobin, white cell count (WBCs), platelet count, and bone marrow infiltration) were not significantly different between NHL subgroup with cyclin Dl over expression and that with normal cyclin Dl expression. Serum lactic dehydrogenase (LDH) levels and lymphadenopathy were significantly higher in NHL group with cyclin D1 over expression as compared to those without. Also, cyclin D1 over expression is associated with poor outcome of NHL patients. Cyclin Dl over expression was evident among all cases of MCL and few cases of large B-cell lymphoma. Cyclin Dl over expression might be used as adjuvant tool for diagnosis of MCL; has role in NHL biology and is bad prognostic index in NHL. PMID:17607588

  1. Hair cell damage recruited Lgr5-expressing cells are hair cell progenitors in neonatal mouse utricle.

    PubMed

    Lin, Jinchao; Zhang, Xiaodong; Wu, Fengfang; Lin, Weinian

    2015-01-01

    Damage-activated stem/progenitor cells play important roles in regenerating lost cells and in tissue repair. Previous studies reported that the mouse utricle has limited hair cell regeneration ability after hair cell ablation. However, the potential progenitor cell population regenerating new hair cells remains undiscovered. In this study, we first found that Lgr5, a Wnt target gene that is not usually expressed in the neonatal mouse utricle, can be activated by 24 h neomycin treatment in a sub-population of supporting cells in the striolar region of the neonatal mouse utricle. Lineage tracing demonstrated that these Lgr5-positive supporting cells could regenerate new hair cells in explant culture. We isolated the damage-activated Lgr5-positive cells with flow cytometry and found that these Lgr5-positive supporting cells could regenerate hair cells in vitro, and self-renew to form spheres, which maintained the capacity to differentiate into hair cells over seven generations of passages. Our results suggest that damage-activated Lgr5-positive supporting cells act as hair cell progenitors in the neonatal mouse utricle, which may help to uncover a potential route to regenerate hair cell in mammals.

  2. VISA is required for B cell expression of TLR7.

    PubMed

    Xu, Liang-Guo; Jin, Lei; Zhang, Bi-Cheng; Akerlund, Linda J; Shu, Hong-Bing; Cambier, John C

    2012-01-01

    B cells play a critical role in the initialization and development of the systemic lupus erythematosus that is dependent on the expression of the endosomal ssRNA receptor TLR7. Previous studies have established that B cell expression of TLR7 is controlled by the type I IFN secreted by plasmacytoid dendritic cells. In this article, we report that VISA, also known as MAVS, IPS-1, and CardIf, essential for RIG-I/MDA5-mediated signaling following sensing of cytosolic RNA, regulate B cell expression of TLR7 and CD23. We found that B cells from a VISA(-/-) mouse express reduced TLR7 but normal basal levels of type I IFN. We also show that although IFN-β and TLR7 agonists synergize to promote TLR7 expression in VISA(-/-) B cells, they do not fully complement the defect seen in VISA(-/-) cells. Cell transfer experiments revealed that the observed effects of VISA(-/-) are B cell intrinsic. The reduced TLR7 expression in B cells is correlated with impaired TLR7 agonist-induced upregulation of activation markers CD69 and CD86, cell proliferation, production of IFN-α, TNF, and IL-12, and NF-κB activation. Finally, studies indicate that genetic background may influence the observed phenotype of our VISA(-/-) mice, because VISA(-/-) B cells differ in CD23 and TLR7 expression when on C57BL/6 versus 129Sv-C57BL/6 background. Thus, our findings suggest an unexpected link between VISA-mediated cytosolic RLR signaling and autoimmunity.

  3. Expression of MIF and CD74 in leukemic cell lines: correlation to DR expression destiny.

    PubMed

    Georgouli, Mirella; Papadimitriou, Lina; Glymenaki, Maria; Patsaki, Valia; Athanassakis, Irene

    2016-06-01

    Invariant chain (Ii) or CD74 is a non-polymorphic glycoprotein, which apart from its role as a chaperone dedicated to MHCII molecules, is known to be a high-affinity receptor for macrophage migration inhibitory factor (MIF). The present study aimed to define the roles of CD74 and MIF in the immune surveillance escape process. Towards this direction, the cell lines HL-60, Raji, K562 and primary pre-B leukemic cells were examined for expression and secretion of MIF. Flow cytometry analysis detected high levels of MIF and intracellular/membrane CD74 expression in all leukemic cells tested, while MIF secretion was shown to be inversely proportional to intracellular HLA-DR (DR) expression. In the MHCII-negative cells, IFN-γ increased MIF expression and induced its secretion in HL-60 and K562 cells, respectively. In K562 cells, CD74 (Iip33Iip35) was shown to co-precipitate with HLA-DOβ (DOβ), inhibiting thus MIF or DR binding. Induced expression of DOα in K562 (DOα-DOβ+) cells in different transfection combinations decreased MIF expression and secretion, while increasing surface DR expression. Thus, MIF could indeed be part of the antigen presentation process.

  4. Muscimol prevents long-lasting potentiation of dorsal horn field potentials in rats with chronic constriction injury exhibiting decreased levels of the GABA transporter GAT-1.

    PubMed

    Miletic, Gordana; Draganic, Pero; Pankratz, Matthew T; Miletic, Vjekoslav

    2003-09-01

    The inhibitory activity of gamma-aminobutyric acid (GABA) is considered critical in setting the conditions for synaptic plasticity, and many studies support an important role of GABA in the suppression of nociceptive transmission in the dorsal horn. Consequently, any injury-induced modification of the GABA action has the potential to critically modify spinal synaptic plasticity. We have previously reported that chronic constriction injury of the sciatic nerve was accompanied by long-lasting potentiation of superficial spinal dorsal horn field potentials following high-frequency tetanus. In this study we examined whether the GABA-A receptor agonist muscimol would modify post-tetanic responses in rats with chronic constriction injury. In animals exhibiting maximal thermal hyperalgesia as one sign of neuropathic pain 7 days after loose ligation of the sciatic nerve, spinal application of muscimol (5, 10 or 20 microg) before the high-frequency (50 Hz) tetanus produced a long-lasting depression (rather than potentiation) of spinal dorsal horn field potentials. In separate but related Western immunoblot experiments, we also established that the chronic constriction injury was accompanied by significant decreases in the content of the GABA transporter GAT-1. These data demonstrated that GABA-A receptor agonists may effectively influence the expression of long-lasting synaptic plasticity in the spinal dorsal horn, and that an injury-induced loss in GABA transporter content may have contributed to a depletion of GABA from its terminals within the spinal dorsal horn. These data lent further support to the notion that the loss of GABA inhibition may have important consequences for the development of neuropathic pain. PMID:14499453

  5. Manganese accumulation in membrane fractions of primary astrocytes is associated with decreased γ-aminobutyric acid (GABA) uptake, and is exacerbated by oleic acid and palmitate.

    PubMed

    Fordahl, Steve C; Erikson, Keith M

    2014-05-01

    Manganese (Mn) exposure interferes with GABA uptake; however, the effects of Mn on GABA transport proteins (GATs) have not been identified. We sought to characterize how Mn impairs GAT function in primary rat astrocytes. Astrocytes exposed to Mn (500 μM) had significantly reduced (3)H-GABA uptake despite no change in membrane or cytosolic GAT3 protein levels. Co-treatment with 100 μM oleic or palmitic acids (both known to be elevated in Mn neurotoxicity), exacerbated the Mn-induced decline in (3)H-GABA uptake. Mn accumulation in the membrane fraction of astrocytes was enhanced with fatty acid administration, and was negatively correlated with (3)H-GABA uptake. Furthermore, control cells exposed to Mn only during the experimental uptake had significantly reduced (3)H-GABA uptake, and the addition of GABA (50 μM) blunted cytosolic Mn accumulation. These data indicate that reduced GAT function in astrocytes is influenced by Mn and fatty acids accumulating at or interacting with the plasma membrane.

  6. Biased Allelic Expression in Human Primary Fibroblast Single Cells

    PubMed Central

    Borel, Christelle; Ferreira, Pedro G.; Santoni, Federico; Delaneau, Olivier; Fort, Alexandre; Popadin, Konstantin Y.; Garieri, Marco; Falconnet, Emilie; Ribaux, Pascale; Guipponi, Michel; Padioleau, Ismael; Carninci, Piero; Dermitzakis, Emmanouil T.; Antonarakis, Stylianos E.

    2015-01-01

    The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability. PMID:25557783

  7. Optimizing transient recombinant protein expression in mammalian cells.

    PubMed

    Hopkins, Ralph F; Wall, Vanessa E; Esposito, Dominic

    2012-01-01

    Transient gene expression (TGE) in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells. The rapidly increasing need for recombinant proteins requires further improvements in TGE technology. While a great deal of focus has been directed toward optimizing the secretion of antibodies and other naturally secreted targets, much less work has been done on ways to improve cytoplasmic expression in mammalian cells. The benefits to protein production in mammalian cells, particularly for eukaryotic proteins, should be very significant - glycosylation and other posttranslational modifications will likely be native or near-native, solubility and protein folding would likely improve overexpression in heterologous hosts, and expression of proteins in their proper intracellular compartments is much more likely to occur. Improvements in this area have been slow, however, due to limited development of the cell culture processes needed for low-cost, higher-throughput expression in mammalian cells, and the relatively low diversity of DNA vectors for protein production in these systems. Here, we describe how the use of recombinational cloning, coupled with improvements in transfection protocols which increase speed and lower cost, can be combined to make mammalian cells much more amenable for routine recombinant protein expression. PMID:21987258

  8. VTA Projection Neurons Releasing GABA and Glutamate in the Dentate Gyrus.

    PubMed

    Ntamati, Niels R; Lüscher, Christian

    2016-01-01

    Both dopamine and nondopamine neurons from the ventral tegmental area (VTA) project to a variety of brain regions. Here we examine nondopaminergic neurons in the mouse VTA that send long-range projections to the hippocampus. Using a combination of retrograde tracers, optogenetic tools, and electrophysiological recordings, we show that VTA GABAergic axons make synaptic contacts in the granule cell layer of the dentate gyrus, where we can elicit small postsynaptic currents. Surprisingly, the currents displayed a partial sensitivity to both bicuculline and NBQX, suggesting that these mesohippocampal neurons corelease both GABA and glutamate. Finally, we show that this projection is functional in vivo and its stimulation reduces granule cell-firing rates under anesthesia. Altogether, the present results describe a novel connection between GABA and glutamate coreleasing of cells of the VTA and the dentate gyrus. This connection could be relevant for a variety of functions, including reward-related memory and neurogenesis. PMID:27648470

  9. VTA Projection Neurons Releasing GABA and Glutamate in the Dentate Gyrus

    PubMed Central

    2016-01-01

    Abstract Both dopamine and nondopamine neurons from the ventral tegmental area (VTA) project to a variety of brain regions. Here we examine nondopaminergic neurons in the mouse VTA that send long-range projections to the hippocampus. Using a combination of retrograde tracers, optogenetic tools, and electrophysiological recordings, we show that VTA GABAergic axons make synaptic contacts in the granule cell layer of the dentate gyrus, where we can elicit small postsynaptic currents. Surprisingly, the currents displayed a partial sensitivity to both bicuculline and NBQX, suggesting that these mesohippocampal neurons corelease both GABA and glutamate. Finally, we show that this projection is functional in vivo and its stimulation reduces granule cell-firing rates under anesthesia. Altogether, the present results describe a novel connection between GABA and glutamate coreleasing of cells of the VTA and the dentate gyrus. This connection could be relevant for a variety of functions, including reward-related memory and neurogenesis. PMID:27648470

  10. VTA Projection Neurons Releasing GABA and Glutamate in the Dentate Gyrus

    PubMed Central

    2016-01-01

    Abstract Both dopamine and nondopamine neurons from the ventral tegmental area (VTA) project to a variety of brain regions. Here we examine nondopaminergic neurons in the mouse VTA that send long-range projections to the hippocampus. Using a combination of retrograde tracers, optogenetic tools, and electrophysiological recordings, we show that VTA GABAergic axons make synaptic contacts in the granule cell layer of the dentate gyrus, where we can elicit small postsynaptic currents. Surprisingly, the currents displayed a partial sensitivity to both bicuculline and NBQX, suggesting that these mesohippocampal neurons corelease both GABA and glutamate. Finally, we show that this projection is functional in vivo and its stimulation reduces granule cell-firing rates under anesthesia. Altogether, the present results describe a novel connection between GABA and glutamate coreleasing of cells of the VTA and the dentate gyrus. This connection could be relevant for a variety of functions, including reward-related memory and neurogenesis.

  11. Regulated expression of erythropoietin by two human hepatoma cell lines

    SciTech Connect

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  12. [GABA(A)-Coupled Cl-/HCO3(-)-ATPase: Candidate for an Novel Primary Active Transporter in Neuronal Membranes].

    PubMed

    Menzikov, S A

    2015-01-01

    Cl(-)-transport systems in cell membranes from various origins (including neurons) play an important role in different processes of their vital functions. Various transport mechanisms involved in the maintenance of intracellular concentration of Cl- that differs from concentration equilibrium have been considered. This review provides the biochemical properties of the GABA(A)-coupled Cl-/HCO3(-)-ATPase which is a candidate for an novel primary active system in neuronal membranes. Special emphasis has been placed on a review of the prerequisites for the existence of the GABA(A)-coupled ATPase. This work provides data for the benefit not only functional but also the alleged structural coupling of the enzyme with GABA(A)-receptors. It is concluded on the importance of the found ATPase in primary active transport processes across the plasma membrane of neuronal cells with different level of the organization.

  13. ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells

    PubMed Central

    Moreb, Jan S; Baker, Henry V; Chang, Lung-Ji; Amaya, Maria; Lopez, M Cecilia; Ostmark, Blanca; Chou, Wayne

    2008-01-01

    Background Aldehyde dehydrogenase isozymes ALDH1A1 and ALDH3A1 are highly expressed in non small cell lung cancer. Neither the mechanisms nor the biologic significance for such over expression have been studied. Methods We have employed oligonucleotide microarrays to analyze changes in gene profiles in A549 lung cancer cell line in which ALDH activity was reduced by up to 95% using lentiviral mediated expression of siRNA against both isozymes (Lenti 1+3). Stringent analysis methods were used to identify gene expression patterns that are specific to the knock down of ALDH activity and significantly different in comparison to wild type A549 cells (WT) or cells similarly transduced with green fluorescent protein (GFP) siRNA. Results We confirmed significant and specific down regulation of ALDH1A1 and ALDH3A1 in Lenti 1+3 cells and in comparison to 12 other ALDH genes detected. The results of the microarray analysis were validated by real time RT-PCR on RNA obtained from Lenti 1+3 or WT cells treated with ALDH activity inhibitors. Detailed functional analysis was performed on 101 genes that were significantly different (P < 0.001) and their expression changed by ≥ 2 folds in the Lenti 1+3 group versus the control groups. There were 75 down regulated and 26 up regulated genes. Protein binding, organ development, signal transduction, transcription, lipid metabolism, and cell migration and adhesion were among the most affected pathways. Conclusion These molecular effects of the ALDH knock-down are associated with in vitro functional changes in the proliferation and motility of these cells and demonstrate the significance of ALDH enzymes in cell homeostasis with a potentially significant impact on the treatment of lung cancer. PMID:19025616

  14. Bmi1 is expressed in vivo in intestinal stem cells

    PubMed Central

    Sangiorgi, Eugenio; Capecchi, Mario R

    2010-01-01

    Bmi1 plays an essential part in the self-renewal of hematopoietic and neural stem cells. To investigate its role in other adult stem cell populations, we generated a mouse expressing a tamoxifen-inducible Cre from the Bmi1 locus. We found that Bmi1 is expressed in discrete cells located near the bottom of crypts in the small intestine, predominantly four cells above the base of the crypt (+4 position). Over time, these cells proliferate, expand, self-renew and give rise to all the differentiated cell lineages of the small intestine epithelium. The induction of a stable form of b-catenin in these cells was sufficient to rapidly generate adenomas. Moreover, ablation of Bmi1+ cells using a Rosa26 conditional allele, expressing diphtheria toxin, led to crypt loss. These experiments identify Bmi1 as an intestinal stem cell marker in vivo. Unexpectedly, the distribution of Bmi1-expressing stem cells along the length of the small intestine suggested that mammals use more than one molecularly distinguishable adult stem cell subpopulation to maintain organ homeostasis. PMID:18536716

  15. Regulation of global gene expression and cell proliferation by APP

    PubMed Central

    Wu, Yili; Zhang, Si; Xu, Qin; Zou, Haiyan; Zhou, Weihui; Cai, Fang; Li, Tingyu; Song, Weihong

    2016-01-01

    Down syndrome (DS), caused by trisomy of chromosome 21, is one of the most common genetic disorders. Patients with DS display growth retardation and inevitably develop characteristic Alzheimer’s disease (AD) neuropathology, including neurofibrillary tangles and neuritic plaques. The expression of amyloid precursor protein (APP) is increased in both DS and AD patients. To reveal the function of APP and elucidate the pathogenic role of increased APP expression in DS and AD, we performed gene expression profiling using microarray method in human cells overexpressing APP. A set of genes are significantly altered, which are involved in cell cycle, cell proliferation and p53 signaling. We found that overexpression of APP inhibits cell proliferation. Furthermore, we confirmed that the downregulation of two validated genes, PSMA5 and PSMB7, inhibits cell proliferation, suggesting that the downregulation of PSMA5 and PSMB7 is involved in APP-induced cell proliferation impairment. Taken together, this study suggests that APP regulates global gene expression and increased APP expression inhibits cell proliferation. Our study provides a novel insight that APP overexpression may contribute to the growth impairment in DS patients and promote AD pathogenesis by inhibiting cell proliferation including neural stem cell proliferation and neurogenesis. PMID:26936520

  16. Expression of pleiotrophin in small cell lung cancer.

    PubMed

    Wang, H Q; Wang, J

    2015-01-01

    Pleiotrophin (PTN) is a kind of heparin binding growth factor closely related to tumor progression. This study aimed to discuss the significance of the expression of PTN in benign and malignant lung cancer tissues, especially small cell lung cancer. Lung cancer samples were collected for study and lung tissue samples with benign lesions were taken as controls. The expression of PTN was detected using tissue chip combined with the immunohistochemical method, and the differences of small cell lung cancer with non-small cell lung cancer and benign lesion tissue were compared. It was found that PTN expression was mainly located in the cytoplasm and membrane of cells; PTN expression in the lung cancer group was higher than that in the control group (p < 0.01), and PTN expression in the small cell cancer group was higher than that in the squamous carcinoma group and glandular cancer group (p < 0.05). In addition, PTN expression quantity in patients with lung cancer were in close correlation with TNM staging, pathological type and tumor differentiation degree (p < 0.05). PTN was found to express abnormally high in lung cancer, especially small cell lung cancer tissue. PTN is most likely to be a new tumor marker for diagnosis and prognosis of lung cancer. PMID:25864755

  17. Effects of c-myc expression on cell cycle progression.

    PubMed Central

    Hanson, K D; Shichiri, M; Follansbee, M R; Sedivy, J M

    1994-01-01

    We used targeted homologous recombination to disrupt one c-myc gene copy in a diploid fibroblast cell line and found that a twofold reduction in Myc expression resulted in lower exponential growth rates and a lengthening of the G0-to-S-phase transition (M. Shichiri, K. D. Hanson and J. M. Sedivy, Cell Growth Differ. 4:93-104, 1993). Myc is a transcription factor, and the number of target genes whose regulation could result in differential growth rates may be very large. We have approached this problem by examining effects of reduced c-myc expression in three broad areas: (i) secretion of growth factors, (ii) expression of growth factor receptors, and (iii) intracellular signal transduction between Myc and components of the intrinsic cell cycle clock. We have found no evidence that differential medium conditioning can account for the growth phenotypes. Likewise, the expression of receptors for platelet-derived growth factor, epidermal growth factor, basic fibroblast growth factor, and insulin-like growth factor I was the same in diploid and heterozygous cells (platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, and insulin-like growth factor are the sole growth factors required by these cells for growth in serum-free medium). In contrast, expression of cyclin E, cyclin A, and Rb phosphorylation were delayed when quiescent c-myc heterozygous cells were stimulated to enter the cell cycle. Expression of cyclin D1, cyclin D3, and Cdk2 was not affected. The timing of cyclin E induction was the earliest observable effect of reduced Myc expression. Our data indicate that Myc contributes to regulation of proliferation by a cell-autonomous mechanism that involves the modulation of cyclin E expression and, consequently, progression through the restriction point of the cell cycle. Images PMID:8065309

  18. 6,3'-Dinitroflavone is a low efficacy modulator of GABA(A) receptors.

    PubMed

    Furtmueller, Roman; Furtmueller, Birgit; Ramerstorfer, Joachim; Paladini, Alejandro C; Wasowski, Cristina; Marder, Mariel; Huck, Sigismund; Sieghart, Werner

    2008-09-01

    6,3'-Dinitroflavone (6,3'-DNF) is a synthetic flavone derivative that exerts anxiolytic effects in the elevated plus maze. Based on the finding that this effect is blocked by Ro15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate) which is a specific antagonist at the benzodiazepine binding site of GABA(A) receptors we investigated the interaction of 6,3'-DNF with several recombinant GABA(A) receptor subtypes. Inhibition of [(3)H]flunitrazepam binding to recombinant GABA(A) receptors in transiently transfected HEK293 cells indicated that 6,3'-DNF exhibited the highest affinity for GABA(A) receptors composed of alpha1beta2gamma2 subunits and a 2-20 fold lower affinity for homologous receptors containing alpha2, alpha3, or alpha5 subunits. Two-electrode voltage-clamp experiments in Xenopus oocytes indicated that 6,3'-DNF does not induce chloride flux in the absence of GABA, but exerts low efficacy inverse agonistic modulatory effects on GABA-elicited currents in the GABA(A) receptor subtypes alpha1beta2gamma2 and alpha5beta2gamma2. In the subtypes alpha2beta2gamma2, alpha3beta2gamma2, alpha4beta2gamma2, alpha6beta2gamma2 or alpha4beta2delta and alpha4beta3delta, 6,3'-DNF exerts either none or very low efficacy positive modulatory effects. In contrast, 100 nM Ro15-1788 exhibited weak to moderate partial agonistic effects on each receptor investigated. These data indicate that Ro15-1788 only can antagonize the weak inverse agonist effects of 6,3'-DNF on alpha1beta2gamma2 and alpha5beta2gamma2 receptors, but will enhance the weak agonistic effects on the other receptor subtypes investigated. The possible mechanism of the Ro15-1788 sensitive anxiolytic effect of 6,3'-DNF is discussed.

  19. Expression of heparanase in basal cell carcinoma and squamous cell carcinoma*

    PubMed Central

    Pinhal, Maria Aparecida Silva; Almeida, Maria Carolina Leal; Costa, Alessandra Scorse; Theodoro, Thérèse Rachell; Serrano, Rodrigo Lorenzetti; Machado Filho, Carlos D'Apparecida Santos

    2016-01-01

    Background Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. Objectives Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). Methods Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). Results The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. Conclusion The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.

  20. Cytoskeletal Expression and Remodeling in Pluripotent Stem Cells

    PubMed Central

    Boraas, Liana C.; Guidry, Julia B.; Pineda, Emma T.; Ahsan, Tabassum

    2016-01-01

    Many emerging cell-based therapies are based on pluripotent stem cells, though complete understanding of the properties of these cells is lacking. In these cells, much is still unknown about the cytoskeletal network, which governs the mechanoresponse. The objective of this study was to determine the cytoskeletal state in undifferentiated pluripotent stem cells and remodeling with differentiation. Mouse embryonic stem cells (ESCs) and reprogrammed induced pluripotent stem cells (iPSCs), as well as the original un-reprogrammed embryonic fibroblasts (MEFs), were evaluated for expression of cytoskeletal markers. We found that pluripotent stem cells overall have a less developed cytoskeleton compared to fibroblasts. Gene and protein expression of smooth muscle cell actin, vimentin, lamin A, and nestin were markedly lower for ESCs than MEFs. Whereas, iPSC samples were heterogeneous with most cells expressing patterns of cytoskeletal proteins similar to ESCs with a small subpopulation similar to MEFs. This indicates that dedifferentiation during reprogramming is associated with cytoskeletal remodeling to a less developed state. In differentiation studies, it was found that shear stress-mediated differentiation resulted in an increase in expression of cytoskeletal intermediate filaments in ESCs, but not in iPSC samples. In the embryoid body model of spontaneous differentiation of pluripotent stem cells, however, both ESCs and iPSCs had similar gene expression for cytoskeletal proteins during early differentiation. With further differentiation, however, gene levels were significantly higher for iPSCs compared to ESCs. These results indicate that reprogrammed iPSCs more readily reacquire cytoskeletal proteins compared to the ESCs that need to form the network de novo. The strategic selection of the parental phenotype is thus critical not only in the context of reprogramming but also the ultimate functionality of the iPSC-differentiated cell population. Overall, this