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Splicing of human GABAB receptor subunit 1 (GABAB1) in non-alcoholic and alcoholic brainsLee, Chang Hoon 26 January 2012 (has links)
Gamma-aminobutyric acid type B (GABAB) receptor is a G protein coupled receptor (GPCR) that mediates decreased neural activity. It has two subunits, GABAB1 and GABAB2. Previous complementary DNA (cDNA) microarray data showed strong GABAB1 signals from human prefrontal cortex using an intron 4 region probe, and these studies indicated that novel intron 4 containing GABAB1 splicing variants exist. We cloned GABAB1k, l, m, and n including mouse GABAB1j. Expression of these variants are much lower than other major known splicing variants, but GABAB1k, l, m, and n levels are similar across brain tissues. GABAB1l and GABAB1m impair GABAB receptor induced function. To better define GABAB1 splicing in alcoholic brains, whole transcriptome shortgun sequencing (RNA-seq) experiments were proposed. Due to the complexity of GABAB1 splicing, we used gene specific libraries as well as whole transcriptome libraries to maximize GABAB1 specific splicing junction search. The splicing junction search data found that GABAB1 gene is 2 to 3 times longer than the previous known gene length. Extremely low expression at 5’ end exons was found, and GABAB1 exons were grouped based on expression levels. Chronic alcohol altered exon/intron expression and splicing junctions more than overall gene expression. Decreased exon expression at a GABA binding site, a transmembrane domain (TM), and a microRNA (miRNA) binding site may diminish the normal GABAB1 transcript population and compromise signal transduction following chronic alcohol exposure. This may explain why GABAB receptor agonists have therapeutic benefit in treating alcoholism. During the sequence mapping, read pile-ups and gaps were found from whole transcriptome libraries in known exons. These may prevent single nucleotide polymorphism (SNP) and splicing junction identification and gene expression calculations. Sequence analysis found sequence biases from their mapped reads. The major sequence biases were from RNaseIII RNA fragmentation and T4 polynucleotide kinase (T4PNK) reaction. Heat fragmentation and OptiKinase treatment removed the read pile-ups and gaps including the sequence biases. The identification of RNaseIII target sequences can be incorporated into methods of miRNA gene prediction. These data showed the complexity of GABAB1 receptor splicing and the perturbation of splicing by chronic alcohol abuse demonstrate the power of RNA-seq to provide new insight into gene expression and the role of GABAB receptors in alcoholism. In addition, many other important brain genes may have unexplored splicing variants which will be important for alcoholism and other psychiatric diseases. Also, new RNA-seq library constructions improved the quality of gene expression studies. / text
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The story of alpha-conotoxins, Vc1.1 and RgIA, on their journey to becoming therapeuticsReena Halai Unknown Date (has links)
Abstract The broad aim of this thesis is to structurally and functionally explore two α-conotoxins, from venomous sea snails, Vc1.1 and RgIA, in the hope of improving their journey to becoming analgesic therapeutics (introduction to conotoxins in Chapter 1). Vc1.1 is a two-disulfide peptide that is of interest as a potential therapeutic for the treatment of neuropathic pain. Despite investigations, limited structure-activity relationships have been conducted on this α-conotoxin. Consequently there is restricted insight into the interaction of this peptide with one of its analgesic targets, the α9α10 nicotinic acetylcholine receptor (nAChR). Late in this PhD project, the GABAB receptor was implicated as the possible target for conotoxins in neuropathic pain relief. However, there is still debate in the literature with regard to the true target of Vc1.1 and the α9α10 nAChR is still believed to be the target by some groups. This thesis predominantly focuses on the α9α10 nicotinic acetylcholine receptor. Chapter 4 of this thesis presents an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp or Lys (materials and methods described in Chapter 3) and examined using NMR spectroscopy (theory of NMR presented in Chapter 2), to determine the effects of the mutations on the structure of Vc1.1. The structural fold was found to be preserved in all peptides except where Pro was substituted. Chapter 5 explores the effect of these mutations on the blocking of acetylcholine (ACh)-evoked membrane currents at the α9α10 nAChR. Electrophysiological studies showed that the key residues for Vc1.1’s activity are Asp5-Arg7 and Asp11-Ile15, as changes at these positions resulted in the loss of activity at the α9α10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. Hence, Chapter 6 describes a second generation of mutants that was synthesized, namely N9G, N9I, N9L, S4R and S4K+N9A, all of which were more potent than Vc1.1 at both the rat α9α10 and the human α9/rat α10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the α3β2, α3β4 and α7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the α9α10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the α9α10 nAChR subtype and will help in the further development of Vc1.1 or its analogs as drugs. However, despite peptides exhibiting high degrees of potency and selectivity, such as Vc1.1 and RgIA, they are potentially hindered in their development as drugs due to their stability and bioavailability limitations, leading to invasive delivery techniques. Chapter 7 presents a range of cyclic RgIA analogs, tested at their targets the α9α10 nAChR and the GABAB receptor, that retain their activity and increase their stability in human serum relative to non-cyclic RgIA. NMR spectroscopy was used to determine the structure of the non-cyclic peptide and the cyclic peptide to confirm similarities in the global fold of the peptide. Structural perturbations and reduced activities were observed for cyclic RgIA analogs cyclized via linkers composed of three and four residues. Analogs with five, six and seven residues showed no structural perturbations, but differences in their activities at the different receptors. Because cRgIA-6 showed high potency for the GABAB receptor and lower potency for the α9α10 nAChR, this study has identified a GABAB selective peptide. Additionally, because the cRgIA-7 showed high potency for the α9α10 nAChR and low potency for the GABAB receptor, a α9α10 nAChR selective analog has also been identified. With improvements in these peptides against enzymatic attack, they show great potential on their path to becoming orally available analgesics as they may be able to withstand enzymatic conditions in the stomach.
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Lokalizace GABAB receptoru v dorzálním kochleárním jádře a sluchové kůře myši za fyziologických a patologických podmínek / Localization of GABAB receptor in the mouse dorsal cochlear nucleus and auditory cortex under physiological and pathological conditionsMelichar, Adolf January 2018 (has links)
GABAB receptors play an important role in regulation of neuronal excitability and stability of neural microcircuits. It is well known that dysregulation of slow GABAergic signalisation can lead to many pathological conditions (epilepsy, anxiety etc.). Current research indicates that the imbalance in the inhibitory transfer, caused by changes in the expression of GABABR in the auditory system could play an important role in the progression of tinnitus. The goal of the present thesis was to determine the distribution of the GABAB receptor and its auxiliary subunit KCTD12 in the mouse auditory cortex and the dorsal cochlear nucleus (DCN). Furthermore, a change in GABAB receptor localization in the DCN was observed in mice exposed to an acoustic stress. The GABAB receptor was expressed across the entire auditory cortex, both on the body and on the neuronal fibres. On the contrary, KCTD12 was found only in a particular subgroup of neurons that includes VIP (vasoactive intestinal peptide) and cholecystokinin positive interneurons., GABABR and KCTD12 protein were found in all layers and in all studied cells types (fusiform, cartwheel and stellate) of the DCN. Acoustic trauma of the WT mice resulted in GABAB receptor internalization specifically in fusiform cells that are the main projection neurons of the...
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Modulation of Neurotransmission by the GABAB ReceptorKantamneni, Sriharsha 20 December 2016 (has links)
No / Most inhibitory signals are mediated via γ-aminobutyric acid (GABA) receptors whereas glutamate receptors mediate most excitatory signals (Trends Neurosci 14:515–519, 1991; Annu Rev Neurosci 17:31–108, 1994). Many factors influence the regulation of excitatory and inhibitory synaptic inputs on a given neuron. One important factor is the subtype of neurotransmitter receptor present not only at the correct location to receive the appropriate signals but also their abundance at synapses (Pharmacol Rev 51: 7–61, 1999; Cold Spring Harb Perspect Biol 3, 2011). GABAB receptors are G-protein-coupled receptors and different subunits dimerise to form a functional receptor. GABAB receptor subunits are widely expressed in the brain and by assembling different isoform combinations and accessory proteins they produce variety of physiological and pharmacological profiles in mediating both inhibitory and excitatory neurotransmission. This chapter will describe the understanding of the molecular mechanisms underlying GABAB receptor regulation of glutamate and GABAA receptors and how they modulate excitatory and inhibitory neurotransmission.
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Association Between Gamma Aminobutyric Acid (gaba) Type B Receptors Gene Polymorphisms And Idiopathic Generalized EpilepsyEroglu, Ezgi 01 February 2012 (has links) (PDF)
Epilepsy is neurological disorder affecting 0.5 to 1% of the population all around the world. It is characterized by the seizures, which are the sudden alterations of behavior due to a temporary change in electrical functioning of the brain. Idiopathic generalized epilepsy (IGE) accounts for one-fifth of all the other epilepsy types, and several gene mutations were identified as the causes of IGE. In general, voltage-gated and ligand-gated ion channel mutations are linked with seizure formation. Gamma amino butyric acid (GABA), the most important inhibitory neurotransmitter of the central nervous system, and its receptors are commonly mentioned in the pathophysiology of epilepsies. Decrease in the inhibitory effect of GABA in neurons causes epileptic discharges resulting in seizure development.
The study population consisted of a total of 176 idiopathic generalized epilepsy (IGE) patients, 83 subjects having psychogenic non-epileptic seizures (PNES), 86 non-epileptic control subjects from Turkey. Total blood samples were obtained from Gü / lhane Military Medical Academy Hospital Neurology Department, Ankara. There was no statistically difference between the patient and control groups in terms of age. Genomic DNA isolations were performed and genotyping of G1465A and C59T polymorphisms of GABAB1 gene / rs1999501, rs967932, rs3780428 and rs944688 polymorphisms of GABAB2 gene were determined by PCR-RFLP technique.
In this study, GABAB1 G1465A polymorphic allele was not observed in Turkish population. For GABAB1 C59T polymorphism, polymorphic allele frequencies were found as 0.097 in IGE patients / 0.072 in PNES subjects and 0.105 in non-epileptic control subjects. No significant difference is identified for C59T polymorphism in all three groups.
Four SNPs of GABAB2 were studied / rs967932 was found to increase the risk of IGE 3.6-fold (P=0.031) compared to PNES subjects, polymorphic allele frequencies were found as 0.060 in IGE patients / 0.018 in PNES subjects and 0.035 in non-epileptic control subjects. For rs1999501 polymorphism, polymorphic allele frequencies were found as 0.077 in IGE patients / 0.048 in PNES subjects and 0.093 in non-epileptic control subjects. For rs3780428 polymorphism, polymorphic allele frequencies were found as 0.267 in IGE patients / 0.235 in PNES subjects and 0.256 in non-epileptic control subjects. For rs944688 polymorphism, polymorphic allele frequencies were found as 0.196 in IGE patients / 0.260 in PNES subjects and 0.227 in non-epileptic control subjects. No significant difference was identified for rs1999501, rs3780428 and rs944688 polymorphisms among IGE patients, PNES subjects and non-epileptic control groups.
IGE risk was 6.54-fold higher for subjects having combined GA genotype for rs967932 and GG genotype for rs3780428 when compared with PNES subjects (P=0.042). The combination of CC genotype for rs1999501, GG genotype for rs967932 and TT genotype for rs944688 had around 9-fold protective effect against IGE when both compared with PNES subjects (P=0.038) and non-epileptic control subjects (P=0.041).
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Maturation of GABAergic signaling during brainstem development / Die Änderung der GABAerger Signalwege in der HirnstammreifungTantalaki, Evangelia 05 July 2007 (has links)
No description available.
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Differential regulation of GABAB receptor trafficking by different modes of N-methyl-D-aspartate (NMDA) receptor signalingKantamneni, Sriharsha, Gonzàlez-Gonzàlez, I.M., Luo, J., Cimarosti, H., Jacobs, S.C., Jaafari, N., Henley, J.M. 2013 December 1924 (has links)
Yes / Inhibitory GABAB receptors (GABABRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABABRs are heterodimers of GABAB1 and GABAB2 subunits and here we show that the trafficking and surface expression of GABABRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABABR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABABRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABAB1 but decreases GABAB2 surface expression. The increase in surface GABAB1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABAB2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABABR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival. / BBSRC, MRC and the European Research Council
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Régulation des sous-types d’hétérodimères du récepteur GABAB dans la moelle épinière en conditions de douleurs neuropathiques : rôle des protéines partenairesPapon, Marie-Amélie 04 December 2009 (has links)
Dans le système nerveux central, le récepteur inhibiteur GABAB est un archétype des RCPGs hétérodimériques. Il est composé en effet de deux sous-unités, la sous-unité GABAB1 (B1a ou B1b) qui lie l’agoniste et la sous-unité GABAB2 couplée aux protéines G. L’activation de ce récepteur a un effet antinociceptif bien établi concernant les douleurs aiguës mais son effet reste cependant très limité en cas de douleurs neuropathiques. Notre hypothèse est que son activation et sa signalisation peuvent être altérées par des protéines partenaires, aboutissant à des processus de désinhibition dans la moelle épinière en conditions de neuropathie. Nos résultats mettent en évidence le rôle de deux protéines partenaires qui sont surexprimées en conditions douloureuses et qui diminuent l’activation du récepteur GABAB via deux mécanismes différents. D’un part, la protéine cytosolique 14-3-3? induit la dissociation de l’hétérodimère B1b/B2. Cette action a lieu principalement dans les compartiments post-synaptiques. D’autre part, la fibuline-2, protéine de la matrice extracellulaire diminue l’activation de l’hétérodimère B1a/B2. Il s’agit cette fois préférentiellement d’une action dans les compartiments pré-synaptiques. Des stratégies anti-sens (siRNA anti-14-3-3? ou anti-fibuline-2) ou des peptides de compétition sélectifs de l’interaction B1b/14-3-3? permettent de potentialiser les effets antinociceptifs d’un agoniste du récepteur sur un modèle animal de neuropathie. L’ensemble de ces résultats suggèrent que l’état d’oligomérisation des RCPGs peut être modulé in vivo par des protéines partenaires endogènes impliquées dans le développement ou le maintien d’états pathologiques de sensibilisation à la douleur. / In the central nervous system, the inhibitory GABAB receptor is an obligate heterodimeric GPCR that requires the association between GABAB1 (B1a or B1b) and GABAB2 subunits. The heterodimeric GABAB receptor activation has a well-known antinociceptive action in acute pain but its effect appears limited in pathological states. Our hypothesis is that the GABAB activation and signaling could be altered by partner proteins, thus resulting in desinhibition processes in the spinal cord. In the present study, we investigated the role of two partner proteins overexpressed in neuropathic states which decrease GABAB activation through two different mechanisms. On the one hand, the cytosolic 14-3-3? protein induces the dissociation of the heterodimer B1b/B2. This effect occurs in post-synaptic compartments. On the other hand, fibulin-2, an extracellular matrix protein, which decreases the activation of the heterodimer B1a/B2 localized preferentially in presynaptic compartments. Anti-sens strategies (anti-14-3-3? or anti-fibulin-2 siRNA) or competing peptides specific of 14-3-3?/B1b interaction, potentiate the antinociceptive effects of GABAB agonist in an animal model of neuropathic pain. Taken together, our data suggest that GPCR oligomeric state can be modulated in vivo by endogenous partners proteins that are involved in the development and the maintenance of pain sensitization.
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Neuronal hypothalamic plasticity in chickenSallagundala, Nagaraja 05 April 2007 (has links)
Aufgabe der elektrophysiologischen Studie zur Charakterisierung der neuronalen hypothalamischen Plastizität beim Haushuhn war es, den Einfluss des Alters sowie GABAerger Substanzen auf die Feuerrate und die Temperatursensitivität (thermischer Koeffizient: TC) von Hypothalamusneuronen mittels extrazellulärer Ableitungen in Hirnschnitten zu untersuchen. Im Vergleich zu adulten Vögeln und Säugetieren wurde bei juvenilen Hühnern eine hohe neuronale Kältesensitivität nachgewiesen, die offensichtlich eine spezifische Eigenschaft juveniler Vögel ist. Die Ontogenese der neuronalen hypothalamischen Thermosensitivität ist deutlich artspezifisch. Einige Neurone wiesen eine inherente Kältesensitivität auf. Eine mögliche zentrale Rolle kältesensitiver Neurone im Rahmen der Thermoregulation juveniler Hühner wurde postuliert. Muscimol und Baclofen hemmen signifikant die Feuerrate der Hypothalamusneurone, unabhängig von der jeweiligen Thermosensitivität. Demgegenüber bewirken Bicucullin und CGP35348 einem Anstieg der Feuerrate. Nur bei kältesensitiven Neuronen wurde der TC signifikant durch GABAB-Rezeptor-Liganden verändert (signifikant erhöht durch Baclofen und durch CGP35348 gehemmt). Der Effekt von Muscimol und Baclofen auf Feuerrate und TC wurde durch Co-Perfusion mit einer 10-fach höheren Konzentration der entsprechenden Antagonisten Bicucullin und CGP35348 aufgehoben. Der wesentliche GABAerge Einfluss auf thermosensitive und –insensitive Hypothalamusneurone ist mit dem bei Säugetieren nachgewiesenen vergleichbar. Der einzige Unterschied betrifft die GABAB-Rezeptor vermittelte Änderung des TC. Beim Hühnerküken betraf dies die kältesensitiven und beim Säugetier die wärmesensitiven Neurone. Der grundlegende Mechanismus der GABAergen Beeinflussung thermosensitiver und –insensitiver Neurone scheint einen älteren evolutionären Ursprung zu haben. Eine funktionelle Rolle GABAerger Substanzen im Rahmen der zentralen Kontrolle der Körpertemperatur beim Vogel ist möglich. / In the present electrophysiological studies, characterization of neuronal hypothalamic plasticity in the chicken aims to investigate the influence of age during development by extracellular recordings. High neuronal cold sensitivity has been found in juvenile chicken in contrast to adult mammals and birds. High hypothalamic cold sensitivity seems to be a specific characteristic feature in juvenile birds. Between species a species specificity of the early development of neuronal hypothalamic thermosensitivity could be clearly demonstrated. Existence of inherent nature to a certain degree suggests a possible thermoregulatory role of cold-sensitive neurons in chicken. The effects of the GABAergic substances on neuronal tonic activity (firing rate) and temperature sensitivity (temperature coefficient) in hypothalamic neurons have been examined. Muscimol and baclofen in equimolar concentrations significantly inhibited tonic activity, regardless of their type of thermosensitivity. In contrast bicuculline and CGP 35348 increased firing rate. Temperature coefficient was significantly changed by ligands of GABAB receptors, restricted to cold-sensitive neurons. The TC was significantly increased by baclofen and significantly decreased by CGP 35348. Effects of muscimol and baclofen on firing rate and TC were prevented by co-perfusion of appropriate antagonists bicuculline and CGP 35348, respectively in tenfold higher concentration. Thus the main effects of GABA in chicken are similar with that described in mammals. The only difference is in respect of the GABAB receptors mediated change restricted to cold-sensitive neurons in chicken but in mammals only seen in warm-sensitive neurons. However, the results indicate that the fundamental mechanism of GABAergic influence in chicken are conserved during evolution. The response of hypothalamic neurons to temperature changes suggest a possible functional role of GABAergic substances in the control of body temperature in birds.
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Postnatale Entwicklung des GABAergen Systems im Gehirn der Maus / Postnatal development of the GABAergic system in mouse brainRitter, Barbara 27 June 2001 (has links)
No description available.
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