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George P. Hess - One of the best experts on this subject based on the ideXlab platform.
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Rapid chemical kinetic techniques for investigations of Neurotransmitter Receptors expressed in Xenopus oocytes
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Li Niu, Raymond W. Vazquez, Georg Nagel, Thomas Friedrich, Ernst Bamberg, Robert E. Oswald, George P. HessAbstract:Xenopus laevis oocytes have been used extensively during the past decade to express and study Neurotransmitter Receptors of various origins and subunit composition and also to express and study Receptors altered by site-specific mutations. Interpretations of the effects of structural differences on receptor mechanisms were, however, hampered by a lack of rapid chemical reaction techniques suitable for use with oocytes. Here we describe flow and photolysis techniques, with 2-ms and 100-μs time resolution, respectively, for studying Neurotransmitter Receptors in giant (≈20-μm diameter) patches of oocyte membranes, using muscle and neuronal acetylcholine Receptors as examples. With these techniques, we find that the muscle receptor in BC3H1 cells and the same receptor expressed in oocytes have comparable kinetic properties. This finding is in contrast to previous studies and raises questions regarding the interpretations of the many studies of Receptors expressed in oocytes in which an insufficient time resolution was available. The results obtained indicate that the rapid reaction techniques described here, in conjunction with the oocyte expression system, will be useful in answering many outstanding questions regarding the structure and function of diverse Neurotransmitter Receptors.
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Determination of the chemical mechanism of Neurotransmitter receptor-mediated reactions by rapid chemical kinetic techniques.
Biochemistry, 1993Co-Authors: George P. HessAbstract:Rapid chemical reaction techniques play an important role in unraveling the mechanism of reactions mediated by soluble proteins, including enzymes involved in the regulation of intracellular processes and the biosynthesis of proteins and nucleic acids. Regulatory proteins change conformation rapidly and must, therefore, be studied in the microsecond-to-millisecond time region. Similar techniques for investigating reactions mediated by membrane-bound Neurotransmitter Receptors were not available, and the mechanisms of the reactions are poorly understood. These proteins must be studied in a membrane-bound form in cells or vesicles, over a wide range of reactant concentrations, and in the microsecond-to-millisecond time region. Four rapid-mixing techniques for making kinetic measurements in the microsecond and millisecond time regions have now been developed for investigating Neurotransmitter Receptors in the membranes of neurons and muscle cells, thus extending chemical kinetic approaches to membrane-bound proteins and intercellular processes. Neurotransmitter Receptors regulate transmission of signals between neurons (approximately 10(12) in the human nervous system), thereby allowing perception of stimuli, integration and storage of information, and reaction to the environment. Six structurally related Neurotransmitter Receptors, and many isoforms, have been identified by use of recombinant DNA technology. Modern electrophysiological techniques show that these different proteins, upon binding a specific chemical signal (Neurotransmitter), transiently open transmembrane channels, which are characterized by their ion selectivity, conductance, and lifetime. To be able to account for the receptor-mediated voltage changes that trigger signal transmission between cells, we still need to know the concentration of open receptor-channels. This concentration changes with time and is affected by the concentration of Neurotransmitter. Rapid reaction techniques are particularly suitable for determining the relationship between Neurotransmitter concentration and the time-dependent concentration of the open receptor-channels. The four rapid reaction techniques adapted or developed for studying receptor mechanisms are quench- and stopped-flow, adapted for use with vesicles, and cell-flow and laser-pulse photolysis for use with single cells. The approach was initiated when it was found that the Neurotransmitter Receptors desensitize (become transiently inactive) faster, by almost two orders of magnitude, than was believed. Before fast reaction techniques were used, the chemical properties of only desensitized forms were investigated, although this was not recognized. So far, the chemical mechanism(s) of the excitatory (cation-specific) acetylcholine receptor in membrane vesicles, electroplax cells, and single clonal cells, and the inhibitory (anion-specific) gamma-aminobutyric acid (GABA) receptor in primary cerebral cortical cells have been investigated with the new techniques.(ABSTRACT TRUNCATED AT 400 WORDS)
Ricardo Miledi - One of the best experts on this subject based on the ideXlab platform.
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Profiling Neurotransmitter receptor expression in the Ambystoma mexicanum brain
Neuroscience Letters, 2013Co-Authors: Jorge Mauricio Reyes-ruiz, Matthew J. Korn, Paul A. Nakamura, Nicole J. Shirkey, Agenor Limon, Jamie K. Wong, Ricardo MilediAbstract:Abstract Ability to regenerate limbs and central nervous system (CNS) is unique to few vertebrates, most notably the axolotl ( Ambystoma sp.). However, despite the fact the Neurotransmitter Receptors are involved in axonal regeneration, little is known regarding its expression profile. In this project, RT-PCR and qPCR were performed to gain insight into the Neurotransmitter Receptors present in Ambystoma . Its functional ability was studied by expressing axolotl Receptors in Xenopus laevis oocytes by either injection of mRNA or by direct microtransplantation of brain membranes. Oocytes injected with axolotl mRNA expressed ionotropic Receptors activated by GABA, aspartate + glycine and kainate, as well as metabotropic Receptors activated by acetylcholine and glutamate. Interestingly, we did not see responses following the application of serotonin. Membranes from the axolotl brain were efficiently microtransplanted into Xenopus oocytes and two types of native GABA Receptors that differed in the temporal course of their responses and affinities to GABA were observed. Results of this study are necessary for further characterization of axolotl Neurotransmitter Receptors and may be useful for guiding experiments aimed at understanding activity-dependant limb and CNS regeneration.
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microtransplantation of Neurotransmitter Receptors from postmortem autistic brains to xenopus oocytes
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Agenor Limon, Ricardo Miledi, Jorge Mauricio ReyesruizAbstract:Autism is a complex disorder that arises from the pervasive action of genetic and epigenetic factors that alter synaptic connectivity of the brain. Although GABA and glutamate Receptors seem to be two of those factors, very little is known about the functional properties of the autistic Receptors. Autistic tissue samples stored in brain banks usually have relatively long postmortem times, and it is highly desirable to know whether Neurotransmitter Receptors in such tissues are still functional. Here we demonstrate that native Receptors microtransplanted from autistic brains, as well as de novo mRNA-expressed Receptors, are still functional and susceptible to detailed electrophysiological characterization even after long postmortem intervals. The opportunity to study the properties of human Receptors present in diseased brains not only opens new avenues toward understanding autism and other neurological disorders, but it also makes the microtransplantation method a useful translational system to evaluate and develop novel medicinal drugs.
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expression of caenorhabditis elegans Neurotransmitter Receptors and ion channels in xenopus oocytes
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Ataulfo Martineztorres, Ricardo MilediAbstract:Injection of Caenorhabditis elegans polyA RNA into Xenopus laevis oocytes led to the expression of Neurotransmitter Receptors that generated some unique responses, including ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid Receptors as well as Receptors that coupled to G proteins, such as those to octopamine, norepinephrine, and angiotensin, which activated the oocyte’s own phosphatidylinositol system and calcium-gated chloride channels. The oocytes also expressed chloride-conducting glutamate Receptors, muscarinic acetylcholine Receptors, and voltage-operated calcium channels. Unexpectedly, serotonin (5-hydroxytryptamine), dopamine, GABA, and kainate did not generate ionic currents, suggesting that the corresponding Receptors were not expressed or were not functional in the oocytes. The use of X. laevis oocytes for expressing worm RNA demonstrates that there are many molecular components whose role remains to be clarified in the nematode. Among them are the nature of the endogenous agonists for the octopamine and angiotensin Receptors and the subunits that compose the ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid Receptors and the norepinephrine Receptors that couple to the phosphoinositide cascade.
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Microtransplantation of Neurotransmitter Receptors From Cells to Xenopus Oocyte Membranes
Methods in molecular biology (Clifton N.J.), 2006Co-Authors: Ricardo Miledi, Eleonora Palma, Fabrizio EusebiAbstract:The Xenopus oocyte is largely used as a cell expression system for studying both structure and function of transmitter Receptors and ion channels. Messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional ion channels. A new method was developed further to transplant Neurotransmitter Receptors from human brain or cultured cell lines to the membrane of Xenopus oocytes. This method represents a modification of the method used many years ago of injecting into oocytes membrane vesicles from Torpedo electroplaques, yielding the expression of functional Torpedo acetylcholine Receptors. We describe this approach by extracting membrane vesicles from human hippocampus or temporal neocortex and from mammalian cell lines stably expressing glutamate or neuronal nicotinic Receptors. Because the human Neurotransmitter Receptors are "microtransplanted" with their native cell membranes, this method extends the usefulness of Xenopus oocytes as an expression system for addressing issues in many fields, including channelopathies.
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microtransplantation of membranes from cultured cells to xenopus oocytes a method to study Neurotransmitter Receptors embedded in native lipids
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Eleonora Palma, Ricardo Miledi, Flavia Trettel, Sergio Fucile, Massimiliano Renzi, Fabrizio EusebiAbstract:The Xenopus oocyte is used as a convenient cell expression system to study the structure and function of heterogenic transmitter Receptors and ion channels. Recently, we introduced a method to microtransplant already assembled Neurotransmitter Receptors from the human brain to the plasma membrane of Xenopus oocytes. The same approach was used here to transplant Neurotransmitter Receptors expressed from cultured cells to the oocytes. Membrane vesicles prepared from a human embryonic kidney cell line (HEK293) stably expressing the rat glutamate receptor 1 were injected into oocytes, and, within a few hours, the oocyte plasma membrane acquired α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate Receptors, which had the same properties as those expressed in the original HEK cells. Analogously, oocytes injected with membranes prepared from rat pituitary GH(4)C1 cells, stably expressing homomeric human neuronal α7 nicotinic acetylcholine Receptors (α7-AcChoRs), incorporated in their plasma membrane AcChoRs that behaved as those expressed in GH(4)C1 cells. Similar results were obtained with HEK cells stably expressing heteromeric human neuronal α4β2-AcChoRs. All this makes the Xenopus oocyte a powerful tool for detailed investigations of Receptors and other proteins expressed in the membrane of cultured cells.
Laurent Groc - One of the best experts on this subject based on the ideXlab platform.
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surface trafficking of Neurotransmitter Receptors from cultured neurons to intact brain preparations
Neuropharmacology, 2020Co-Authors: Julien P. Dupuis, Laurent GrocAbstract:Abstract Over the last decade, developments in single molecule imaging have changed our vision of synaptic physiology. By providing high spatio-temporal resolution maps of the molecular actors of neurotransmissions, these techniques have revealed that pre- and post-synaptic proteins are not randomly distributed but precisely organized at the nanoscale, and that this specific organization is dynamically regulated. At the centre of synaptic transmissions, Neurotransmitter Receptors have been shown to form nanodomains at synapses and to dynamically move in and out of these confinement areas through lateral diffusion within the membrane plane on millisecond timescales, thereby directly contributing to the regulation of synaptic transmission and plasticity. Since the vast majority of these discoveries originated from observations made on dissociated neurons lacking several features of brain tissue (e.g. three-dimensional organization, tissue density), they were initially considered with caution. However, the recent implementation of single-particle tracking (SPT) approaches in cultured and acute brain preparations confirmed that early findings on the dynamic properties of Receptors at the surface of neurons can be extended to more physiological conditions. Taking example of dopamine D1 and NMDA glutamate Receptors we here review our current knowledge of the features of Neurotransmitter receptor surface diffusion in intact brain tissue. Through detailed comparison with cultured neurons, we also discuss how these biophysical properties are influenced by the complexity of the extracellular environment. This article is part of the special issue entitled ‘Mobility and trafficking of neuronal membrane proteins’.
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Dynamics of surface Neurotransmitter Receptors and transporters in glial cells: Single molecule insights.
Cell Calcium, 2017Co-Authors: Silvia Ciappelloni, Ciaran Murphy-royal, Julien P. Dupuis, Stéphane H. R. Oliet, Laurent GrocAbstract:Abstract The surface dynamics of Neurotransmitter Receptors and transporters, as well as ion channels, has been well-documented in neurons, revealing complex molecular behaviour and key physiological functions. However, our understanding of the membrane trafficking and dynamics of the signalling molecules located at the plasma membrane of glial cells is still in its infancy. Yet, recent breakthroughs in the field of glial cells have been obtained using combination of superresolution microscopy, single molecule imaging, and electrophysiological recordings. Here, we review our current knowledge on the surface dynamics of Neurotransmitter Receptors, transporters and ion channels, in glial cells. It has emerged that the brain cell network activity, synaptic activity, and calcium signalling, regulate the surface distribution and dynamics of these molecules. Remarkably, the dynamics of a given Neurotransmitter receptor/transporter at the plasma membrane of a glial cell or neuron is unique, revealing the existence of cell-type specific regulatory pathways. Thus, investigating the dynamics of signalling proteins at the surface of glial cells will likely shed new light on our understanding of glial cell physiology and pathology.
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Lateral diffusion of excitatory Neurotransmitter Receptors during synaptogenesis
Molecular Mechanisms of Synaptogenesis, 2006Co-Authors: Laurent Groc, Brahim Lounis, Martin Heine, Daniel ChoquetAbstract:The understanding of how Receptors accumulate within the developing synapse has captured a lot of attention. Lateral diffusion of surface Neurotransmitter Receptors has emerged as a key pathway to regulate receptor trafficking and surface distribution, in addition to the receptor cycling between intracellular and plasma membrane pools. The Neurotransmitter receptor lateral diffusion depends on several factors, such as interactions with other proteins highly enriched within the synaptic structure. During synaptogenesis, Receptors aggregate and cluster within developing synapses. As described in this chapter, lateral diffusion of Receptors is likely to play an important role in such process since receptor lateral diffusion is high during synaptogenesis, providing favourable conditions for the “capture” of Receptors within synaptic contacts. Indeed, the “diffusion-trap” model for receptor accumulation in developing synapses has now gained experimental support from excitatory synapses, although direct evidence to test this model is still lacking due to the absence of adequate tools to precisely control extrasynaptic receptor lateral diffusion. Within the developing synapse, it also emerges that Neurotransmitter release generates surface instability of the Receptors and their stabilization requires additional factors.
Jennifer M. Pocock - One of the best experts on this subject based on the ideXlab platform.
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Microglial Neurotransmitter Receptors trigger superoxide production in microglia; consequences for microglial-neuronal interactions.
Journal of neurochemistry, 2012Co-Authors: Emma Mead, Lucianne Dobson, Simon J. R. Heales, Simon Eaton, Angelina J. Mosley, Jennifer M. PocockAbstract:J. Neurochem. (2012) 121, 287–301. Abstract Microglia express three isoforms of the NADPH oxidase, Nox1, Nox2 and Nox4, with the potential to produce superoxide (O2˙−). Microglia also express Neurotransmitter Receptors, which can modulate microglial responses. In this study, microglial activity of Nox1, Nox2 and Nox4 in primary rat cultured microglia or the rodent BV2 cell line were altered by microglial Neurotransmitter receptor modulation. Glutamate, GABA or ATP triggered microglial O2˙− production via Nox activation. Nox activation was elicited by agonists of metabotropic mGlu3 Receptors and by group III Receptors, by GABAA but not GABAB Receptors, and by purinergic P2X7 or P2Y2/4 Receptors but not P2Y1 Receptors, and inhibited by metabotropic glutamate receptor 5 antagonists. The Neurotransmitters also modulated Nox mRNA expression and NADPH activity. The activation of Nox by BzATP or GABA promoted a neuroprotective phenotype whilst the activation of Nox by glutamate promoted a neurotoxic phenotype. Taken together, these data indicate that microglial Neurotransmitter Receptors can signal via Nox to promote neuroprotection or neurotoxicity. This has implications for the subsequent neurotoxic profile of microglia when Neurotransmitter levels may become skewed in neurodegeneration.
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Microglial Neurotransmitter Receptors trigger superoxide production in microglia; consequences for microglial–neuronal interactions
Journal of Neurochemistry, 2012Co-Authors: Emma L. Mead, Angelina Mosley, Lucianne Dobson, Simon J. R. Heales, Simon Eaton, Jennifer M. PocockAbstract:J. Neurochem. (2012) 121, 287–301. Abstract Microglia express three isoforms of the NADPH oxidase, Nox1, Nox2 and Nox4, with the potential to produce superoxide (O2˙−). Microglia also express Neurotransmitter Receptors, which can modulate microglial responses. In this study, microglial activity of Nox1, Nox2 and Nox4 in primary rat cultured microglia or the rodent BV2 cell line were altered by microglial Neurotransmitter receptor modulation. Glutamate, GABA or ATP triggered microglial O2˙− production via Nox activation. Nox activation was elicited by agonists of metabotropic mGlu3 Receptors and by group III Receptors, by GABAA but not GABAB Receptors, and by purinergic P2X7 or P2Y2/4 Receptors but not P2Y1 Receptors, and inhibited by metabotropic glutamate receptor 5 antagonists. The Neurotransmitters also modulated Nox mRNA expression and NADPH activity. The activation of Nox by BzATP or GABA promoted a neuroprotective phenotype whilst the activation of Nox by glutamate promoted a neurotoxic phenotype. Taken together, these data indicate that microglial Neurotransmitter Receptors can signal via Nox to promote neuroprotection or neurotoxicity. This has implications for the subsequent neurotoxic profile of microglia when Neurotransmitter levels may become skewed in neurodegeneration.
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Neurotransmitter Receptors on microglia.
Trends in Neurosciences, 2007Co-Authors: Jennifer M. Pocock, Helmut KettenmannAbstract:Microglia are the intrinsic immune cells of the brain and express chemokine and cytokine Receptors that interact with the peripheral immune cells. Recent studies have indicated that microglia also respond to the brain's classical signalling substances, the Neurotransmitters. Here, we review the evidence for the expression of Neurotransmitter Receptors on microglia and the consequences of this receptor activation for microglial behaviour. It is evident that Neurotransmitters instruct microglia to perform distinct types of responses, such as triggering an inflammatory cascade or acquiring a neuroprotective phenotype. Understanding how microglia respond to different Neurotransmitters will thus have important implications for controlling the reactivity of these cells in acute injury, as well as for treating chronic neurodegenerative diseases.
Li Niu - One of the best experts on this subject based on the ideXlab platform.
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Rapid chemical kinetic techniques for investigations of Neurotransmitter Receptors expressed in Xenopus oocytes
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Li Niu, Raymond W. Vazquez, Georg Nagel, Thomas Friedrich, Ernst Bamberg, Robert E. Oswald, George P. HessAbstract:Xenopus laevis oocytes have been used extensively during the past decade to express and study Neurotransmitter Receptors of various origins and subunit composition and also to express and study Receptors altered by site-specific mutations. Interpretations of the effects of structural differences on receptor mechanisms were, however, hampered by a lack of rapid chemical reaction techniques suitable for use with oocytes. Here we describe flow and photolysis techniques, with 2-ms and 100-μs time resolution, respectively, for studying Neurotransmitter Receptors in giant (≈20-μm diameter) patches of oocyte membranes, using muscle and neuronal acetylcholine Receptors as examples. With these techniques, we find that the muscle receptor in BC3H1 cells and the same receptor expressed in oocytes have comparable kinetic properties. This finding is in contrast to previous studies and raises questions regarding the interpretations of the many studies of Receptors expressed in oocytes in which an insufficient time resolution was available. The results obtained indicate that the rapid reaction techniques described here, in conjunction with the oocyte expression system, will be useful in answering many outstanding questions regarding the structure and function of diverse Neurotransmitter Receptors.