The chemoconvulsant pilocarpine induces astrocytic connexin43 hemichannel opening, which might contribute to its seizurogenic effect

Abstract

Event Abstract Back to Event The chemoconvulsant pilocarpine induces astrocytic connexin43 hemichannel opening, which might contribute to its seizurogenic effect Laura Walrave1*, Mathieu Vinken2, Luc Leybaert3 and Ilse J. Smolders1* 1 Vrije Universiteit Brussel, Pharmaceutical Chemistry, Drug Analysis and Drug Information, Center for Neurosciences, Belgium 2 Vrije Universiteit Brussel, In Vitro Toxicology and Dermato-Cosmetology, Belgium 3 Universiteit Gent, Basic Medical Sciences, Ghent University, Ghent, Belgium, Belgium This project tends to elucidate the role of astrocytic connexin43 (Cx43) hemichannels (HCs) in limbic seizures and epilepsy. Cx43 HCs are formed by six Cx43 proteins and are located between the cytosol of an astrocyte and the extracellular environment. We hypothesized that Cx43 HCs might contribute to seizures, as it is known that various pathological stimuli can activate Cx43 HC opening, leading to the release of small signaling molecules (e.g. glutamate, D-serine, ATP), disturbed transmembrane ion fluxes and loss of essential metabolites. To study this, we recently performed microdialysis experiments combined with electrocorticography monitoring, in which we perfused a chemoconvulsant (pilocarpine) in the hippocampus of mice. These experiments revealed that TAT-Gap19, a selective Cx43 HC inhibitor, has an anticonvulsant effect in this model. However, by performing these experiments, the mechanism of action is still not fully elucidated. It is known that pilocarpine induces seizures via muscarinic M1 receptor agonism and that seizures are maintained via activation of NMDA receptors, but no literature is available on the effect of pilocarpine on the open/closed state of Cx43 HCs. We therefore wanted to investigate whether pilocarpine has an effect on astrocytic Cx43 HC activity. To this end, pilocarpine was incubated on acute hippocampal slices of eGFP-GFAP mice. In these mice, astrocytes are labeled by the enhanced green fluorescent protein (eGFP) under the control of the human glial fibrillary acidic protein (GFAP) promotor, to be able to visualize astrocytes by direct fluorescence microscopy. Next, ethidium bromide (EtBr) uptake experiments were performed in order to study the open probability of Cx43 HCs following pilocarpine treatment. EtBr passes from the extracellular environment to the nuclei of cells through HCs and the uptake is proportional to the number of active (open) HCs. The experiments were repeated in the presence of TAT-Gap19, so we could correlate the increase in EtBr uptake with Cx43 HC opening, as EtBr can also pass other Cx HCs. We also used carbenoxolone (CBX) as a positive control, as this is a well-known (but non-selective) Cx and pannexin HC inhibitor. Additionally, the linker protein “TAT” and a scrambled version of TAT-Gap19 (scrambled TAT-Gap19) were used as negative control peptides. After exposure to pilocarpine, the EtBr signal in astrocytes was on average twice stronger than the uptake in control conditions. This effect was suppressed by CBX, demonstrating that EtBr uptake occurred through HCs. The pilocarpine-induced EtBr uptake in astrocytes was also significantly decreased when TAT-Gap19 was used, implicating that pilocarpine increases Cx43 HC activity. There were no significant differences between the pilocarpine-treated slices and slices that were pre-treated with one of the control peptides for TAT-Gap19 (i.e. TAT and scrambled TAT-Gap19). Altogether these observations indicate that the commonly used chemoconvulsant pilocarpine activates astrocytic Cx43 HCs and that CBX and TAT-Gap19 can significantly decrease this activation. This mechanism of action is likely to be responsible for the anticonvulsant effect of TAT-Gap19 in our in vivo animal models. Keywords: connexin43 hemichannels, ethidium bromide uptake, Pilocarpine, TAT-Gap19, Seizures Conference: 12th National Congress of the Belgian Society for Neuroscience, Gent, Belgium, 22 May - 22 May, 2017. Presentation Type: Oral Presentation Topic: Neural Excitability, Synapses, and Glia: Cellular Mechanisms Citation: Walrave L, Vinken M, Leybaert L and Smolders IJ (2019). The chemoconvulsant pilocarpine induces astrocytic connexin43 hemichannel opening, which might contribute to its seizurogenic effect. Front. Neurosci. Conference Abstract: 12th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2017.94.00026 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 24 Apr 2017; Published Online: 25 Jan 2019. * Correspondence: Miss. Laura Walrave, Vrije Universiteit Brussel, Pharmaceutical Chemistry, Drug Analysis and Drug Information, Center for Neurosciences, Jette, 1090, Belgium, laura.walrave@vub.ac.be Prof. Ilse J Smolders, Vrije Universiteit Brussel, Pharmaceutical Chemistry, Drug Analysis and Drug Information, Center for Neurosciences, Jette, 1090, Belgium, ilse.smolders@vub.ac.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Laura Walrave Mathieu Vinken Luc Leybaert Ilse J Smolders Google Laura Walrave Mathieu Vinken Luc Leybaert Ilse J Smolders Google Scholar Laura Walrave Mathieu Vinken Luc Leybaert Ilse J Smolders PubMed Laura Walrave Mathieu Vinken Luc Leybaert Ilse J Smolders Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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