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Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into Xenopus Oocytes for Their Functional Analysis

Metabotropic glutamate receptors (mGluRs) are membrane receptors that play a central role in the modulation of synaptic transmission and neuronal excitability and whose dysregulation is implicated in diverse neurological disorders. Most current understanding about the electrophysiological properties...

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Autores principales: Miller, Brice, Moreno, Naomi, Gutierrez, Berenice A., Limon, Agenor
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9609105/
https://www.ncbi.nlm.nih.gov/pubmed/36295690
http://dx.doi.org/10.3390/membranes12100931
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author Miller, Brice
Moreno, Naomi
Gutierrez, Berenice A.
Limon, Agenor
author_facet Miller, Brice
Moreno, Naomi
Gutierrez, Berenice A.
Limon, Agenor
author_sort Miller, Brice
collection PubMed
description Metabotropic glutamate receptors (mGluRs) are membrane receptors that play a central role in the modulation of synaptic transmission and neuronal excitability and whose dysregulation is implicated in diverse neurological disorders. Most current understanding about the electrophysiological properties of such receptors has been determined using recombinant proteins. However, recombinant receptors do not necessarily recapitulate the properties of native receptors due to the lack of obligated accessory proteins, some of which are differentially expressed as function of developmental stage and brain region. To overcome this limitation, we sought to microtransplant entire synaptosome membranes from frozen rat cortex into Xenopus oocytes, and directly analyze the responses elicited by native mGluRs. We recorded ion currents elicited by 1 mM glutamate using two electrodes voltage clamp. Glutamate produced a fast ionotropic response (6 ± 0.3 nA) in all microtransplanted oocytes (n = 218 oocytes) and a delayed oscillatory response (52 ± 7 nA) in 73% of them. The participation of Group 1 mGluRs was confirmed by the presence of metabotropic oscillations during the administration of (±)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD; Group 1 mGluR agonist), and the absence of oscillations during co-administration of N-(1-adamantyl)quinoxaline-2-carboxamide (NPS 2390; Group 1 mGluR antagonist). Since both mGluR1 and mGluR5 belong to Group 1 mGluRs, further investigation revealed that mGluR1 antagonism with LY 456236 has little effect on metabotropic oscillations, while mGluR5 antagonism with 100 µM AZD 9272 has significant reduction of metabotropic currents elicited by ACPD and glutamate. We confirmed the expression of mGluR1 and mGluR5 in native synaptosomes by immunoblots, both of which are enhanced when compared to their counterpart proteins in rat cortex tissue lysates. Finally, these results demonstrate the merit of using microtransplantation of native synaptosomes for the study of mGluRs and the contribution of mGluR5 to the metabotropic glutamate signaling, providing a better tool for the understanding of the role of these receptors in neurological disorders.
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spelling pubmed-96091052022-10-28 Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into Xenopus Oocytes for Their Functional Analysis Miller, Brice Moreno, Naomi Gutierrez, Berenice A. Limon, Agenor Membranes (Basel) Article Metabotropic glutamate receptors (mGluRs) are membrane receptors that play a central role in the modulation of synaptic transmission and neuronal excitability and whose dysregulation is implicated in diverse neurological disorders. Most current understanding about the electrophysiological properties of such receptors has been determined using recombinant proteins. However, recombinant receptors do not necessarily recapitulate the properties of native receptors due to the lack of obligated accessory proteins, some of which are differentially expressed as function of developmental stage and brain region. To overcome this limitation, we sought to microtransplant entire synaptosome membranes from frozen rat cortex into Xenopus oocytes, and directly analyze the responses elicited by native mGluRs. We recorded ion currents elicited by 1 mM glutamate using two electrodes voltage clamp. Glutamate produced a fast ionotropic response (6 ± 0.3 nA) in all microtransplanted oocytes (n = 218 oocytes) and a delayed oscillatory response (52 ± 7 nA) in 73% of them. The participation of Group 1 mGluRs was confirmed by the presence of metabotropic oscillations during the administration of (±)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD; Group 1 mGluR agonist), and the absence of oscillations during co-administration of N-(1-adamantyl)quinoxaline-2-carboxamide (NPS 2390; Group 1 mGluR antagonist). Since both mGluR1 and mGluR5 belong to Group 1 mGluRs, further investigation revealed that mGluR1 antagonism with LY 456236 has little effect on metabotropic oscillations, while mGluR5 antagonism with 100 µM AZD 9272 has significant reduction of metabotropic currents elicited by ACPD and glutamate. We confirmed the expression of mGluR1 and mGluR5 in native synaptosomes by immunoblots, both of which are enhanced when compared to their counterpart proteins in rat cortex tissue lysates. Finally, these results demonstrate the merit of using microtransplantation of native synaptosomes for the study of mGluRs and the contribution of mGluR5 to the metabotropic glutamate signaling, providing a better tool for the understanding of the role of these receptors in neurological disorders. MDPI 2022-09-26 /pmc/articles/PMC9609105/ /pubmed/36295690 http://dx.doi.org/10.3390/membranes12100931 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Miller, Brice
Moreno, Naomi
Gutierrez, Berenice A.
Limon, Agenor
Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into Xenopus Oocytes for Their Functional Analysis
title Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into Xenopus Oocytes for Their Functional Analysis
title_full Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into Xenopus Oocytes for Their Functional Analysis
title_fullStr Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into Xenopus Oocytes for Their Functional Analysis
title_full_unstemmed Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into Xenopus Oocytes for Their Functional Analysis
title_short Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into Xenopus Oocytes for Their Functional Analysis
title_sort microtransplantation of postmortem native synaptic mglurs receptors into xenopus oocytes for their functional analysis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9609105/
https://www.ncbi.nlm.nih.gov/pubmed/36295690
http://dx.doi.org/10.3390/membranes12100931
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