Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into <i>Xenopus</i> 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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| Format: | Article |
| Language: | English |
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MDPI AG
2022-09-01
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| Series: | Membranes |
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| Online Access: | https://www.mdpi.com/2077-0375/12/10/931 |
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| author | Brice Miller Naomi Moreno Berenice A. Gutierrez Agenor Limon |
| author_facet | Brice Miller Naomi Moreno Berenice A. Gutierrez Agenor Limon |
| author_sort | Brice Miller |
| collection | DOAJ |
| 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 <i>Xenopus</i> 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 (<i>n</i> = 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. |
| first_indexed | 2024-03-09T19:50:24Z |
| format | Article |
| id | doaj.art-b7c4f5de471c4814b57f400d6fef88ef |
| institution | Directory Open Access Journal |
| issn | 2077-0375 |
| language | English |
| last_indexed | 2024-03-09T19:50:24Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Membranes |
| spelling | doaj.art-b7c4f5de471c4814b57f400d6fef88ef2023-11-24T01:12:54ZengMDPI AGMembranes2077-03752022-09-01121093110.3390/membranes12100931Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into <i>Xenopus</i> Oocytes for Their Functional AnalysisBrice Miller0Naomi Moreno1Berenice A. Gutierrez2Agenor Limon3Mitchell Center for Neurodegenerative Diseases, Department of Neurology, The University of Texas Medical Branch, Galveston, TX 77555, USAMitchell Center for Neurodegenerative Diseases, Department of Neurology, The University of Texas Medical Branch, Galveston, TX 77555, USAMitchell Center for Neurodegenerative Diseases, Department of Neurology, The University of Texas Medical Branch, Galveston, TX 77555, USAMitchell Center for Neurodegenerative Diseases, Department of Neurology, The University of Texas Medical Branch, Galveston, TX 77555, USAMetabotropic 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 <i>Xenopus</i> 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 (<i>n</i> = 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.https://www.mdpi.com/2077-0375/12/10/931G-protein coupled receptorspost-mortemphospholipase CmGluRmicrotransplantation of synaptic membranes |
| spellingShingle | Brice Miller Naomi Moreno Berenice A. Gutierrez Agenor Limon Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into <i>Xenopus</i> Oocytes for Their Functional Analysis Membranes G-protein coupled receptors post-mortem phospholipase C mGluR microtransplantation of synaptic membranes |
| title | Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into <i>Xenopus</i> Oocytes for Their Functional Analysis |
| title_full | Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into <i>Xenopus</i> Oocytes for Their Functional Analysis |
| title_fullStr | Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into <i>Xenopus</i> Oocytes for Their Functional Analysis |
| title_full_unstemmed | Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into <i>Xenopus</i> Oocytes for Their Functional Analysis |
| title_short | Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into <i>Xenopus</i> Oocytes for Their Functional Analysis |
| title_sort | microtransplantation of postmortem native synaptic mglurs receptors into i xenopus i oocytes for their functional analysis |
| topic | G-protein coupled receptors post-mortem phospholipase C mGluR microtransplantation of synaptic membranes |
| url | https://www.mdpi.com/2077-0375/12/10/931 |
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