The Experts below are selected from a list of 66 Experts worldwide ranked by ideXlab platform
John M Clark - One of the best experts on this subject based on the ideXlab platform.
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Neurolemma injected xenopus oocytes an innovative ex vivo approach to study the effects of pyrethroids on ion channels in their native state
Engineering, 2020Co-Authors: John M Clark, Steve SymingtonAbstract:Abstract Microtransplantation of rat brain Neurolemma into the plasma membrane of Xenopus laevis oocytes is an ex vivo method used to study channels and receptors in their native state using standard electrophysiological approaches. In this review, we show that oocytes injected with adult rat brain Neurolemma elicited tetrodotoxin-sensitive inward ion currents upon membrane depolarization, which were increased in a concentration-dependent manner by treatment with the pyrethroid insecticides permethrin and deltamethrin. Under our initial protocols, oocyte health was reduced over time and Neurolemma incorporation varied between batches of oocytes from different frogs, limiting the usefulness of the assay for regulatory issues. A collection of changes to the assay procedure, data acceptance criteria, and analysis method yield substantially improved precision and, hence, assay performance. These changes established this ex vivo approach as a toxicologically relevant assay to study the toxicodynamic action of pyrethroids on ion channels in their native state using Neurolemma fragments prepared from juvenile and adult rat brains.
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evaluation of microtransplantation of rat brain Neurolemma into xenopus laevis oocytes as a technique to study the effect of neurotoxicants on endogenous voltage sensitive ion channels
Neurotoxicology, 2017Co-Authors: Edwin Murenzi, Abigail C Toltin, Steven B Symington, Molly M Morgan, John M ClarkAbstract:Microtransplantation of mammalian brain Neurolemma into the plasma membrane of Xenopus oocytes is used to study ion channels in their native form as they appear in the central nervous system. Use of microtransplanted Neurolemma is advantageous for various reasons: tissue can be obtained from various sources and at different developmental stages; ion channels and receptors are present in their native configuration in their proper lipid environment along with appropriate auxiliary subunits; allowing the evaluation of numerous channelpathies caused by neurotoxicants in an ex vivo state. Here we show that Xenopus oocytes injected with post-natal day 90 (PND90) rat brain Neurolemma fragments successfully express functional ion channels. Using a high throughput two electrode voltage clamp (TEVC) electrophysiological system, currents that were sensitive to tetrodotoxin, ω-conotoxin MVIIC, and tetraethylammonium were detected, indicating the presence of multiple voltage-sensitive ion channels (voltage-sensitive sodium (VSSC), calcium and potassium channels, respectively). The protein expression pattern for nine different VSSC isoforms (Nav1.1-Nav1.9) was determined in Neurolemma using automated western blotting, with the predominant isoforms expressed being Nav1.2 and Nav1.6. VSSC were also successfully detected in the plasma membrane of Xenopus oocytes microtransplanted with Neurolemma. Using this approach, a "proof-of-principle" experiment was conducted where a well-established structure-activity relationship between the neurotoxicant, 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) and its non-neurotoxic metabolite, 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (DDE) was examined. A differential sensitivity of DDT and DDE on Neurolemma-injected oocytes was determined where DDT elicited a concentration-dependent increase in TTX-sensitive inward sodium current upon pulse-depolarization whereas DDE resulted in no significant effect. Additionally, DDT resulted in a slowing of sodium channel inactivation kinetics whereas DDE was without effect. These results are consistent with the findings obtained using heterologous expression of single isoforms of rat brain VSSCs in Xenopus oocytes and with many other electrophysiological approaches, validating the use of the microtransplantation procedure as a toxicologically-relevant ex vivo assay. Once fully characterized, it is likely that this approach could be expanded to study the role of environmental toxicants and contaminants on various target tissues (e.g. neural, reproductive, developmental) from many species.
Edwin Murenzi - One of the best experts on this subject based on the ideXlab platform.
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evaluation of microtransplantation of rat brain Neurolemma into xenopus laevis oocytes as a technique to study the effect of neurotoxicants on endogenous voltage sensitive ion channels
Neurotoxicology, 2017Co-Authors: Edwin Murenzi, Abigail C Toltin, Steven B Symington, Molly M Morgan, John M ClarkAbstract:Microtransplantation of mammalian brain Neurolemma into the plasma membrane of Xenopus oocytes is used to study ion channels in their native form as they appear in the central nervous system. Use of microtransplanted Neurolemma is advantageous for various reasons: tissue can be obtained from various sources and at different developmental stages; ion channels and receptors are present in their native configuration in their proper lipid environment along with appropriate auxiliary subunits; allowing the evaluation of numerous channelpathies caused by neurotoxicants in an ex vivo state. Here we show that Xenopus oocytes injected with post-natal day 90 (PND90) rat brain Neurolemma fragments successfully express functional ion channels. Using a high throughput two electrode voltage clamp (TEVC) electrophysiological system, currents that were sensitive to tetrodotoxin, ω-conotoxin MVIIC, and tetraethylammonium were detected, indicating the presence of multiple voltage-sensitive ion channels (voltage-sensitive sodium (VSSC), calcium and potassium channels, respectively). The protein expression pattern for nine different VSSC isoforms (Nav1.1-Nav1.9) was determined in Neurolemma using automated western blotting, with the predominant isoforms expressed being Nav1.2 and Nav1.6. VSSC were also successfully detected in the plasma membrane of Xenopus oocytes microtransplanted with Neurolemma. Using this approach, a "proof-of-principle" experiment was conducted where a well-established structure-activity relationship between the neurotoxicant, 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) and its non-neurotoxic metabolite, 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (DDE) was examined. A differential sensitivity of DDT and DDE on Neurolemma-injected oocytes was determined where DDT elicited a concentration-dependent increase in TTX-sensitive inward sodium current upon pulse-depolarization whereas DDE resulted in no significant effect. Additionally, DDT resulted in a slowing of sodium channel inactivation kinetics whereas DDE was without effect. These results are consistent with the findings obtained using heterologous expression of single isoforms of rat brain VSSCs in Xenopus oocytes and with many other electrophysiological approaches, validating the use of the microtransplantation procedure as a toxicologically-relevant ex vivo assay. Once fully characterized, it is likely that this approach could be expanded to study the role of environmental toxicants and contaminants on various target tissues (e.g. neural, reproductive, developmental) from many species.
Molly M Morgan - One of the best experts on this subject based on the ideXlab platform.
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evaluation of microtransplantation of rat brain Neurolemma into xenopus laevis oocytes as a technique to study the effect of neurotoxicants on endogenous voltage sensitive ion channels
Neurotoxicology, 2017Co-Authors: Edwin Murenzi, Abigail C Toltin, Steven B Symington, Molly M Morgan, John M ClarkAbstract:Microtransplantation of mammalian brain Neurolemma into the plasma membrane of Xenopus oocytes is used to study ion channels in their native form as they appear in the central nervous system. Use of microtransplanted Neurolemma is advantageous for various reasons: tissue can be obtained from various sources and at different developmental stages; ion channels and receptors are present in their native configuration in their proper lipid environment along with appropriate auxiliary subunits; allowing the evaluation of numerous channelpathies caused by neurotoxicants in an ex vivo state. Here we show that Xenopus oocytes injected with post-natal day 90 (PND90) rat brain Neurolemma fragments successfully express functional ion channels. Using a high throughput two electrode voltage clamp (TEVC) electrophysiological system, currents that were sensitive to tetrodotoxin, ω-conotoxin MVIIC, and tetraethylammonium were detected, indicating the presence of multiple voltage-sensitive ion channels (voltage-sensitive sodium (VSSC), calcium and potassium channels, respectively). The protein expression pattern for nine different VSSC isoforms (Nav1.1-Nav1.9) was determined in Neurolemma using automated western blotting, with the predominant isoforms expressed being Nav1.2 and Nav1.6. VSSC were also successfully detected in the plasma membrane of Xenopus oocytes microtransplanted with Neurolemma. Using this approach, a "proof-of-principle" experiment was conducted where a well-established structure-activity relationship between the neurotoxicant, 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) and its non-neurotoxic metabolite, 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (DDE) was examined. A differential sensitivity of DDT and DDE on Neurolemma-injected oocytes was determined where DDT elicited a concentration-dependent increase in TTX-sensitive inward sodium current upon pulse-depolarization whereas DDE resulted in no significant effect. Additionally, DDT resulted in a slowing of sodium channel inactivation kinetics whereas DDE was without effect. These results are consistent with the findings obtained using heterologous expression of single isoforms of rat brain VSSCs in Xenopus oocytes and with many other electrophysiological approaches, validating the use of the microtransplantation procedure as a toxicologically-relevant ex vivo assay. Once fully characterized, it is likely that this approach could be expanded to study the role of environmental toxicants and contaminants on various target tissues (e.g. neural, reproductive, developmental) from many species.
Abigail C Toltin - One of the best experts on this subject based on the ideXlab platform.
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evaluation of microtransplantation of rat brain Neurolemma into xenopus laevis oocytes as a technique to study the effect of neurotoxicants on endogenous voltage sensitive ion channels
Neurotoxicology, 2017Co-Authors: Edwin Murenzi, Abigail C Toltin, Steven B Symington, Molly M Morgan, John M ClarkAbstract:Microtransplantation of mammalian brain Neurolemma into the plasma membrane of Xenopus oocytes is used to study ion channels in their native form as they appear in the central nervous system. Use of microtransplanted Neurolemma is advantageous for various reasons: tissue can be obtained from various sources and at different developmental stages; ion channels and receptors are present in their native configuration in their proper lipid environment along with appropriate auxiliary subunits; allowing the evaluation of numerous channelpathies caused by neurotoxicants in an ex vivo state. Here we show that Xenopus oocytes injected with post-natal day 90 (PND90) rat brain Neurolemma fragments successfully express functional ion channels. Using a high throughput two electrode voltage clamp (TEVC) electrophysiological system, currents that were sensitive to tetrodotoxin, ω-conotoxin MVIIC, and tetraethylammonium were detected, indicating the presence of multiple voltage-sensitive ion channels (voltage-sensitive sodium (VSSC), calcium and potassium channels, respectively). The protein expression pattern for nine different VSSC isoforms (Nav1.1-Nav1.9) was determined in Neurolemma using automated western blotting, with the predominant isoforms expressed being Nav1.2 and Nav1.6. VSSC were also successfully detected in the plasma membrane of Xenopus oocytes microtransplanted with Neurolemma. Using this approach, a "proof-of-principle" experiment was conducted where a well-established structure-activity relationship between the neurotoxicant, 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) and its non-neurotoxic metabolite, 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (DDE) was examined. A differential sensitivity of DDT and DDE on Neurolemma-injected oocytes was determined where DDT elicited a concentration-dependent increase in TTX-sensitive inward sodium current upon pulse-depolarization whereas DDE resulted in no significant effect. Additionally, DDT resulted in a slowing of sodium channel inactivation kinetics whereas DDE was without effect. These results are consistent with the findings obtained using heterologous expression of single isoforms of rat brain VSSCs in Xenopus oocytes and with many other electrophysiological approaches, validating the use of the microtransplantation procedure as a toxicologically-relevant ex vivo assay. Once fully characterized, it is likely that this approach could be expanded to study the role of environmental toxicants and contaminants on various target tissues (e.g. neural, reproductive, developmental) from many species.
Steven B Symington - One of the best experts on this subject based on the ideXlab platform.
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evaluation of microtransplantation of rat brain Neurolemma into xenopus laevis oocytes as a technique to study the effect of neurotoxicants on endogenous voltage sensitive ion channels
Neurotoxicology, 2017Co-Authors: Edwin Murenzi, Abigail C Toltin, Steven B Symington, Molly M Morgan, John M ClarkAbstract:Microtransplantation of mammalian brain Neurolemma into the plasma membrane of Xenopus oocytes is used to study ion channels in their native form as they appear in the central nervous system. Use of microtransplanted Neurolemma is advantageous for various reasons: tissue can be obtained from various sources and at different developmental stages; ion channels and receptors are present in their native configuration in their proper lipid environment along with appropriate auxiliary subunits; allowing the evaluation of numerous channelpathies caused by neurotoxicants in an ex vivo state. Here we show that Xenopus oocytes injected with post-natal day 90 (PND90) rat brain Neurolemma fragments successfully express functional ion channels. Using a high throughput two electrode voltage clamp (TEVC) electrophysiological system, currents that were sensitive to tetrodotoxin, ω-conotoxin MVIIC, and tetraethylammonium were detected, indicating the presence of multiple voltage-sensitive ion channels (voltage-sensitive sodium (VSSC), calcium and potassium channels, respectively). The protein expression pattern for nine different VSSC isoforms (Nav1.1-Nav1.9) was determined in Neurolemma using automated western blotting, with the predominant isoforms expressed being Nav1.2 and Nav1.6. VSSC were also successfully detected in the plasma membrane of Xenopus oocytes microtransplanted with Neurolemma. Using this approach, a "proof-of-principle" experiment was conducted where a well-established structure-activity relationship between the neurotoxicant, 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) and its non-neurotoxic metabolite, 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (DDE) was examined. A differential sensitivity of DDT and DDE on Neurolemma-injected oocytes was determined where DDT elicited a concentration-dependent increase in TTX-sensitive inward sodium current upon pulse-depolarization whereas DDE resulted in no significant effect. Additionally, DDT resulted in a slowing of sodium channel inactivation kinetics whereas DDE was without effect. These results are consistent with the findings obtained using heterologous expression of single isoforms of rat brain VSSCs in Xenopus oocytes and with many other electrophysiological approaches, validating the use of the microtransplantation procedure as a toxicologically-relevant ex vivo assay. Once fully characterized, it is likely that this approach could be expanded to study the role of environmental toxicants and contaminants on various target tissues (e.g. neural, reproductive, developmental) from many species.