Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation
Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators, have been used to explore...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2015-04-01
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| Series: | Frontiers in Molecular Neuroscience |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fnmol.2015.00010/full |
| _version_ | 1818850570072489984 |
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| author | J. Michael eGee J. Michael eGee J. Michael eGee Meredith B. Gibbons Meredith B. Gibbons Meredith B. Gibbons Marsa eTaheri Marsa eTaheri Sierra ePalumbos S. Craig eMorris S. Craig eMorris Roy M. Smeal Roy M. Smeal Katherine F. Flynn Katherine F. Flynn Michael N. Economo Michael N. Economo Christian G. Cizek Christian G. Cizek Mario R. Capecchi Mario R. Capecchi Petr eTvrdik Karen S. Wilcox Karen S. Wilcox John A. White John A. White |
| author_facet | J. Michael eGee J. Michael eGee J. Michael eGee Meredith B. Gibbons Meredith B. Gibbons Meredith B. Gibbons Marsa eTaheri Marsa eTaheri Sierra ePalumbos S. Craig eMorris S. Craig eMorris Roy M. Smeal Roy M. Smeal Katherine F. Flynn Katherine F. Flynn Michael N. Economo Michael N. Economo Christian G. Cizek Christian G. Cizek Mario R. Capecchi Mario R. Capecchi Petr eTvrdik Karen S. Wilcox Karen S. Wilcox John A. White John A. White |
| author_sort | J. Michael eGee |
| collection | DOAJ |
| description | Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators, have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson’s disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (< 5 kb). In utero electroporation offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5-14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal activity in the rat brain. |
| first_indexed | 2024-12-19T06:51:14Z |
| format | Article |
| id | doaj.art-3807f5026071472590f4793874944f74 |
| institution | Directory Open Access Journal |
| issn | 1662-5099 |
| language | English |
| last_indexed | 2024-12-19T06:51:14Z |
| publishDate | 2015-04-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Molecular Neuroscience |
| spelling | doaj.art-3807f5026071472590f4793874944f742022-12-21T20:31:42ZengFrontiers Media S.A.Frontiers in Molecular Neuroscience1662-50992015-04-01810.3389/fnmol.2015.00010138273Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporationJ. Michael eGee0J. Michael eGee1J. Michael eGee2Meredith B. Gibbons3Meredith B. Gibbons4Meredith B. Gibbons5Marsa eTaheri6Marsa eTaheri7Sierra ePalumbos8S. Craig eMorris9S. Craig eMorris10Roy M. Smeal11Roy M. Smeal12Katherine F. Flynn13Katherine F. Flynn14Michael N. Economo15Michael N. Economo16Christian G. Cizek17Christian G. Cizek18Mario R. Capecchi19Mario R. Capecchi20Petr eTvrdik21Karen S. Wilcox22Karen S. Wilcox23John A. White24John A. White25University of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahHoward Hughes Medical InstituteUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahUniversity of UtahComplex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators, have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson’s disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (< 5 kb). In utero electroporation offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5-14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal activity in the rat brain.http://journal.frontiersin.org/Journal/10.3389/fnmol.2015.00010/fullneural networkscalcium imagingastrogliarat modeltdTomatogene delivery |
| spellingShingle | J. Michael eGee J. Michael eGee J. Michael eGee Meredith B. Gibbons Meredith B. Gibbons Meredith B. Gibbons Marsa eTaheri Marsa eTaheri Sierra ePalumbos S. Craig eMorris S. Craig eMorris Roy M. Smeal Roy M. Smeal Katherine F. Flynn Katherine F. Flynn Michael N. Economo Michael N. Economo Christian G. Cizek Christian G. Cizek Mario R. Capecchi Mario R. Capecchi Petr eTvrdik Karen S. Wilcox Karen S. Wilcox John A. White John A. White Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation Frontiers in Molecular Neuroscience neural networks calcium imaging astroglia rat model tdTomato gene delivery |
| title | Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation |
| title_full | Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation |
| title_fullStr | Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation |
| title_full_unstemmed | Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation |
| title_short | Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation |
| title_sort | imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation |
| topic | neural networks calcium imaging astroglia rat model tdTomato gene delivery |
| url | http://journal.frontiersin.org/Journal/10.3389/fnmol.2015.00010/full |
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