Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation.
Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation.
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DOI：
10.3389/fnmol.2015.00010
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发表时间：
2015
影响因子：
4.8
通讯作者：
White JA
中科院分区：
文献类型：
--
作者：
Gee JM;Gibbons MB;Taheri M;Palumbos S;Morris SC;Smeal RM;Flynn KF;Economo MN;Cizek CG;Capecchi MR;Tvrdik P;Wilcox KS;White JA
星形胶质细胞和神经元网络之间的复杂相互作用开始受到重视，但仍然知之甚少。表达细胞活性荧光蛋白报告基因的转基因小鼠，如遗传编码钙指示剂（GECIs）的GCaMP家族，已被用于探索网络行为。然而，在某些情况下，可能需要使用长期建立的大鼠模型，这些模型密切模拟人类病症的特定方面，例如帕金森病和癫痫持续状态后癫痫的发展。用于在大鼠脑中表达报告蛋白的方法相对有限。转基因老鼠技术已经存在，但还相当不成熟。病毒介导的表达是稳健的但不稳定的，需要侵入性注射，并且仅对相当小的基因（<5 kb）有效。子宫内电穿孔（IUE）提供了一种有价值的替代方法。IUE是一种成熟的方法，用于在大鼠脑中分离星形胶质细胞和神经元群体，而对转基因大小没有严格限制。我们构建了一套IUE质粒工具集，这些质粒携带由CAG驱动的GCaMP变体3、6s或6 f，并靶向细胞溶质或质膜。因为GCaMP的低基线荧光可阻碍转染细胞的鉴定，所以我们包括共表达胞质tdTomato蛋白的选项。采用由携带piggyBac反向末端重复（ITR）侧翼的CAG-GCaMP-IRES-tdTomato盒的质粒和编码表达piggyBac转座酶的单独质粒组成的二元系统来稳定表达GCaMP和tdTomato。在胚胎第13.5-14.5天共电穿孔质粒，随后在从皮层或海马制备的急性或培养的脑切片中对星形胶质细胞和神经元活性成像。在从不同年龄的大鼠（最多127天）获得的切片中检测到大的自发瞬变。在这份报告中，我们证明了这个工具集的实用性，询问星形胶质细胞和神经元的活动在大鼠大脑。
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 (GECIs), 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 (IUE) 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 (ITR)-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.
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影响因子：
16.2
作者：
Haustein MD;Kracun S;Lu XH;Shih T;Jackson-Weaver O;Tong X;Xu J;Yang XW;O'Dell TJ;Marvin JS;Ellisman MH;Bushong EA;Looger LL;Khakh BS
通讯作者：
Khakh BS
影响因子：
--
作者：
Chen, Fuyi;Maher, Brady J;LoTurco, Joseph J
通讯作者：
LoTurco, Joseph J
影响因子：
48
作者：
Kralj, Joel M.;Douglass, Adam D.;Hochbaum, Daniel R.;Maclaurin, Dougal;Cohen, Adam E.
通讯作者：
Cohen, Adam E.
DOI：
10.1007/978-1-61779-452-0_36
发表时间：
2012
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Ding, Shinghua
通讯作者：
Ding, Shinghua
影响因子：
3
作者：
Chen F;LoTurco J
通讯作者：
LoTurco J