Université Pierre et Marie Curie-Paris 6, Neurobiologie des Processus Adaptatifs, CNRS UMR 7102, Paris, France.
J Neurosci Res. 2010 Mar;88(4):695-711. doi: 10.1002/jnr.22249.
Responses of three bioluminescent Ca(2+) sensors were studied in vitro and in neurons from brain slices. These sensors consisted of tandem fusions of green fluorescent protein (GFP) with the photoproteins aequorin, obelin, or a mutant aequorin with high Ca(2+) sensitivity. Kinetics of GFP-obelin responses to a saturating Ca(2+) concentration were faster than those of GFP-aequorin at all Mg(2+) concentrations tested, whereas GFP-mutant aequorin responses were the slowest. GFP-photoproteins were efficiently expressed in pyramidal neurons following overnight incubation of acute neocortical slices with recombinant Sindbis viruses. Expression of GFP-photoproteins did not result in conspicuous modification of morphological or electrophysiological properties of layer V pyramidal cells. The three sensors allowed the detection of Ca(2+) transients associated with action potential discharge in single layer V pyramidal neurons. In these neurons, depolarizing steps of increasing amplitude elicited action potential discharge of increasing frequency. Bioluminescent responses of the three sensors were similar in several respects: detection thresholds, an exponential increase with stimulus intensity, photoprotein consumptions, and kinetic properties. These responses, which were markedly slower than kinetics measured in vitro, increased linearly during the action potential discharge and decayed exponentially at the end of the discharge. Onset slopes increased with stimulus intensity, whereas decay kinetics remained constant. Dendritic light emission contributed to whole-field responses, but the spatial resolution of bioluminescence imaging was limited to the soma and proximal apical dendrite. Nonetheless, the high signal-to-background ratio of GFP-photoproteins allowed the detection of Ca(2+) transients associated with 5 action potentials in single neurons upon whole-field bioluminescence recordings.
三种生物发光钙离子传感器在体外和脑片神经元中的反应进行了研究。这些传感器由绿色荧光蛋白（GFP）与光蛋白水母发光蛋白、小眼发光蛋白或具有高钙离子敏感性的突变水母发光蛋白串联融合而成。在所有测试的镁离子浓度下，GFP-小眼发光蛋白对饱和钙离子浓度的 GFP 响应动力学比 GFP-水母发光蛋白快，而 GFP-突变水母发光蛋白的响应最慢。在急性新皮层切片与重组辛德毕斯病毒孵育过夜后，GFP-光蛋白在锥体神经元中有效表达。GFP-光蛋白的表达并未导致 V 层锥体神经元形态或电生理特性的明显改变。这三种传感器允许检测与单个 V 层锥体神经元动作电位放电相关的钙离子瞬变。在这些神经元中，幅度逐渐增加的去极化步骤引发频率逐渐增加的动作电位放电。三种传感器的生物发光反应在几个方面相似：检测阈值、与刺激强度呈指数增加、光蛋白消耗和动力学特性。这些反应明显比体外测量的动力学慢，在动作电位放电期间线性增加，并在放电结束时指数衰减。起始斜率随刺激强度增加而增加，而衰减动力学保持不变。树突光发射对全场反应有贡献，但生物发光成像的空间分辨率仅限于胞体和近端顶树突。尽管如此，GFP-光蛋白的高信噪比允许在全场生物发光记录中检测与单个神经元中 5 个动作电位相关的钙离子瞬变。
