Optogenetic probing and manipulation of the calyx-type presynaptic terminal in the embryonic chick ciliary ganglion.
Optogenetic probing and manipulation of the calyx-type presynaptic terminal in the embryonic chick ciliary ganglion.
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DOI：
10.1371/journal.pone.0059179
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Yawo H
中科院分区：
文献类型：
--
作者：
Egawa R;Hososhima S;Hou X;Katow H;Ishizuka T;Nakamura H;Yawo H
鸡睫状神经节(CG)的帽状突触作为突触发育、形态和生理的模型系统，已被广泛研究了几十年。尽管最近光遗传学在探测和/或操纵递质释放的基本步骤，如膜去极化和钙离子升高等方面取得了进展，但目前的基因操纵方法并不适用于特定靶向于帽状突触前终末。在这里，我们评估了一种操纵该模型突触的突触前终末的分子和功能组织的方法。我们在胚胎第2天(E2)通过体内电穿孔的方法将EGFP表达载体导入Edinger-Westphal(EW)核神经元的前体细胞，并在E8-14检测CG。我们发现数十个帽型突触前终末和轴突被选择性地标记为EGFP荧光。当将包含膜系留荧光蛋白m-CFP、m-YFP和m-RFP的Brainrow结构与Cre表达结构一起引入时，每个突触前轴突的颜色编码促进了相互纠缠的投射之间的区分，特别是在突触连接的发育重组期间。同时表达通道视紫红质的一个嵌合变体--通道视紫红质快速受体(ChRFR)和红移的荧光钙传感器R-GECO1，在直接光刺激突触前终末的情况下，用光学方法测量钙的升高。虽然这种光诱发的钙升高主要依赖于动作电位，但即使在没有细胞外钙的情况下，仍有一个重要的成分存在。提示ChRFR的光激活直接或间接促进了细胞内钙离子的释放。上述系统通过促进对帽状突触前终末的分子研究，将为揭示突触形态、生理和发育的分子机制提供一个实验平台。
The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite recent advances in optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca2+ elevation, the current gene-manipulating methods are not suitable for targeting specifically the calyx-type presynaptic terminals. Here, we evaluated a method for manipulating the molecular and functional organization of the presynaptic terminals of this model synapse. We transfected progenitors of the Edinger-Westphal (EW) nucleus neurons with an EGFP expression vector by in ovo electroporation at embryonic day 2 (E2) and examined the CG at E8–14. We found that dozens of the calyx-type presynaptic terminals and axons were selectively labeled with EGFP fluorescence. When a Brainbow construct containing the membrane-tethered fluorescent proteins m-CFP, m-YFP and m-RFP, was introduced together with a Cre expression construct, the color coding of each presynaptic axon facilitated discrimination among inter-tangled projections, particularly during the developmental re-organization period of synaptic connections. With the simultaneous expression of one of the chimeric variants of channelrhodopsins, channelrhodopsin-fast receiver (ChRFR), and R-GECO1, a red-shifted fluorescent Ca2+-sensor, the Ca2+ elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca2+ elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca2+. It is suggested that the photo-activation of ChRFR facilitated the release of Ca2+ from intracellular Ca2+ stores directly or indirectly. The above system, by facilitating the molecular study of the calyx-type presynaptic terminal, would provide an experimental platform for unveiling the molecular mechanisms underlying the morphology, physiology and development of synapses.
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DOI：
10.1523/jneurosci.4190-10.2010
发表时间：
2010-11-10
期刊：
The Journal of neuroscience : the official journal of the Society for Neuroscience
影响因子：
--
作者：
Knöpfel T;Lin MZ;Levskaya A;Tian L;Lin JY;Boyden ES
通讯作者：
Boyden ES
影响因子：
3.7
作者：
Card JP;Kobiler O;Ludmir EB;Desai V;Sved AF;Enquist LW
通讯作者：
Enquist LW
影响因子：
2.5
作者：
FUJIWARA, T;NAGAKURO, C
通讯作者：
NAGAKURO, C
影响因子：
48
作者：
Hampel, Stefanie;Chung, Phuong;McKellar, Claire E.;Hall, Donald;Looger, Loren L.;Simpson, Julie H.
通讯作者：
Simpson, Julie H.
影响因子：
5.5
作者：
LANDMESSER, L;PILAR, G
通讯作者：
PILAR, G