Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses.
Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses.
复制标题
DOI：
10.1073/pnas.1720648115
复制
发表时间：
2018-05-22
影响因子：
11.1
通讯作者：
Török K
中科院分区：
文献类型：
--
作者：
Helassa N;Dürst CD;Coates C;Kerruth S;Arif U;Schulze C;Wiegert JS;Geeves M;Oertner TG;Török K
兴奋性突触将突触前动作电位转化为化学信号，由突触后谷氨酸受体感知。为了窃听突触传递，遗传编码的谷氨酸荧光传感器已经被开发出来。然而，即使是最好的传感器也落后于突触间隙中谷氨酸的快速动态。在这里，我们报道了一种超快的遗传编码谷氨酸传感器iGluu的发展，它使我们能够在100 hz脉冲序列中成像谷氨酸清除和突触抑制。我们发现，只有显示成对脉冲促进的钮扣才能迅速从抑郁中恢复过来。因此，突触前钮扣作为频率特异性过滤器，将尖峰序列的选择特征传递给特定的突触后细胞。谷氨酸能突触在许多不同的时间尺度上表现出丰富的可塑性机制，包括递质释放效率的动态变化以及突触后谷氨酸受体数量和功能的变化。基因编码的谷氨酸传感器iGluSnFR能够以高达~ 10hz的频率可视化谷氨酸从突触前末端释放。然而，为了解决谷氨酸在高频爆发期间的动态，需要更快的指标。在这里，我们报道了快速（iGluf）和超快速（iGluu）变体的发展，它们具有相当的亮度，但谷氨酸的Kd增加（分别为137 μM和600 μM）。与iGluSnFR相比，iGluu的体外解离速率快6倍，突触动力学快5倍。将三态模型拟合到动力学数据中，我们确定谷氨酸结合后的大构象变化是限速步骤。在100hz刺激的大鼠海马片培养中，我们发现iGluu足够快地解决单个谷氨酸释放事件，表明谷氨酸从突触间隙中迅速清除。100hz训练期间iGluu反应的抑制与突触后epsp的抑制相关，表明高频刺激期间的抑制纯粹是突触前的。在单个钮扣处，可以通过第二个脉冲（成对脉冲促进/抑制）释放的谷氨酸量来预测抑郁的恢复，这表明Schaffer侧扣处的脉冲串具有不同频率依赖的滤波。
Excitatory synapses convert presynaptic action potentials into chemical signals that are sensed by postsynaptic glutamate receptors. To eavesdrop on synaptic transmission, genetically encoded fluorescent sensors for glutamate have been developed. However, even the best available sensors lag behind the very fast glutamate dynamics in the synaptic cleft. Here, we report the development of an ultrafast genetically encoded glutamate sensor, iGluu, which allowed us to image glutamate clearance and synaptic depression during 100-Hz spike trains. We found that only boutons showing paired-pulse facilitation were able to rapidly recover from depression. Thus, presynaptic boutons act as frequency-specific filters to transmit select features of the spike train to specific postsynaptic cells. Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to ∼10 Hz. However, to resolve glutamate dynamics during high-frequency bursts, faster indicators are required. Here, we report the development of fast (iGluf) and ultrafast (iGluu) variants with comparable brightness but increased Kd for glutamate (137 μM and 600 μM, respectively). Compared with iGluSnFR, iGluu has a sixfold faster dissociation rate in vitro and fivefold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGluu is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGluu responses during 100-Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high-frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons.
登录
查看更多内容
影响因子：
5.1
作者：
Scimemi, Annalisa;Beato, Marco
通讯作者：
Beato, Marco
影响因子：
--
作者：
Gee, Christine E;Ohmert, Iris;Oertner, Thomas G
通讯作者：
Oertner, Thomas G
影响因子：
4.6
作者：
Zheng, Kaiyu;Jensen, Thomas P.;Rusakov, Dmitri A.
通讯作者：
Rusakov, Dmitri A.
DOI：
10.1073/pnas.0712008105
发表时间：
2008-03-18
影响因子：
11.1
作者：
Hires, Samuel Andrew;Zhu, Yongling;Tsien, Roger Y.
通讯作者：
Tsien, Roger Y.
影响因子：
3.3
作者：
Jenkins, Meagan A.;Traynelis, Stephen F.
通讯作者：
Traynelis, Stephen F.