Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes.
Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes.
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具有不同尺寸荧光探针的突触和突触外AMPA受体的超分辨率成像。
DOI：
10.7554/elife.27744
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发表时间：
2017-07-27
期刊：
影响因子：
7.7
通讯作者：
Selvin PR
中科院分区：
文献类型：
--
作者：
Lee SH;Jin C;Cai E;Ge P;Ishitsuka Y;Teng KW;de Thomaz AA;Nall D;Baday M;Jeyifous O;Demonte D;Dundas CM;Park S;Delgado JY;Green WN;Selvin PR
以往跟踪AMPA受体（AMPAR）在突触中的扩散的研究发现，突触外存在一个大的移动AMPAR池。使用超分辨率显微镜，我们研究了荧光团大小和光稳定性如何影响大鼠突触后密度（psd）内外的AMPAR运输。有机荧光染料（≈4 nm），量子点，或小（≈10 nm直径；sQDs）或大（约20 nm； bQDs），通过不同尺寸的连接剂耦合到ampar上。我们发现，90%的荧光染料标记的ampar在psd的受限纳米结构域中扩散，并在15分钟或更长时间内保持稳定。不到10%的sQD-AMPARs是突触外和高度移动的。相比之下，如先前观察到的，5-10%的bQD-AMPARs发生在psd中，90-95%发生在突触外。与AMPAR进入受开放PSD“槽”占用限制的假设相反，我们的研究结果表明，AMPAR在PSD中快速进入稳定的“纳米结构域”，其寿命为bbbb15分钟，并且不会在突触外膜中积累。遗忘是我们日常生活中常见的经历。然而，关于我们是如何记忆的，以及为什么我们的记忆有时会让我们失望，还有很多未知之处。大脑包含800到1000亿个神经细胞或神经元，它们在称为突触的连接处相互交流。在突触中，一个神经元释放化学信息，该信息必须通过一个小间隙扩散，然后激活另一个神经元上称为受体的蛋白质。如果第一个神经元反复激活第二个神经元，第二个细胞就会在突触的膜上插入额外的受体。这加强了两个神经元之间的联系。突触的增强被认为是学习的关键机制之一。为了证实这一点，能够监测突触中单个受体的运动和位置将是有帮助的。然而，突触上两个神经细胞之间的间隙，称为突触间隙，宽度不超过40纳米。它比人的头发细25倍，而且太小了，无法用光学显微镜看到。电子显微镜可以看到突触，但在活体组织中不起作用。唯一的另一种选择是将荧光标记——一种染料或一种称为量子点的人造晶体——附着在突触中发现的蛋白质上，并监测由此产生的荧光。不过探针必须足够小，才能穿过突触间隙。利用荧光显微镜，研究人员检查了一种叫做AMPA受体的蛋白质在突触中的分布，这种蛋白质在记忆中起着关键作用。多项研究表明，AMPA受体聚集在突触外。这导致了一种假说，即在学习过程中，AMPA受体在突触外等待，直到突触膜内有空间可用。然而，这一点还没有得到直接证实，部分原因是传统的荧光染料和量子点太大，无法在与受体结合时进入突触间隙。Lee等人现在已经开发出一种量子点，它只有10纳米宽，因此足够小，可以带着AMPA受体进入突触间隙。这些小量子点随后被用来标记从大鼠身上收集的神经元中的AMPA受体，然后在培养皿中培养，这为突触提供了一个全新的视角。图像显示，神经元中的大多数AMPA受体在突触内部的受限区域内循环，有点像握笔，而不是像之前假设的那样在外部等待。用更小的4纳米宽的荧光标签标记受体也会产生类似的效果。AMPA受体是如何进入突触并产生新记忆的，还需要进一步的研究。
Previous studies tracking AMPA receptor (AMPAR) diffusion at synapses observed a large mobile extrasynaptic AMPAR pool. Using super-resolution microscopy, we examined how fluorophore size and photostability affected AMPAR trafficking outside of, and within, post-synaptic densities (PSDs) from rats. Organic fluorescent dyes (≈4 nm), quantum dots, either small (≈10 nm diameter; sQDs) or big (>20 nm; bQDs), were coupled to AMPARs via different-sized linkers. We find that >90% of AMPARs labeled with fluorescent dyes or sQDs were diffusing in confined nanodomains in PSDs, which were stable for 15 min or longer. Less than 10% of sQD-AMPARs were extrasynaptic and highly mobile. In contrast, 5–10% of bQD-AMPARs were in PSDs and 90–95% were extrasynaptic as previously observed. Contrary to the hypothesis that AMPAR entry is limited by the occupancy of open PSD ‘slots’, our findings suggest that AMPARs rapidly enter stable ‘nanodomains’ in PSDs with lifetime >15 min, and do not accumulate in extrasynaptic membranes. Forgetting is a common experience in our everyday life. Yet much remains unknown about how we remember, and about why our memories sometimes fail us. The brain contains 80 to 100 billion nerve cells or neurons, which communicate with one another at junctions called synapses. At a synapse, one neuron releases a chemical message, which must diffuse across a small gap, and then activate proteins called receptors on another neuron. If the first neuron activates the second repeatedly, the second cell responds by inserting additional receptors into its membrane at the synapse. This strengthens the connection between the two neurons. Strengthening of synapses is thought to be one of the key mechanisms underlying learning. To confirm this, it would be helpful to be able to monitor the movement and position of individual receptors at synapses. However, the space between the two nerve cells at at synapse, called the synaptic cleft, is no more than 40 nanometers wide. This is about 25 times thinner than a human hair, and too small to be seen with light microscopy. Electron microscopy can visualize synapses, but does not work in living tissue. The only other option is to attach a fluorescent label – either a dye or a man-made crystal called a quantum dot – to a protein found in synapses and monitor the resulting fluorescence. Though the probe must be small enough to pass through the synaptic cleft to do this. Using fluorescence microscopy, researchers have examined the distribution in synapses of proteins called AMPA receptors, which have a key role in memory. Multiple studies have shown groups of AMPA receptors gathered outside synapses. This has led to the suggestion that during learning, AMPA receptors wait outside the synapse until a space becomes available within the synapse’s membrane. However, this has yet to be confirmed directly, in part because conventional fluorescent dyes and quantum dots are too bulky to enter synaptic clefts when bound to a receptor. Lee et al. have now developed a quantum dot that is only 10 nanometers wide and therefore small enough to enter the synaptic cleft with an AMPA receptor attached. These small quantum dots were then used to label AMPA receptors in neurons collected from rats and then grown in a petri dish, which provided a completely new view of synapses. The images show that the majority of AMPA receptors in neurons circulate within confined domains – a little like holding pens – inside the synapse, rather than waiting outside as previously assumed. Labeling the receptors with smaller 4-nanometer-wide fluorescent tags produces a similar picture. Further work is still need to determine how AMPA receptors get into the synapse and contribute to new memories.