Developing a strategy for 4-color in vivo two-photon imaging

许多神经发育和神经退行性脑障碍表现为突触完整性、稳定性 和经验依赖性选择，导致连接缺陷和功能紊乱。然而我们的能力 研究这些疾病如何影响突触结构和功能受到以下困难的严重限制： 可视化活体大脑中的突触并追踪它们的各种蛋白质成分。我们建议发展 并测试新的标签和显微镜配置，使同步实时跟踪多达 在完整的小鼠大脑中的四种细胞蛋白质。下面的目标提出了两个四色星座 是为了解决不同类别的实验问题而设计的。对双光子的调整 能够适应这两个目标的显微镜设计将主要在检测路径中。 开发和实施光谱分辨双光子显微镜，用于同时跟踪多达 位于突触间隙两侧的四种蛋白质。这个目标中的4色星座被设计成 除了两种突触后蛋白质和一种突触前传入标记外， 突触后细胞填充我们将标记两个被认为是相互排斥的兴奋性突触后标记， PSD 95和PSD 93，在丘脑传入遗传标记的背景下。在这种情况下，tdTomato标记丘脑 传入，eYFP充当细胞填充物，PSD 95-teal标记成熟的兴奋性突触，PSD 93融合到远侧突触， 红色荧光团（iRFP 682）标记未成熟的兴奋性突触。有了这个标签，人们可以问这样的问题， 成熟PSD 95阳性棘与未成熟PSD 93阳性棘的丘脑神经支配的比率和动力学是什么 积极的脊柱？这种4色组合也可以用于监测任何两个突触后标记， 与任何细胞类型特异性传入标记组合。目标2：开发和实施光谱分辨 双光子显微镜用于同时跟踪多达四个突触后荧光团，其中一个 是绿色。在绿色范围内有几类荧光团特别有用 与结构标记联合收割机。最值得注意的是，pH敏感的GFP突变体，超级黄道pHluorin（SEP） 用于标记和跟踪突触受体，以及广泛用于监测 神经元活动不幸的是，绿色荧光团与大多数蓝色和黄色标记不相容，这是由于 它们的发射光谱重叠。在这里，锥体神经元将共同表达：红细胞填充（mScarlet）以标记 树突状形态、PSD 95-iRFP 682和PSD 93-BFP（短波长蓝色）以标记成熟和未成熟 兴奋性突触，分别，和SEP-GluR 1。我们可以问这样的问题， AMPA受体插入到成熟的PSD 95阳性和未成熟的PSD 93阳性棘中的比例？这4色 组合也可用于用SEP标记和追踪NMDA或GABA受体亚基。 用于GCaMP钙传感器的SEP标记的受体将进一步扩展这种四色荧光的可能性。 通过使活性报告子与突触标记整合，可以构建一个新的星座。

Many neurodevelopmental and neurodegenerative brain disorders manifest impaired synaptic integrity, stability, and experience-dependent selection, resulting in wiring deficits and perturbed function. Yet our ability to investigate how such disorders affect synaptic structure and function is severely limited by the difficulty of visualizing synapses in the living brain and tracking their varied protein components. We propose developing and testing new labeling and microscope configurations that would enable simultaneous live tracking of up to four cellular proteins in the context of the intact mouse brain. The aims below put forth two 4-color constellations that are designed to address distinct classes of experimental questions. Adjustments to the two-photon microscope design that could accommodate both aims would primarily be in the detection path. Aim 1: To develop and implement spectrally resolved two-photon microscopy for simultaneous tracking of up to four proteins situated on both sides of the synaptic cleft. The 4-color constellation in this aim is designed to enable in vivo monitoring of a presynaptic afferent label in addition to two postsynaptic proteins and a postsynaptic cell fill. We will label two excitatory post-synaptic markers that are considered mutually exclusive, PSD95 and PSD93, in the context of a thalamic afferent genetic label. In this scenario, tdTomato labels thalamic afferents, eYFP serves as a cell fill, PSD95-teal labels mature excitatory synapses, and PSD93 fused to a far red fluorophore (iRFP682) labels immature excitatory synapses. With this labeling one could ask questions such as, what is the ratio and dynamics of thalamic innervation to mature PSD95 positive spines vs immature PSD93 positive spines? This 4-color combination could also be used to monitor any two postsynaptic labels in combination with any cell-type specific afferent label. Aim 2: To develop and implement spectrally resolved two-photon microscopy for simultaneous tracking of up to four postsynaptic fluorophores, one of these being green. There are several categories of fluorophores in the green range that would be particularly useful to combine with structural markers. Most notably, the pH-sensitive GFP mutant, Super ecliptic pHluorin (SEP) used to tag and track synaptic receptors, and the GCaMP family of calcium sensors, broadly used to monitor neuronal activity. Unfortunately, a green fluorophore is incompatible with most blue and yellow labels due to overlap of their emission spectra. Here pyramidal neurons would co-express: a red cell fill (mScarlet) to label dendritic morphology, PSD95-iRFP682 and PSD93-BFP (a short wavelength blue) to label mature and immature excitatory synapses, respectively, and SEP-GluR1. We could then ask questions such as, what are the dynamics of AMPA receptor insertion into mature PSD95 positive vs immature PSD93 positive spines? This 4-color combination could also be used to tag and track NMDA or GABA receptor subunits with SEP. Swapping the SEP-tagged receptor for a GCaMP calcium sensor would further expand the possibilities of this 4-color constellation by enabling the integration of an activity reporter with synaptic labeling.