2 photon imaging in visual cortex of awake monkey

 描述（由申请人提供）：双光子钙成像（2PCI）允许同时可视化和生理表征一小片皮质内的数百个神经元，具有单个神经元分辨率。随着遗传编码Ca2+指标（GECIs）的出现，现在可以长时间识别和跟踪神经元，长达一个月或更长时间。这为神经元的彻底表征和研究学习的神经基础的纵向研究开辟了新的机会。虽然清醒2 PCI是一种在小型动物中成熟的技术，但其发展仍处于起步阶段， 灵长类动物我们的目标是在清醒的猕猴中成功地实施2PCI，并将其建立为灵长类系统神经科学家武器库中的强大工具。该建议解决了在清醒猕猴中成功进行双光子成像的两个主要挑战。首先，必须植入具有清晰透明窗口和与2P显微镜物镜的合适接口的成像室，并在清醒动物中长时间保持无组织生长。其次，必须为猕猴开发一种可靠表达GECI的方案。我们将在目标1和目标2中独立应对每一项挑战，并将在目标3中结合联合收割机所产生的技术。在目标1中，我们将植入一个定制设计的低切迹腔室，在无血手术中进行开颅术和硬脑膜切开术，并植入人工硬脑膜。然后，我们将改进技术，通过在一个微妙的无血程序中去除新膜再生，使腔室保持数月。我们将在室内表达GFP，并在数月内评估2P成像的质量。我们还将实施用于图像稳定和对齐的硬件和软件策略，在会话内校正运动伪影并在会话中识别匹配的神经元。在目标2中，我们将通过使用各种参数进行注射并评估死后组织中的表达来鉴定适当的AAV衣壳血清型、启动子和注射程序以表达GCaMP6。我们还将评估GCaMP信号随时间的稳定性、信噪比、线性度、对细胞的毒性以及激光对麻醉动物细胞的毒性。最后，在目标3中，我们将在 清醒的动物，使用目标2中改进的程序表达GECI。我们将量化V1神经元的基本生理参数（方向，空间频率等）的调谐。以确定信号是否足够稳定，以允许对神经元进行长期研究。我们的实验将首次详细评估清醒猕猴的2P成像是否可以作为神经元纵向研究和可视化的有力工具，我们的研究结果将为实施这项技术提供一个配方，可以很容易地移植到其他实验室。最终，这项技术可以帮助我们了解神经元网络是如何支撑学习和复杂行为的，它可以帮助我们制定策略来缓解这些能力受损的大脑疾病。

 DESCRIPTION (provided by applicant): Two-photon calcium imaging (2PCI) allows the simultaneous visualization and physiological characterization of hundreds of neurons within a small patch of cortex with single neuron resolution. With the advent of genetically-encoded Ca2+ indicators (GECIs), it is now possible to identify and track neurons for extended periods, up to a month or longer. This opens up new opportunities for the thorough characterization of neurons and for longitudinal studies that examine the neural bases of learning. While awake 2PCI is a well-established technique in smaller animals, its development is still in its infancy in the primate. Our goal is to successfully implement 2PCI in the awake behaving macaque and establish it as a powerful tool in the arsenal of primate systems neuroscientist. This proposal addresses the two major challenges to successful 2-photon imaging in the awake macaque. First, an imaging chamber with a clear, transparent window and a suitable interface to the 2P microscope objective must be implanted and maintained free of tissue growth for a prolonged period in the awake animal. Second, a protocol for the reliable expression of a GECI must be developed for the macaque. We will work independently on each of these challenges in Aims 1 and 2 and will combine the resulting technologies in Aim 3. In Aim 1, we will implant a custom-designed low profile chamber, perform a craniotomy and durotomy in a bloodless surgery and implant an artificial dura. We will then refine the technique to maintain the chamber over months by removing the neomembrane regrowth in a delicate bloodless procedure. We will express GFP within the chamber and assess the quality of 2P imaging over the course of months. We will also implement hardware and software strategies for image stabilization and alignment, both within session to correct for motion artifacts and to identify matched neurons across sessions. In Aim 2, we will identify the appropriate AAV capsid serotypes, promotors, and injection procedures to express GCaMP6 by performing injections with a variety of parameters and assessing expression in postmortem tissue. We will also assess the stability of the GCaMP signal over time, its signal-to-noise ratio, its linearity, its toxicity to cells and the toxicity f the laser to the cells in anesthetized animals. Finally, in Aim 3 we will conduct 2P experiments in the awake animal with the GECI expressed using the procedures refined in Aim 2. We will quantify the tuning of V1 neurons for basic physiological parameters (orientation, spatial frequency, etc.) across weeks and months to determine whether the signals are sufficiently stable to allow long-term studies of neurons. Our experiments will provide the first detailed evaluation of whether 2P imaging in the awake macaque can serve as a powerful tool for longitudinal studies and visualization of neurons, and our results will provide a recipe for implementation of this technique that can be easily transplanted to other labs. Ultimately, this technique can help us understand how networks of neurons underlie learning and complex behavior, and it can aid in devising strategies to alleviate brain disorders in which these capacities are impaired.