2 photon imaging in visual cortex of awake monkey

2 清醒猴视觉皮层的光子成像

• 批准号: 9117239
• 负责人: Wyeth Daniel Bair
• 金额: $ 26.7万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9117239
• 关键词: Address; Afferent Neurons; Anesthesia procedures; Animals; Antibodies; Artificial Implants; Autopsy; Behavior; Behavioral; Blood Vessels; Brain Diseases; Calcium; Capsid; Cells; Chronic; Complex; Computer software; Craniotomy; Custom; Development; Dura Mater; Equilibrium; Evaluation; Fluorescence; Frequencies; Goals; Growth; Health; Image; Imagery; Implant; Injection of therapeutic agent; Investigation; Laser Scanning Microscopy; Lasers; Learning; Longevity; Longitudinal Studies; Macaca; Maintenance; Measures; Methodology; Methods; Microscope; Monkeys; Morphologic artifacts; Motion; Movement; Neuroglia; Neurons; Neurosciences; Noise; Operative Surgical Procedures; Optics; Photons; Physiological; Population; Positioning Attribute; Primates; Procedures; Property; Protocols documentation; Recipe; Research; Resolution; Serotyping; Shapes; Signal Transduction; Space Perception; Staining method; Stains; Stimulus; System; Techniques; Technology; Testing; Time; Tissues; Toxic effect; Training; Transplantation; V1 neuron; Visual Cortex; Washington; Work; animal tissue; awake; base; cranium; design; impaired capacity; implantation; infancy; learned behavior; neurophysiology; novel; programs; promoter; public health relevance; relating to nervous system; research study; response; skull implant; tool; two-photon

 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.

 描述（由应用程序提供）：两光子钙成像（2pci）允许在具有单个神经元分辨率的小皮质斑块中同时可视化和物理表征数百个神经元。随着一般编码的Ca2+指标（GECIS）的推进，现在可以识别和跟踪长期一个月或更长时间的神经元。这为神经元的彻底表征和研究神经元的纵向研究打开了新的机会。虽然Awake 2pci是较小动物的一项公认的技术，但其发展仍处于起步阶段 灵长类动物。我们的目标是成功实现2pci在醒目的表现中，并将其确立为Prime Systems神经科学家武器库中的强大工具。该提议解决了在清醒猕猴中成功进行2光量成像的两个主要挑战。首先，在清醒动物中，必须将带有清晰，透明的窗户的成像室和与2p显微镜物镜的合适界面植入，并在长时间内没有组织生长。其次，必须为猕猴开发用于可靠表达的协议。我们将在AIM 1和2中独立处理这些挑战，并将最终的技术结合在AIM 3中。在AIM 1中，我们将植入定制设计的低调腔室，在无血肿的手术中进行颅骨切开术和硬脑膜切开术，并植入人造硬皮。然后，我们将通过在专门的无血液程序中去除新膜改革来完善几个月来维护腔室的技术。我们将在会议厅内表达GFP，并在几个月内评估2P成像的质量。我们还将在会话内实施硬件和软件策略，以进行图像稳定和对齐方式，以纠正运动伪像，并在会话中识别匹配的神经元。在AIM 2中，我们将通过对具有多种参数的注射并评估在事后组织中的表达来确定适当的AAV CAPSID血清型，启动子和注射程序来表达GCAMP6。我们还将评估GCAMP信号随时间的稳定性，其信噪比，其线性，对细胞的毒性以及对麻醉动物中细胞激光的毒性。最后，在AIM 3中，我们将在 使用AIM 2中精制的程序表达的GECI清醒动物。我们将在几周和几个月内量化V1神经元的调整，以确定信号是否足够稳定以允许对神经元进行长期研究。我们的实验将对清醒猕猴中的2P成像进行首次详细评估，以作为纵向研究和可视化神经元的强大工具，我们的结果将为实施此技术提供秘诀，可以轻松地将其移植到其他实验室。最终，这项技术可以帮助我们了解神经元网络如何构成学习和复杂行为的基础，并且可以帮助制定策略来减轻这些能力受损的脑部疾病。