Developing next generation multiphoton systems to reveal cortico-thalamic interactions underlying short-term memory in behaving mice

开发下一代多光子系统以揭示行为小鼠短期记忆背后的皮质-丘脑相互作用

• 批准号: 10680577
• 负责人: Murat Yildirim
• 金额: $ 24.9万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680577
• 关键词: Ablation; Animals; Arousal; Behavior; Behavioral Paradigm; Brain; Brain imaging; Brain region; Cells; Communication; Coupling; Custom; Detection; Elements; Etiology; Functional Imaging; Goals; Image; Individual; Label; Lasers; Lateral; Lateral Geniculate Body; Length; Location; Methods; Microscope; Mus; Neurons; Neurosciences; Optics; Pathology; Penetration; Performance; Photons; Physiology; Protocols documentation; Resolution; Rewards; Sensory; Short-Term Memory; Site; Speed; System; Technology; Tissues; Visual; area striata; attenuation; awake; design; detector; expectation; extrastriate visual cortex; improved; instrument; multi-photon; multiphoton microscopy; neural; next generation; novel; optogenetics; response; three photon microscopy; two photon microscopy; two-photon; visual stimulus

One of the goals of systems neuroscience is to understand how sensory information is transformed into goal- directed behavior via diverse brain regions and circuits. To achieve this aim, it is critical to elucidate computations performed within specific layers of the cortex by specific cell classes and the communication dynamics between multiple brain regions. Two-photon microscopy has been used successfully to perform functional brain imaging at the single-cell level mice, but its penetration is limited by tissue scattering to the top layers of the cortex. I have developed a 3-photon microscope to overcome this challenge. Today, the main drawback of 3-photon microscope is its relatively modest speed, limiting its use for multi-site imaging. Optimizing instrument design and imaging protocol to overcome this limitation is required for broad end-user acceptance. In this proposal, I will construct and optimize a combined 2-photon and 3-photon microscope for multi-site, superficial and deep brain imaging at single-cell resolution. Specifically, I have first developed a custom-made 3-photon microscope with optimized laser and microscope parameters (Aim 1a). Optimizing these parameters can improve imaging speed and imaging depth while lowering the average laser power to avoid damage in the live mouse brain. The microscope performance improvement has been validated by performing functional imaging in the primary visual cortex of GCaMP6 mice to characterize visual responses of each cortical layer and subplate. In addition, I will characterize the effective attenuation lengths (EAL) of higher visual areas in awake mice with label-free imaging and laser-ablation methods. Then, I will demonstrate the microscope’s performance by examining cell-specific differences within a layer 6 (L6) of V1. Since neuronal responses to visual stimuli are modulated by the cortical state such as arousal, or reward expectation, I will image adjacent sets of neurons with distinct projections to the lateral geniculate nucleus (LGN) and lateral posterior (LP) regions (e.g., cortico-cortical [CC] and cortico-thalamic [CT] neurons in L6) in primary and higher visual areas to reveal circuit-based response types within a single cortical layer using retrobead-based tracing methods (Aim 1b). Next, I have developed custom-made 2-photon wide-field microscope to perform neuronal recordings and manipulations in the primary visual cortex and higher visual areas (Aim 2a). I have improved imaging speed and field of view by implementing multifocal multiphoton microscopy (MMM). Multiple foci two-photon excitation efficiency will be optimized by coupling a diffractive element (DOE) with customized intermediate optics. High sensitivity single-photon counting detection will be achieved using a novel avalanche photodiode array detector. To demonstrate microscope performance and which brain regions are necessary for a well-established goal-directed behavioral paradigm, I will perform SLM- based two-photon optogenetics while imaging expert animals (Aim 2b). In addition to imaging and stimulating neuronal activity across superficial depths at single regions and at multiple regions, it is necessary to image and optogenetically manipulate neuronal activity at multiple depths, at targeted locations, and for identified neurons, in order to determine the causality of neuronal subpopulations in behavior. Here, I will design and implement two- and three-photon MMM systems to extend the depth performance of MMM for multi-site neuronal recording across multiple regions and multiple layers and integrate this system with the 2-photon optogenetics system implemented in Aim 2a (Aim 3a). I will use this technology for modulating specific components of the cortico- cortical and cortico-thalamo-cortical projections of V1-V2-PPC-MC circuit (Aim 3b).

系统神经科学的目标之一是了解感觉信息如何转化为目标 通过不同的大脑区域和回路来指导行为。为了实现这一目标，阐明计算至关重要 由特定的细胞类别和之间的通信动态在皮层的特定层内执行 多个大脑区域。双光子显微镜已成功用于进行功能性脑成像 在单细胞水平的小鼠中，但其渗透受到分散到皮质顶层的组织的限制。我有 开发了三光子显微镜来克服这一挑战。今天，3光子的主要缺点 显微镜的缺点是其速度相对适中，限制了其多部位成像的使用。优化仪器设计 为了获得广泛的最终用户接受，需要克服这一限制的成像协议。在这个提案中，我 将构建和优化用于多位点、浅层和深层的组合 2 光子和 3 光子显微镜 单细胞分辨率的脑成像。具体来说，我首先开发了一个定制的三光子显微镜 具有优化的激光和显微镜参数（目标 1a）。优化这些参数可以改善成像 速度和成像深度，同时降低平均激光功率以避免对活体小鼠大脑造成损害。这 通过在初级视觉中进行功能成像，显微镜性能的改进已得到验证 GCaMP6 小鼠的皮质，以表征每个皮质层和亚板的视觉反应。另外，我会 通过无标记成像表征清醒小鼠较高视觉区域的有效衰减长度 (EAL) 和激光烧蚀方法。然后，我将通过检查细胞特异性来展示显微镜的性能 V1 的第 6 层 (L6) 内的差异。由于神经元对视觉刺激的反应是由皮层调节的 状态，例如唤醒或奖励期望，我将对相邻的神经元组进行成像，并对其进行不同的投影 外侧膝状核 (LGN) 和外侧后部 (LP) 区域（例如皮质-皮质 [CC] 和皮质-丘脑 [CT] L6 中的神经元）位于初级和高级视觉区域，以揭示单个视觉区域内基于回路的反应类型 使用基于逆转录珠的追踪方法（目标 1b）。接下来，我开发了定制的 2 光子 宽视野显微镜可在初级视觉皮层和更高级别进行神经元记录和操作 视觉区域（目标 2a）。我通过实施多焦多光子提高了成像速度和视野 显微镜（MMM）。多焦点双光子激发效率将通过耦合衍射来优化 元件（DOE）与定制的中间光学器件。高灵敏度单光子计数检测将 使用新型雪崩光电二极管阵列探测器实现。展示显微镜性能和 哪些大脑区域对于一个完善的目标导向行为范式是必需的，我将执行 SLM- 基于双光子光遗传学，同时对专业动物进行成像（目标 2b）。除了成像和刺激 为了了解单个区域和多个区域浅层深度的神经元活动，有必要对其进行成像和 光遗传学操纵多个深度、目标位置和已识别神经元的神经元活动， 为了确定神经元亚群行为的因果关系。在这里，我将设计并实现 二光子和三光子 MMM 系统可扩展 MMM 的深度性能以进行多位点神经元记录 跨多个区域和多个层，并将该系统与 2 光子光遗传学系统集成 在目标 2a（目标 3a）中实施。我将使用这项技术来调节皮质的特定成分 V1-V2-PPC-MC 回路的皮质和皮质-丘脑-皮质投影（目标 3b）。