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: 10671180
• 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/10671180
• 关键词: Developing; next; generation; multiphoton; systems

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是如何转化为目标的-- 通过不同的大脑区域和回路引导行为。为了实现这一目标，阐明计算是至关重要的 通过特定的细胞类别在皮质的特定层内执行，以及之间的通信动力学 多个脑区。双光子显微镜已被成功地用于进行脑功能成像 在单细胞水平的小鼠身上，但其穿透受到组织散射到皮层顶层的限制。我有过 开发了一种3光子显微镜来克服这一挑战。今天，三光子的主要缺点是 显微镜的速度相对较慢，限制了其在多点成像中的使用。优化仪器设计 而克服这一限制的成像协议是被终端用户广泛接受所必需的。在这项建议中，我 将构建和优化多点、浅层和深层的双光子和三光子组合显微镜 以单细胞分辨率进行脑部成像。具体地说，我首先开发了一种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 同时降低平均激光功率以避免对活体小鼠大脑造成损害。这个 显微镜性能的改善已通过在初级视觉中执行功能成像得到验证 GCaMP6小鼠的大脑皮质，以表征每一皮层和亚板的视觉反应。另外，我会 无标记成像表征清醒小鼠高视觉区域的有效衰减长度 以及激光消融方法。然后，我将通过检查特定细胞来演示显微镜的性能 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大脑皮层 状态，如觉醒，或奖励期望，我会想象相邻的几组神经元，它们有不同的投影到 外侧膝状核(LGN)和外侧后部(LP)区域(如皮质[CC]和丘脑皮质 [CT]L6)初级和高级视觉区域的神经元，以揭示单个 使用基于珠子的追踪法追踪皮质层(目标1b)。接下来，我开发了定制的双光子 在初级视觉皮质和更高级别执行神经元记录和操作的广视场显微镜 视觉区域(目标2a)。我通过实施多焦点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一个衍射光来优化 元件(DOE)，带有定制的中间光学元件。高灵敏度的单光子计数检测将被 采用一种新颖的雪崩光电二极管阵列探测器实现。展示显微镜性能和 目标导向的行为范式需要哪些大脑区域，我将进行SLM- 以双光子光遗传学为基础，同时为专家动物成像(目标2b)。除了成像和刺激 在单个区域和多个区域的浅层神经元活动，有必要成像和 光遗传学控制神经元在多个深度、目标位置的活动，以及识别出的神经元， 以确定行为中神经元亚群的因果关系。在这里，我将设计和实现 用于多点神经元记录的双光子和三光子MMM系统扩展MMM的深度性能 跨越多区域、多层次，并将该系统与双光子光遗传学系统集成 在目标2a(目标3a)中执行。我将使用这项技术来调节皮质的特定成分- V1-V2-PPC-MC环路的皮质和皮质-丘脑-皮质投射(目标3b)。