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

基本信息

批准号：
9977555
负责人：
Murat Yildirim
金额：
$ 9.12万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2022-04-30
项目状态：
已结题

项目摘要

1 One of the goals of systems neuroscience is to understand how sensory information is transformed into goal- 2 directed behavior via diverse brain regions and circuits. To achieve this aim, it is critical to elucidate computations 3 performed within specific layers of the cortex by specific cell classes and the communication dynamics between 4 multiple brain regions. Two-photon microscopy has been used successfully to perform functional brain imaging 5 at the single-cell level mice, but its penetration is limited by tissue scattering to the top layers of the cortex. I have 6 developed a 3-photon microscope to overcome this challenge. Today, the main drawback of 3-photon 7 microscope is its relatively modest speed, limiting its use for multi-site imaging. Optimizing instrument design 8 and imaging protocol to overcome this limitation is required for broad end-user acceptance. In this proposal, I 9 will construct and optimize a combined 2-photon and 3-photon microscope for multi-site, superficial and deep 10 brain imaging at single-cell resolution. Specifically, I have first developed a custom-made 3-photon microscope 11 with optimized laser and microscope parameters (Aim 1a). Optimizing these parameters can improve imaging 12 speed and imaging depth while lowering the average laser power to avoid damage in the live mouse brain. The 13 microscope performance improvement has been validated by performing functional imaging in the primary visual 14 cortex of GCaMP6 mice to characterize visual responses of each cortical layer and subplate. In addition, I will 15 characterize the effective attenuation lengths (EAL) of higher visual areas in awake mice with label-free imaging 16 and laser-ablation methods. Then, I will demonstrate the microscope’s performance by examining cell-specific 17 differences within a layer 6 (L6) of V1. Since neuronal responses to visual stimuli are modulated by the cortical 18 state such as arousal, or reward expectation, I will image adjacent sets of neurons with distinct projections to the 19 lateral geniculate nucleus (LGN) and lateral posterior (LP) regions (e.g., cortico-cortical [CC] and cortico-thalamic 20 [CT] neurons in L6) in primary and higher visual areas to reveal circuit-based response types within a single 21 cortical layer using retrobead-based tracing methods (Aim 1b). Next, I have developed custom-made 2-photon 22 wide-field microscope to perform neuronal recordings and manipulations in the primary visual cortex and higher 23 visual areas (Aim 2a). I have improved imaging speed and field of view by implementing multifocal multiphoton 24 microscopy (MMM). Multiple foci two-photon excitation efficiency will be optimized by coupling a diffractive 25 element (DOE) with customized intermediate optics. High sensitivity single-photon counting detection will be 26 achieved using a novel avalanche photodiode array detector. To demonstrate microscope performance and 27 which brain regions are necessary for a well-established goal-directed behavioral paradigm, I will perform SLM- 28 based two-photon optogenetics while imaging expert animals (Aim 2b). In addition to imaging and stimulating 29 neuronal activity across superficial depths at single regions and at multiple regions, it is necessary to image and 30 optogenetically manipulate neuronal activity at multiple depths, at targeted locations, and for identified neurons, 31 in order to determine the causality of neuronal subpopulations in behavior. Here, I will design and implement 32 two- and three-photon MMM systems to extend the depth performance of MMM for multi-site neuronal recording 33 across multiple regions and multiple layers and integrate this system with the 2-photon optogenetics system 34 implemented in Aim 2a (Aim 3a). I will use this technology for modulating specific components of the cortico- 35 cortical and cortico-thalamo-cortical projections of V1-V2-PPC-MC circuit (Aim 3b).

1 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—— 2 通过不同的大脑区域和回路进行定向行为为了实现这一目标，阐明计算至关重要。 3 在皮层的特定层内由特定细胞类别和之间的通信动态执行 4 双光子显微镜已成功用于进行功能性脑成像。 5 在单细胞水平小鼠中，但其渗透受到组织分散到皮质顶层的限制。 6 开发了 3 光子显微镜来克服当今 3 光子的主要缺点。 7 显微镜的缺点是其速度相对适中，限制了其用于多部位成像的优化仪器设计。 8 和克服这一限制的成像协议需要广泛的最终用户接受。 9 将构建和优化用于多位点、浅层和深层的组合 2 光子和 3 光子显微镜具体来说，我首先开发了一种定制的 3 光子显微镜。 11 优化激光和显微镜参数（目标 1a）。 12 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伤。 13 显微镜性能的改进已通过在初级视觉中执行功能成像得到验证 14 GCaMP6 小鼠的皮质，以表征每个皮质层和亚板的视觉反应。 15 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) 16 和激光烧蚀方法然后，我将通过检查细胞特异性来演示显微镜的性能。 V1 的第 6 层 (L6) 内有 17 个差异，因为对视觉刺激的神经反应是由皮质调节的。 18 状态，例如唤醒或奖励期望，我将用不同的投影对相邻的神经元组进行成像 19 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 L6 中的 20 个 [CT] 神经元位于初级和高级视觉区域，以揭示单个视觉区域中基于回路的反应类型 21 皮质层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。 22 宽视野显微镜，用于在初级视觉皮层及更高级别进行神经记录和操作 23 个视觉区域（目标 2a）通过实施多焦点多光子提高了成像速度和视野。 24 显微镜（MMM）将通过耦合衍射来优化多焦点双光子激发效率。 25 元件 (DOE) 与中间定制光学器件将成为高灵敏度单光子计数检测。 26 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。 27 哪些大脑区域对于一个完善的目标导向行为范式是必要的，我将执行 SLM- 28 基于双光子光遗传学，同时对专家动物进行成像（目标 2b）。 29 单个区域和多个区域的浅层神经活动，有必要进行成像和 30 多个深度、目标位置和已识别神经元的光遗传学操纵神经活动， 31 为了确定神经亚群在行为中的因果关系，我将在这里设计并实现。 32 个二光子和三光子 MMM 系统，可扩展 MMM 的深度性能以进行多位点神经记录 33 跨越多个区域和多个层，并将该系统与2光子光遗传学系统集成 34 在目标 2a（目标 3a）中实现，我将使用该技术来调节皮质的特定组件。 V1-V2-PPC-MC 回路的 35 个皮质和皮质-丘脑-皮质投影（目标 3b）。