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

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指导行为。为了实现这一目标，阐明计算是至关重要的 3由特定的细胞类别在皮层的特定层内执行， 4多个大脑区域。双光子显微镜已成功地用于进行脑功能成像 5在单细胞水平的小鼠，但它的渗透是有限的组织散射到皮层的顶层。我有 6开发了一种3光子显微镜来克服这一挑战。今天，3光子的主要缺点 7显微镜是其相对适中的速度，限制了其用于多部位成像。优化仪器设计 8和成像协议，以克服这一限制是需要广泛的最终用户接受。在这份提案中，我 9将构建和优化组合的2光子和3光子显微镜，用于多部位，浅层和深层 图10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3光子显微镜 优化激光和显微镜参数（目标1a）。优化这些参数可以改善成像 12速度和成像深度，同时降低平均激光功率，以避免损伤活小鼠大脑。的 13显微镜性能改善已通过在主要视觉中进行功能成像得到验证 14皮质以表征每个皮质层和亚板的视觉反应。此外，我将 图15用无标记成像表征清醒小鼠中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展示显微镜的性能。 V1的层6（L 6）内的17个差异。由于神经元对视觉刺激的反应是由大脑皮层调节的， 18状态，如唤醒，或奖励期望，我将图像相邻的神经元与不同的投影到 19外侧膝状体核（LGN）和外侧后（LP）区域（例如，皮质-皮质[CC]和皮质-丘脑 L 6中的20个[CT]神经元）在初级和高级视觉区域中，以揭示单个 21皮质层，使用基于逆转录珠的追踪方法（目标1b）。接下来，我开发了定制的双光子 22宽视场显微镜进行初级视觉皮层和更高层次的神经元记录和操作 23个视觉区域（目标2a）。我已经通过实现多焦点多光子提高了成像速度和视野 24显微镜（MMM）。多焦点双光子激发效率将通过耦合衍射 25个元件（DOE），具有定制的中间光学器件。高灵敏度的单光子计数探测将是 26实现了使用一种新型的雪崩光电二极管阵列检测器。为了展示显微镜性能， 27大脑区域是必要的一个既定的目标导向的行为范式，我将执行SLM- 28基于双光子光遗传学，同时成像专家动物（目标2b）。除了成像和刺激 29在单个区域和多个区域的浅表深度的神经元活动，有必要成像和 30光遗传学操纵多个深度、靶向位置的神经元活动，并且对于鉴定的神经元， 31，以确定行为中神经元亚群的因果关系。在这里，我将设计并实现 32个双光子和三光子MMM系统，扩展MMM的深度性能，用于多部位神经元记录 33，并将该系统与双光子光遗传学系统集成 34项在目标2a（目标3a）中执行。我将用这项技术来调节皮质的特定成分- 35例V1-V2-PPC-MC回路的皮质和皮质-丘脑-皮质投射（Aim 3b）。

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).