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: 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
• 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
• 来源: https://reporter.nih.gov/project-details/9977555
• 关键词: Ablation; Animals; Area; Arousal; Behavior; Behavioral Paradigm; Brain; Brain imaging; Brain region; Cells; Cerebral cortex; Color; Communication; Coupled; Coupling; Custom; Detection; Discrimination; Elements; Etiology; Functional Imaging; Goals; Heating; Image; Individual; Label; Lasers; Lateral; Lateral Geniculate Body; Length; Location; Locomotion; Maps; Methods; Microscope; Microscopy; Mus; Neurons; Neurosciences; Optics; Pathology; Penetration; Performance; Photons; Physiology; Population; Property; Protocols documentation; Research; Resolution; Rewards; Sensory; Short-Term Memory; Site; Speed; Structure; System; Technology; Thalamic structure; Tissues; Visual; area striata; attenuation; awake; base; calcium indicator; design; detector; expectation; extrastriate visual cortex; imaging study; imaging system; improved; instrument; interest; multiphoton microscopy; neurophysiology; next generation; novel; optogenetics; relating to nervous system; response; retinotopic; three photon microscopy; two photon microscopy; two-dimensional; two-photon; user-friendly; virtual reality system; visual stimulus; white matter

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通过潜水大脑区域和电路的指示行为。为了实现这一目标，阐明计算至关重要 3在皮质的特定层中通过特定的细胞类执行以及之间的通信动力学 4个多个大脑区域。两光子显微镜已成功地用于执行功能性脑成像 5在单细胞水平小鼠处，但其穿透受到组织散射到皮质的顶层的限制。我有 6开发了一个3光子显微镜来克服这一挑战。今天，3-Photon的主要缺点 7显微镜是其相对适度的速度，限制了其用于多站点成像的使用。优化仪器设计 8和要克服此限制的成像协议是广泛的最终用户接受所必需的。在这个建议中，我 9将构建和优化一个合并的2光子和3光子显微镜，用于多站点，浅表和深 单细胞分辨率的10个大脑成像。具体来说，我首先开发了一个定制的3光子显微镜 11具有优化的激光和显微镜参数（AIM 1A）。优化这些参数可以改善想象力 12速度和成像深度，同时降低平均激光功率，以避免活鼠的大脑损害。这 13显微镜性能改善已通过在初级视觉中进行功能成像来验证 14 GCAMP6小鼠的皮质表征每个皮质层和子板的视觉反应。此外，我会 15表征具有无标签成像的清醒小鼠中较高视觉区域的有效衰减长度（EAL） 16和激光燃料方法。然后，我将通过检查细胞特异性来证明显微镜的性能 V1的第6层（L6）中的17个差异。由于神经元对视觉刺激的反应由皮质调节 18个状态，例如唤醒或奖励期望，我将对邻近的神经元组成不同的项目， 19外侧遗传核（LGN）和侧后（LP）区域（例如，皮质皮质[CC]和皮质 - 丘脑丘脑 20 [CT]在L6中的神经元）在初级和较高视觉区域中，以揭示单个基于电路的响应类型 21使用基于反向逆转录的示踪方法的皮质层（AIM 1B）。接下来，我开发了定制的2-Photon 22个宽视野显微镜，在一级视觉皮层中执行神经元记录和操纵 23个视觉区域（AIM 2A）。我通过实现多焦点多光子来提高成像速度和视野 24显微镜（MMM）。通过偶联衍射，将优化多个焦点两光子兴奋效率 25个带有自定义中间光学的元素（DOE）。高灵敏度单光子计数检测将是 26使用新型的雪崩光电二极管阵列检测器实现。展示显微镜性能和 27哪些大脑区域对于建立良好的目标指导行为范式是必需的，我将执行SLM- 在成像专家动物的同时，基于28个两光遗传学（AIM 2B）。除了成像和刺激 29个在单个区域和多个区域的浅表深度之间的神经元活动，必须进行图像和 30在多个深度，靶向位置和确定的神经元处的神经元活性在光源上操纵神经元活性， 31为了确定行为中神经元亚群的因果关系。在这里，我将设计和实施 32个两光子和三光子MMM系统，以扩展多站点神经元记录的MMM的深度性能 33多个区域和多层，并将该系统与2光遗传学系统整合在一起 34在AIM 2A中实施（AIM 3A）。我将使用该技术调节Cortico-的特定组件 35 V1-V2-PPC-MC电路（AIM 3B）的皮质和皮质 - thalamo-cortical项目。