Ulivi Alessandro F, Castello-Waldow Tim P, Weston Ghabiba, Yan Long, Yasuda Ryohei, Chen Alon, Attardo Alessio
Dept. of Stress Neurobiology and Neurogenetics, Max Planck Institute of Psychiatry.
Dept. of Stress Neurobiology and Neurogenetics, Max Planck Institute of Psychiatry; Graduate School of Systemic Neurosciences, Ludwig Maximilians University.
J Vis Exp. 2019 Jun 19(148). doi: 10.3791/59598.
Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.
双光子显微镜是神经科学的一项基础工具，因为它能够在从亚细胞到网络水平的空间尺度以及从毫秒到数周的时间尺度上对活体动物的大脑进行研究。此外，双光子成像可以与各种行为任务相结合，以探索脑功能与行为之间的因果关系。然而，在哺乳动物中，光的穿透和散射受限，使得双光子活体成像大多局限于大脑浅表区域，从而排除了对诸如海马体等深部脑区进行纵向研究的可能性。海马体参与空间导航和情景记忆，是一个长期以来用于研究健康和疾病状态下对学习和记忆至关重要的细胞以及认知过程的模型。在此，详细介绍一种能够对活体小鼠的背侧海马体进行长期光学观察的制备方法。这种制备方法可以与在头部固定、麻醉的活体小鼠中以细胞和亚细胞分辨率进行的双光子光学成像相结合，持续数周时间。这些技术能够对背侧海马体CA1区中数十到数百个神经元的神经元结构或活动诱发的可塑性进行重复成像。此外，这种长期制备方法可以与其他技术如微型内窥镜、头戴式宽视野显微镜或三光子显微镜结合使用，从而极大地扩展了用于研究学习和记忆中涉及的细胞和网络过程的工具库。
