New tools for neuroscience research - from nano to macroscales
|7月28日（日）8:45～9:15 第7会場（朱鷺メッセ 2F 201B）|
Takayasu Mikuni（三國 貴康）1,2
Rapid and precise genome editing using CRISPR-Cas systems is a powerful technology in various scientific fields. Especially, genome editing through homology-directed repair (HDR) provides a versatile approach to precisely introduce insertions, deletions or replacements in the genome. However, it has been considered to be difficult to induce HDR in postmitotic neurons, limiting the application of precise genome editing in the field of neuroscience. Here I present two strategies, SLENDR and vSLENDR, to induce HDR-mediated genome editing in the mammalian brain in vivo (Mikuni* and Nishiyama* et al., Cell. 2016, 165(7), 1803-17; Nishiyama* and Mikuni* et al., Neuron. 2017, 96(4),755-68). In SLENDR, genome editing machinery is delivered into mitotic neuro-progenitors in the embryonic brain through in utero electroporation method, providing a high-throughput and cost-effective approach for HDR-mediated genome editing in the brain in vivo. On the other hand, in vSLENDR, adeno-associated virus vector is used to effectively deliver genome editing machinery to target cells either in vitro or in vivo, enabling highly efficient HDR in virtually any cell type (even in postmitotic neurons), area, and age across the brain. Rapid, precise and efficient genome editing in the brain in vivo using SLENDR and vSLENDR will allow for a wide range of applications in neuroscience research, such as high-throughput imaging of endogenous gene products with high sensitivity, specificity, contrast and resolution in complicated brain tissue.
|7月28日（日）9:15～9:45 第7会場（朱鷺メッセ 2F 201B）|
Yusuke Hirabayashi（平林 祐介）1,2,3，Juan Carlos Tapia（Tapia Juan Carlos）3，Polleux Franck（Franck Polleux）3
3Dept. Neurosci. Columbia University, NY, USA
A challenging aspect of neuroscience revolves around mapping the synaptic connections within neural circuits (connectomics), as well as revealing the intracellular ultrastructural features of neurons over scales spanning several orders of magnitude (nanometers to meters). Despite significant improvements in serial section electron microscopy (SSEM) technologies, several major roadblocks have impaired its general applicability to mammalian neural circuits. In the present study, we introduce a new approach that circumvents some of these roadblocks by adapting a genetically-encoded ascorbate peroxidase (APEX2) as a fusion protein to a membrane-targeted fluorescent reporter (CAAX-Venus), and introduce it in single pyramidal neurons in vivo using extremely sparse in utero cortical electroporation. This approach allows us to perform Correlated Light-SSEM (CoLSSEM), a variant of Correlated Light-EM (CLEM), on individual neurons, reconstructing their dendritic and axonal arborization in a targeted way via combination of high-resolution confocal microscopy, and subsequent imaging of its ultrastructural features and synaptic connections with ATUM-SEM (automated tape-collecting ultramicrotome - scanning electron microscopy) technology. Our method significantly will improve the feasibility of large-scale reconstructions of neurons within a circuit, and permits the description of some ultrastructural features of identified neurons with their functional and/or structural connectivity, one of the main goals of connectomics.
|7月28日（日）9:45～10:15 第7会場（朱鷺メッセ 2F 201B）|
Dissecting behaviorally-relevant circuits at cellular resolution
Hyungbae Kwon（Kwon Hyungbae）
Max Planck Florida Institute
A central question in neuroscience is how neural activity is linked to complex behaviors. However, monitoring activity patterns in the mammalian brain has been particularly challenging because of its complexity and the limited availability of tools with high spatiotemporal precision. Recent developments in electrophysiological and imaging techniques such as multiunit recordings and genetically encoded calcium indicators have significantly improved our understanding of the circuit mechanisms underlying sensory perception and behavior. However, a critical need exists to develop new methods that can convert neural activity to an effector system that directly demonstrates a circuit-behavior relationship.
We recently developed a novel optogenetic technique that can translate neuronal activity to gene expression in vivo at a high spatiotemporal resolution. We have created a dual-control system named Calcium and Light-Induced Gene Handling Toolkit, Cal-Light, that allows gene expression to be initiated by calcium and light. Cal-Light directly links neuronal firing to gene expression, thereby allowing us to map out the activity profile of individual neurons in animals and test their causal relationship with specific behaviors.
We also developed a novel light-gated method to label and manipulate specific neuronal populations activated by neuromodulators in a highly temporally precise manner. We created an inducible dual protein switch system that is turned on and off by not only a ligand but also light. We named this technique iTango2, a light-gated gene expression system that uses beta-arrestin and a light-inducible split tobacco-etch-virus (TEV) protease. We showed that both ligand and light are necessary to induce targeted gene expression using an iTango2 system, indicating that light stimulation can be used to monitor the activity of neuromodulatory ligands in circuits over time.
In this talk, I will present these novel optogenetic methods that label active neuronal ensemble and neuromodulation-sensitive populations and further discuss current ongoing development of new techniques.
|7月28日（日）10:15～10:45 第7会場（朱鷺メッセ 2F 201B）|
Kazuki Tainaka（田井中 一貴）
To examine an entire mammalian body as functional assemblies of individual cells is an important goal in biology and medicine. The systematic identification of cellular properties in their physiological context increases our understanding of complex cellular networks in the body. To this end, three-dimensional (3D) imaging based on tissue clearing technique that are compatible with various fluorescent labeling techniques are promising approaches. The tissue-clearing step is critical for determining the quality of the subsequent acquired image and the feasibility of image processing. Since tissue-clearing methods were first reported by Spalteholz over a century ago, the field has expanded tremendously. However, tissue-clearing techniques still need to be improved for the 3D imaging of larger primate and human organs and mammalian whole bodies. To achieve scalable clearing of such large samples in aqueous media, we considered a series of chemical processes, including delipidation, decoloring, refractive index matching, and decalcification. High-throughput evaluation systems suitable for each chemical process enabled the comprehensive screening of more than 1600 hydrophilic chemicals. The strategic integration of optimal chemical cocktails provided a series of CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) protocols that enabled the efficient clearing of mouse organs, mouse body including bone, and large primate tissue samples. The new CUBIC protocols enabled the scalable imaging of mouse whole body and marmoset whole brain at single-cell resolution. In addition, a fluorescent protein-compatible CUBIC protocol allowed the systematic profiling of various fluorescent labels. CUBIC protocols for human tissues enabled the scalable imaging of large tissue samples over 10 cm3 and exhaustive cell detection.