A high-throughput sequencing and imaging approach to understand the functional basis of olfaction

项目总结 我们建议开发一种理解嗅觉编码的策略，通过将嗅觉编码的分子同一性 气味感受器(OR)在体内对气味的敏感性，并发现处理的神经回路的逻辑 闻。与其他感官形式(即视觉、听觉)不同，人们不了解气味的什么特性 对嗅觉很重要，这些特性是如何由嗅觉神经回路处理的，以及气味如何 受体基因已经进化到优化这种编码。香料的结构与结构之间的关系 (化学空间)，气味受体的序列，潜在的时空活动模式 大脑(神经元空间)和感知气味质量(感知空间)一直难以捉摸。一种高效的 连接嗅觉空间的方法将对嗅觉研究产生范式转换效应。 我们的多学科方法将使用尖端的下一代测序技术(FISSEQ， MAPSeq和RNAseq)与功能广域荧光和体内双光子成像一起确定 表达特定气味受体(ORs)的嗅觉感觉神经元的功能特性 发现它们与单个肾小球嗅球(OB)、二阶OB输出(二尖瓣/簇状)的联系 并将它们的投影统计数据映射到下游的嗅觉处理大脑区域。使用这些工具， 我们将绘制小鼠嗅球中所有3,500个肾小球的身份和空间布局图 或它们从其接收输入的类型。我们将进一步将ORs的分子身份与它们的肾小球联系起来 以OR/气味结合亲和力矩阵的形式响应数百种气味(&gt；500) 在活体内可光学访问的肾小球(~500)。在相同的样本中，我们将跟踪数千人 (&gt；1,000/实验)单个嗅球投射到它们的输入肾小球和它们的目标脑区 通过在活体内通过多光子成像将它们与气味调谐相关的RNA条形码。 我们的方法将弥合ORS的分子生物学和神经生理学之间的差距，并将 开启了一个理解嗅觉功能基础的新时代。它将允许前所未有的分辨率和 用于确定数百种气味的OR-配体相互作用的吞吐量，以及数十种气味的连接性 在一个样本中同时有数千个单个神经元。所获得的数据将使研究或- 配基相互作用，将气味的化学特性与嗅觉联系起来，并构建 用于即时生物医学应用的人造鼻子设备，包括疾病诊断。

PROJECT SUMMARY We propose to develop a strategy for understanding olfactory coding by linking the molecular identity of odorant receptors (OR) to their odor sensitivities in vivo and discovering the logic of neural circuits that process smell. Unlike for other sensory modalities (i.e. vision, audition), it is not understood what properties of odorants are important for olfaction, how these properties are processed by olfactory neural circuits, and how odorant receptor genes have evolved to optimize such an encoding. The relationship between odorant structure (chemical space), the sequence of odorant receptors, the underlying spatial-temporal patterns of activity in the brain (neuronal space) and the perceived odor quality (perceptual space) has been elusive. An efficient method for connecting the olfactory sensory spaces will have a paradigm-shifting effect on olfactory research. Our multidisciplinary approach will use cutting edge next-generation sequencing technologies (FISSEQ, MAPseq and RNAseq) together with functional widefield fluorescence and two photon imaging in vivo to define the functional properties of olfactory sensory neurons that express defined odorant receptors (ORs), to discover their connections to individual glomeruli olfactory bulb (OB), second order OB output (mitral/tufted) cells and map their projection statistics to the downstream olfactory processing brain areas. Using these tools, we will map the identity and spatial layout of all 3,500 glomeruli in the mouse olfactory bulb according to the OR types from which they receive inputs. We will further link the molecular identity of ORs to their glomerular responses to hundreds of odorants (>500) in the form of OR/odorant binding affinity matrices across hundreds of glomeruli (~500) that are optically accessible in vivo. In the same samples, we will track thousands (>1,000/experiment) of individual olfactory bulb projections to their input glomeruli and their target brain areas by RNA-barcoding in relation to their tuning to odorants via multiphoton imaging in vivo. Our approach will bridge the gap between the molecular biology of ORs and neurophysiology and will usher in a new era of understanding the functional basis of olfaction. It will allow unprecedented resolution and throughput for determining OR-ligand interactions across hundreds of odorants, and the connectivity of tens of thousands of single neurons at once in a single specimen. The data obtained will enable the study of OR- ligand interactions, relate the chemical identity of odorants to olfactory perception, and the construction of artificial nose devices for immediate biomedical applications, including disease diagnostics.