Metal-free, genetically encoded reporters for calcium recording with MRI

神经科学中的许多前沿问题，如神经元如何编码经验、改变行为和 退化，需要在活着的动物的整个大脑中监测神经活动。神经元的活动是 与细胞内钙的增加密切相关。因此，神经监测的基石技术 活动包括使用遗传编码的细胞内钙离子荧光记者。在荧光的同时 钙传感工具已被证明对神经科学研究、光学方法具有巨大的变革作用 不允许在全脑覆盖范围内或在任何任意深度监测神经活动。要解决这个问题 挑战，我们将开发一种新型的基因传感器，用于在大脑中可视化累积的钙信号- 使用磁共振成像(MRI)的大范围。为了建造这些传感器，我们将利用水 被称为水通道蛋白的通道。我们将建立在我们早期发现的水通道蛋白可以用来产生 弥散加权磁共振成像通过增加跨细胞膜的水交换速率。不像 常规的磁共振成像记者，基于水通道蛋白的对比不涉及金属的使用，从而允许 完全自主的、高灵敏度的单基因成像。为了实现我们的目标，我们提出了两个内部 相互关联的具体目标。在第一个目标中，我们将开发基于水通道蛋白的钙信号报告(Arcs)。 通过组装合成的多基因簇将刺激诱发的细胞内钙升高偶联到 水通道蛋白表达。ARC将允许神经活动在清醒时定义的刺激时期内整合， 自由行为的动物，随后通过核磁共振读出。在对细胞系进行优化后，我们将验证 初级神经元中弧光的关键性能属性和安全概况。在第二个目标中，我们将建立 通过成像局部和全脑激活反应已建立的ARC的体内功能 涉及化学发生和光发生输入到腹侧被盖区(VTA)的神经调节范例。 同时，我们将根据神经活动的多个互补读数对ARC进行基准测试，包括 血氧水平依赖(BOLD)功能磁共振成像、钙敏感荧光报告和c-fos 免疫组织化学。这个项目的预期结果是一套经过优化和充分验证的基因 这些工具将为神经科学家提供新的途径，以公正地探索参与 协调从感觉功能到行为生成的一切。

Many leading questions in neuroscience such as how neurons encode experience, modify behavior, and degenerate, require neural activity to be monitored throughout the brain in living animals. Neuronal activity is tightly linked to an increase in intracellular calcium. Therefore, a cornerstone technology for monitoring neural activity involves the use of genetically encoded fluorescent reporters of intracellular calcium. While fluorescent tools for calcium sensing have proven immensely transformative for neuroscience research, optical approaches do not allow neural activity to be monitored with brain-wide coverage or at any arbitrary depth. To address this challenge, we will develop a new type of genetic sensor for visualizing cumulative calcium signals at a brain- wide scale using magnetic resonance imaging (MRI). To construct these sensors, we will leverage water channels known as aquaporins. We will build on our earlier discovery that aquaporins can be used to generate diffusion-weighted MRI contrast by increasing the rate of water exchange across the cell membrane. Unlike conventional MRI reporters, aquaporin-based contrast does not involve the use of metals, thereby permitting fully autonomous, single-gene imaging with high sensitivity. To accomplish our goals, we propose two inter- connected specific aims. In the first aim, we will develop aquaporin-based reporters of calcium signaling (ARCS) by assembling a synthetic multi-gene cluster for coupling stimulus-evoked rises in intracellular calcium to aquaporin expression. ARCS will permit neural activity to be integrated over defined stimulation epochs in awake, freely behaving animals and subsequently read out by MRI. Following optimization in cell lines, we will validate key performance attributes and safety profiles of ARCS in primary neurons. In the second aim, we will establish in vivo functionality of ARCS by imaging local and brain-wide activation in response to well-established neuromodulation paradigms involving chemogenetic and optogenetic inputs to the ventral tegmental area (VTA). Concurrently, we will benchmark ARCS against multiple complementary readouts of neural activity including blood oxygenation level dependent (BOLD) fMRI, calcium-sensing fluorescence reporters, and c-fos immunohistochemistry. The anticipated outcome of this project is an optimized and well-validated set of genetic tools that will provide neuroscientists with new avenues for unbiased exploration of neural networks involved in coordinating everything from sensory function to behavior generation.