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

神经科学中的许多主要问题，如神经元如何编码经验，修改行为， 退化，需要在活体动物的整个大脑中监测神经活动。神经元活动是 与细胞内钙离子的增加密切相关。因此，监测神经系统的基础技术 活性涉及使用遗传编码的细胞内钙的荧光报告物。而荧光 钙传感工具已被证明对神经科学研究具有巨大的变革性，光学方法 不允许以全脑覆盖或任意深度监测神经活动。为了解决这个 挑战，我们将开发一种新型的遗传传感器，用于可视化大脑中累积的钙信号- 大规模使用磁共振成像（MRI）。为了建造这些传感器，我们将利用水 称为水通道蛋白。我们将建立在我们早期的发现，水通道蛋白可用于产生 通过增加细胞膜上的水交换速率，增强弥散加权MRI对比度。不像 在常规MRI报告子中，基于水通道蛋白的造影剂不涉及金属的使用，从而允许 全自主、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... 具体目标相联系。第一个目标是开发基于水通道蛋白的钙信号报告基因（ARCS） 通过组装一个合成的多基因簇， 水通道蛋白表达。ARCS将允许神经活动在清醒时的限定刺激时期内被整合， 自由行为的动物，随后通过MRI读出。在细胞系优化后，我们将验证 ARCS在原代神经元中的关键性能属性和安全性特征。在第二个目标中，我们将建立 通过对响应于公认的 神经调节范例涉及到腹侧被盖区（VTA）的化学遗传学和光遗传学输入。 同时，我们将基准ARCS对多个互补的神经活动读数，包括 血氧水平依赖性（BOLD）fMRI、钙敏感荧光报告基因和c-fos 免疫组化该项目的预期成果是一套优化和有效的遗传 这些工具将为神经科学家提供新的途径，用于无偏见地探索参与神经网络的神经网络。 协调从感觉功能到行为产生的一切。

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.