Multiplexed neurochemical methods to understand adenosine neuromodulation

项目总结 腺苷作为神经传递的快速调节器的作用是什么？我们如何利用它的力量 潜在的治疗用途？要回答这样的问题，我们需要能够测量的分析工具 多种神经化学物质同时进行，具有高时间和空间分辨率。我们的实验室率先推出了快速扫描技术 循环伏安法(FSCV)检测腺苷，发现持续的自发的、瞬时的腺苷信号 只有几秒钟。然而，快速腺苷神经调节的范围和影响还不是很清楚。 基因编码的传感器最近被开发出来用于神经递质和钙的检测， 高灵敏度、高选择性和高空间分辨率。虽然它们可以监测各种各样的神经化学物质， 不仅是电活性分子，检测不同分析物的颜色仍然有限。FSCV与 基因编码的传感器将有利于检测神经调节剂腺苷并测量其 下游对多巴胺和谷氨酸的神经传递以及神经元活动的影响。长期的 我的实验室的目标是开发新的工具来实时监测大脑中的神经调节。这样做的目的是 该项目是开发多种工具来了解神经化学相互作用，并将这些工具应用于 了解腺苷对谷氨酸、多巴胺和钙的调节作用。中心假设是如此之快 腺苷的释放为大脑中的神经递质提供了短暂的、但在空间上有局限性的调节。在 首先，我们将开发具有四个电极阵列的多通道FSCV，以确定腺苷有多远 脑片中的扩散及其对多巴胺释放的神经调节作用的范围。在第二个目标中， 我们将结合FSCV和基因编码的荧光传感器来探测 腺苷(用FSCV测量)调节多巴胺(用GRABDA测量)或谷氨酸(测量 使用iGluSnFR)。在第三个目标中，我们将结合多通道FSCV和在体纤维光度测量 基因编码的传感器。我们将演示体内腺苷、多巴胺和钙的检测 改变腺苷对神经传递和神经元活动的调节。这 这项研究意义重大，因为它开发了广泛适用于多路神经递质的工具 和神经调节剂测量，利用FSCV和遗传编码的组合优势 传感器。这也很重要，因为多路复用工具将提供前所未有的时间图像 以及腺苷神经调节的空间动力学。生物影响是理解快速和局部的 腺苷神经调节的性质，这对于设计以腺苷为基础的治疗方法是重要的 腺苷可以起到神经保护作用的疾病，如帕金森氏症、脑缺血或创伤性脑损伤。 这种多用途的工具可以应用于监测许多其他神经化学相互作用，无论是在脑片还是在 活体，并将推动神经化学监测领域超越一次一种神经化学物质的感知。

PROJECT SUMMARY What is the role of adenosine as a rapid modulator of neurotransmission and how can we harness its power for potential therapeutic use? To answer questions such as these, we need analytical tools that can measure multiple neurochemicals simultaneously with high temporal and spatial resolution. Our lab pioneered fast-scan cyclic voltammetry (FSCV) for adenosine, and discovered spontaneous, transient adenosine signaling that lasts only a few seconds. However, the range and effects of rapid adenosine neuromodulation are not well understood. Genetically-encoded sensors have recently been developed for neurotransmitter and calcium detection that offer high sensitivity, selectivity, and spatial resolution. While they can monitor a wide variety of neurochemicals, and not just electroactive molecules, there are still limited colors to detect different analytes. FSCV combined with genetically-encoded sensors would be advantageous to detect the neuromodulator adenosine and measure its downstream effects on dopamine and glutamate neurotransmission, as well as neuronal activity. The long-term goal of my lab is to develop new tools for monitoring real-time neuromodulation in the brain. The goal of this project is to develop multiplexed tools to understand neurochemical interactions and apply these tools to understand adenosine modulation of glutamate, dopamine, and calcium. The central hypothesis is that rapid adenosine release provides transient, but spatially localized, modulation of neurotransmitters in the brain. In the first Aim, we will develop multichannel FSCV, with an array of four electrodes, to determine how far adenosine diffuses in brain slices and the range of its neuromodulatory effects on dopamine release. In the second Aim, we will combine FSCV with genetically-encoded fluorescent sensors to probe the spatial and temporal profile of adenosine (measured with FSCV) modulation of dopamine (measured with GRABDA) or glutamate (measured with iGluSnFR). In the third Aim, we will combine multichannel FSCV and in vivo fiber photometry measurements of genetically-encoded sensors. We will demonstrate in vivo detection of adenosine, dopamine, and calcium changes to probe adenosine neuromodulation of neurotransmission and neuronal activity simultaneously. This research is significant because it develops tools that are broadly applicable for multiplexing neurotransmitter and neuromodulator measurements, harnessing the combined strengths of FSCV and genetically-encoded sensors. It is also significant because multiplexed tools will provide an unprecedented picture of the temporal and spatial dynamics of adenosine neuromodulation. The biological impact is understanding the rapid and local nature of adenosine neuromodulation, which is important for designing adenosine-based therapeutics for diseases such as Parkinson’s, ischemia, or traumatic brain injury where adenosine could be neuroprotective. The multiplexed tools could be applied to monitoring many other neurochemical interactions, in brain slices or in vivo, and will advance the field of neurochemical monitoring beyond one neurochemical at a time sensing.