Multiplexed Sensing and Control of Neuromodulators and Peptides in the Awake Brain

总结 神经调质和其他化学信号的不平衡水平会导致许多神经系统疾病。 然而，以前描述这些效应的研究通常一次只检查一种分子， 对大脑或浸泡所有神经元的脑脊液（CSF）中信号水平的静态描述。在 事实上，数十种信号在不同状态下表现出动态变化，例如安静的清醒状态和社交或非社交状态 性唤起，在疾病中改变。对神经活动模式的跟踪和操纵 对最近的神经科学进展至关重要。我们缺乏类似的工具来估计和控制动态模式 神经调节信号，这可能会彻底改变大脑状态的研究，并有效地恢复健康 神经系统和精神疾病之间的关系此外，我们不明白任何给定的 神经精神药物动态影响内源性神经调质和肽的水平， CSF或脑，从而阻碍了最佳药物递送策略的合理设计，以最大限度地提高疗效， 尽量减少副作用。这些盲点是由于技术限制：而细胞成像和光遗传学 虽然我们已经能够越来越精确地跟踪和操纵脑细胞，但我们缺乏能力， 精确地（i）记录或（ii）同时真实的控制多个神经调节信号。我们 克服第一个挑战，通过开发新的方法，多路复用，定量成像的面板， 疾病相关神经调节信号的基于绿色荧光蛋白的光学传感器（目的1）： 加压素，催产素，生长抑素，多巴胺，去甲肾上腺素，血清素，乙酰胆碱，组胺，褪黑激素， 促肾上腺皮质激素释放因子、血管活性肠肽和腺苷。简而言之，培养的细胞组 将表达单个传感器的3D水凝胶传感器阵列组合到梯度的前面， 折射率（GRIN）透镜，其经由微透镜插入清醒的头部固定小鼠的CSF或脑组织中。 慢性插管。然后，使用传感器阵列的3D双光子成像来估计信号浓度， 通过将相同的传感器阵列事后机器人浸入不同浓度的每种 离体神经调节剂。一旦我们建立了这种方法来跟踪神经调节成分， 小时或天，并跨越行为状态（目标1），我们将使用闭环交付方法来控制动态 在清醒的小鼠大脑中，研究了多达12种神经调节信号的模式，并评估了哪些模式驱动 行为偏好或回避（目标2）。这些实验受益于使用荧光寿命， 以及荧光强度测量，允许定量评估流体成分， 延长时间（数小时至数天），漂白效果最小。总之，这些工具提供了一种新颖的， 研究和控制大脑中多种神经调质的整体框架。敏感的， 小体积信号的实时、多路复用读出补充了微透析， 闭环控制应用于基础和临床神经科学研究的大多数领域。

Summary Imbalanced levels of neuromodulators and other chemical signals contribute to a host of neurological disorders. Yet, previous studies describing these effects often examine only one molecule at a time, and typically provide a static description of signal levels in the brain or in the cerebrospinal fluid (CSF) that bathes all neurons. In reality, dozens of signals exhibit dynamic changes across states such as quiet waking and social or non-social arousal, which are altered in disease. The tracking and manipulation of patterns of neural activity has been critical to recent neuroscience progress. We lack analogous tools for estimation and control of dynamic patterns of neuromodulatory signals, which could revolutionize the study of brain states and effectively restore healthy states across neurologic and psychiatric disorders. Moreover, we do not understand how any given neuropsychiatric drug dynamically influences the levels of endogenous neuromodulators and peptides in the CSF or brain, thus impeding the rational design of optimal drug delivery strategies to maximize efficacy and minimize side effects. These blind spots are due to technical limitations: while cellular imaging and optogenetics have enabled ever-increasing precision in tracking and manipulation of brain cells, we lack the ability to accurately (i) record or (ii) control multiple neuromodulatory signals simultaneously in real time. We are overcoming the first challenge by developing novel methods for multiplexed, quantitative imaging of a panel of green fluorescent protein-based optical sensors of disease-relevant neuromodulatory signals (Aim 1): vasopressin, oxytocin, somatostatin, dopamine, norepinephrine, serotonin, acetylcholine, histamine, melatonin, corticotropin-releasing factor, vasoactive intestinal peptide, and adenosine. Briefly, sets of cultured cells expressing individual sensors are combined in a 3D hydrogel sensor array applied to the front of a gradient refractive index (GRIN) lens, which is inserted into the CSF or brain tissue of an awake, head-fixed mouse via a chronic cannula. Estimates of signal concentration using 3D two-photon imaging of the sensor array are then calibrated via post-hoc robotic dipping of the same sensor array into varying concentrations of each neuromodulator ex vivo. Once we have established this approach to track neuromodulatory composition across hours or days and across behavioral states (Aim 1), we will use closed-loop delivery methods to control dynamic patterns of up to a dozen neuromodulatory signals in the brain in awake mice and evaluate which patterns drive behavioral preference or avoidance (Aim 2).These experiments benefit from the use of fluorescence lifetime and well as fluorescence intensity measurements, allowing quantitative assessment of fluid composition across extended periods of time (hours to days) with minimal effects of bleaching. Together, these tools offer a novel, holistic framework for the study and control of multiple neuromodulators in the brain. The sensitive, real-time, multiplexed readout of signals in small volumes complements microdialysis and enables closed-loop control with applications to most domains of basic and clinical neuroscience research.