Multi-Scale Imaging Core (MSIC)

特定目标-多尺度成像核心(MSIC) 成瘾物质在分子、细胞和电路水平上触发可塑性，表现为持久性 可能导致物质使用障碍的行为变化。以这些变化为目标可能会带来新的 预防或治疗药物使用障碍的策略。然而，我们对分子变化的了解， 构成物质使用的各个方面的细胞过程和异常的电路活动模式 包括强迫症、失去摄入量控制、戒断和复发在内的障碍相当有限。为了促进 更好地理解伴随着药物滥用的分子到电路水平的可塑性，C3A多- Scale成像核心将支持中心调查人员、来自中西部和其他地区的分支机构以及 不同的职业阶段，以获得概念和技术诀窍，并访问最先进的 纳米级分子测量设备，用于亚细胞和超微结构的解剖分析 用于细胞轮廓和大脑回路的中尺度生理成像。C3A多尺度成像核心 将提供前所未有的成像机会来检查多个药物使用障碍的模型 水平，包括：(1)分子和细胞水平成像，具有国际独特的细胞类型和亚细胞 特定舱室相关风暴超分辨率成像及其最新发展 纳米药理学的药物存储扩展；(2)电路级2P成像检查选择性 大细胞群体中神经回路和特定细胞类型的动态生理变化。 目的1.确定细胞和亚细胞隔室特定的纳米级分子和微级 由于长期接触滥用药物而引发的细胞改变。通过使用荧光小 基于分子的药物形态单分子纳米药理学和抗体 免疫断层超分辨率成像，WE和C3A附属研究人员将确定慢性药物 暴露和/或戒断引起持续改变的纳米级分布和重要的 与物质使用障碍最相关的细胞类型和大脑回路中的信号蛋白。通过 将纳米尺度的分子测量与微尺度的共聚焦显微镜数据相关联，我们还将 在已鉴定的亚细胞室中建立相关的形态变化。我们将特别注意 致力于CB1类大麻素和D3多巴胺受体，它们在成瘾的所有阶段都起着至关重要的作用 Cycle及其拮抗剂/负变构调节剂跻身NIDA十大最高药物之列 发展重点。 目的2.表征长程谷氨酸能、多巴胺能和 5-羟色胺能轴突由发育或长期接触滥用药物引起。轴突束 连接遥远的大脑区域遵循不规则的轨迹，因此白质形态很难评估 通过标准脑切片染色。因此，我们将结合我们在量表方法论方面的经验 对整个小鼠大脑进行优化的2P成像。这种方法将被用来确定 发育过程中暴露于THC和其他药物对已识别的远程轴突的完整性和轨迹的影响。 因为出生前接触大麻会改变人类的神经回路，而啮齿动物的研究发现， 长程谷氨酸能轴突对THC特别敏感，我们将初步确定对围产期的影响 将起源于内侧前额叶皮质的谷氨酸能轴突暴露到不同的脑区。 目的3.使用体外和体内2P传感器成像来确定中尺度生理变化 长期接触滥用药物所引发的大脑回路。基因编码技术的最新进展 钙、内源性大麻素和单胺的传感器提供了可视化动态变化的极佳工具 这些信号分子以特定的细胞类型特定的方式实时进行。通过将我们现有的和 急性脑片或清醒行为小鼠(年仅10岁)的钙成像综合方法学 几天前)从外科手术延伸到高性能的数据分析流水线 与Grab-eCB2.1和GRABDA传感器成像一起计算，我们将支持中心和附属机构 科学家将进行纵向2P成像以检查内源性大麻素、多巴胺和网络活动 他们的物质使用障碍的相关模式的变化。我们还将确定围产期是否暴露于THC 干扰内源性大麻素信号的发展与原发灶的钙峰模式相关 从出生早期到断奶日龄清醒行为小鼠的躯体感觉皮层。 目的4.建立成瘾患者荧光寿命成像显微镜(FLIM)的体内检测方法 研究。滥用药物会引起实质性的新陈代谢变化，并扰乱星形胶质细胞与神经元的相互作用。我们 将使用2P-Flim成像来开发活体应用程序，使用基于Flim的传感器来监控能量 代谢、信号级联、蛋白质-蛋白质相互作用和估计星形胶质细胞之间的接近程度 这种物质中的神经元使用由当地和附属成像研究人员建立的障碍模型 核心。

SPECIFIC AIMS-Multiscale Imaging Core (MSIC) Addictive substances trigger plasticity at the molecular, cellular and circuit levels that manifest as persistent behavioral changes that may cause substance use disorders. Targeting these changes may lead to novel strategies for preventing or treating substance use disorders. However, our knowledge of the molecular changes, the cellular processes and the abnormal circuit activity patterns that underlie various aspects of substance use disorders including compulsion, loss of intake control, withdrawal, and relapse is rather limited. To facilitate a better understanding of the molecular to circuit level plasticity accompanying drug abuse, the C3A multi- scale imaging core will support center investigators, affiliates from the Midwest and beyond, and trainees at different career stages to acquire the conceptual and technical know-how, and to access state-of-the-art equipment for nanoscale molecular measurements, for microscale anatomical analysis of subcellular and cellular profiles and for mesoscale physiological imaging of brain circuits. The C3A multi-scale imaging core will provide unprecedented imaging opportunities to examine models of substance use disorders at multiple levels, including: (1) molecular and cellular level imaging with internationally unique cell-type- and subcellular compartment-specific correlated STORM super-resolution imaging, and its recently developed PharmacoSTORM extension for nanoscale pharmacology; (2) circuit level 2P imaging to examine selective neural circuits and cell-type-specific dynamic physiological changes among large cell populations. Aim 1. Determine the cell- and subcellular compartment-specific nanoscale molecular and microscale cellular alterations triggered by chronic exposure to drugs of abuse. By employing fluorescent small molecule-based PharmacoSTORM single-molecule nanoscale pharmacology and antibody-based ImmunoSTORM super-resolution imaging, we and C3A-affiliated researchers will determine if chronic drug exposure and/or withdrawal elicit persistently altered nanoscale distribution and abundance of important signaling proteins in the cell types and brain circuits that are most relevant for substance use disorders. By correlating the nanoscale molecular measurements with microscale confocal microscopy data, we will also establish the associated morphological changes in identified subcellular compartments. Particular attention will be devoted to CB1 cannabinoid and D3 dopamine receptors that have essential roles in all phases of the addiction cycle and whose antagonists/negative allosteric modulators are among NIDA’s ten highest medication development priorities. Aim 2. Characterize the mesoscale circuit rewiring of long-range glutamatergic, dopaminergic and serotonergic axons induced by developmental or chronic exposure to drugs of abuse. Axon tracts connecting distant brain regions follow irregular trajectories, thus white matter morphology is difficult to evaluate by standard brain section staining. Therefore, we will exploit our experience in ScaleS methodology combined with optimized 2P imaging of the entire mouse brain. This approach will be used to determine the impact of developmental exposure to THC and other drugs on the integrity and trajectory of identified long-range axons. Because prenatal cannabis exposure modifies human neural circuits and rodent studies found that developing long-range glutamatergic axons are particularly sensitive to THC, we will initially determine the impact of perinatal THC exposure on glutamatergic axons originating from medial prefrontal cortex to various brain regions. Aim 3. Use in vitro and in vivo 2P sensor imaging to determine the mesoscale physiological changes in brain circuits elicited by chronic exposure to drugs of abuse. Recent advances in genetically encoded sensors for Ca2+, endocannabinoids, and monoamines provide excellent tools to visualize dynamic changes of these signaling molecules in a specific cell-type-specific manner in real-time. By combining our established and comprehensive methodology for Ca2+_imaging in acute brain slices or awake behaving mice (as young as ten days old) extending from the surgical procedure through the data analysis pipeline with High Performance Computing together with GRAB-eCB2.1 and GRABDA sensor imaging, we will support center and affiliated scientists to perform longitudinal 2P imaging to examine endocannabinoid, dopamine, and network activity changes in their relevant models of substance use disorders. We will also determine if perinatal THC exposure perturbs the development of endocannabinoid signaling in association with Ca2+-spike patterns in the primary somatosensory cortex of awake behaving mouse pups from early postnatal to weaning ages. Aim 4. Develop in vivo protocols for Fluorescence Lifetime Imaging Microscopy (FLIM) in addiction research. Drugs of abuse evoke substantial metabolic changes and perturb astrocyte-neuron interactions. We will use 2P-FLIM imaging to develop in vivo applications using FLIM-based sensors to monitor energy metabolism, signaling cascades, protein-protein interactions and to estimate the proximity between astrocytes and neurons in the substance use disorder models established by local and affiliate researchers of the imaging core.