Ex Vivo Assay for In Situ Brain-Wide Mapping of Glutamate/GABA Metabolism

谷氨酸/GABA 代谢原位全脑图谱的离体测定

• 批准号: 8306727
• 负责人: KEVIN L BEHAR
• 金额: $ 51.31万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-22 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8306727
• 关键词: 3-Dimensional; Acute; Address; Algorithms; Alzheimer' s Disease; Amino Acid Neurotransmitters; Amino Acids; Amyotrophic Lateral Sclerosis; Anesthesia procedures; Animals; Astrocytes; Autopsy; Behavioral; Biochemical Reaction; Biological Assay; Biological Markers; Biological Markers; Brain; Cell Culture Techniques; Cells; Computer software; Data; Data Analyses; Data Quality; Data Set; Development; Diffusion; Diffusion Magnetic Resonance Imaging; Disease; Dose; Drug effect disorder; Epilepsy; Functional disorder; Glucose; Glutamates; Glutamine; Human; Image; In Situ; In Vitro; Injection of therapeutic agent; Intravenous infusion procedures; Ketamine; Knowledge; Label; Lead; Libraries; Magnetic Resonance Imaging; Maps; Measurement; Measures; Mental disorders; Metabolic; Metabolism; Methodology; Methods; Modality; Modeling; Mood Disorders; Morphology; National Institute of Mental Health; Neurons; Neurotransmitters; Parkinson Disease; Pathway interactions; Pharmaceutical Preparations; Phosphoproteins; Physiological; Physiology; Preclinical Drug Evaluation; Preparation; Process; Public Health; Rattus; Reporting; Research Personnel; Resolution; Rodent; Rodent Model; Role; Schizophrenia; Screening procedure; Signal Pathway; Signal Transduction; Slice; Strategic Planning; Structure; System; Therapeutic Agents; Therapeutic Effect; Time; Tissues; Tracer; Translating; base; brain tissue; density; gamma-Aminobutyric Acid; image processing; imaging modality; improved; in vivo; interest; irradiation; magnetic resonance spectroscopic imaging; microwave electromagnetic radiation; monoamine; nervous system disorder; neurochemistry; neuroimaging; neuropsychiatry; neurotransmission; neurotransmitter metabolism; novel; oxidation; receptor; research study; response; treatment response

DESCRIPTION (provided by applicant): Evidence of altered brain glutamatergic and GABAergic function is reported in a wide array of psychiatric and neurological disorders. Most current treatments for neuropsychiatric illness target the monoamine systems and have limited efficacy. The acknowledgement of this fact has led to an increased drive to develop novel drugs acting through alternative mechanisms. There is now intense focus on the amino acid neurotransmitter systems (glutamate/GABA/glutamine) as targets for treatment, creating the need to identify reliable biomarker assays. In vitro cell culture and brain slice preparations often fail to predict in vivo responses to glutamate- modulating drugs in humans or unanesthetized animals. There is a pressing need for quantitative assays of glutamate/GABA neurotransmission that reflect the in vivo physiological state, avoids anesthesia or postmortem effects, and can be translated more directly to humans. Recently, a novel ex vivo 3D in situ magnetic resonance spectroscopic imaging (MRSI) approach was introduced, which generates high spatial resolution quantitative maps of numerous neurochemicals from the brain's of rodents euthanized by microwave irradiation, preserving neurochemical levels and microstructure. Applied with 13C labeled tracers, high-spatial resolution 2D and 3D maps of 13C-labeled amino acids can be generated. Appropriately validated, rate maps reflecting neuronal (glutamatergic and GABAergic) and astroglial metabolism, and neurotransmitter cycling can be extracted from the data sets. Combined with other neuroimaging modalities (e.g., T1, T2 diffusion-tensor), quantitative measurements of high information content of multiple endpoints for metabolism, structure, and connectivity can be obtained. The addition of other '-omics' end-point measurements are also possible. With all neuroimaging efficiently acquired in the same brain and coordinate space, the proposed assay has significant potential to reveal altered glutamatergic/GABAergic neuronal and glial pathways, accelerating preclinical drug evaluation and treatment response. Development of this methodology would afford investigators opportunity to obtain thousands of precisely defined neurochemical and anatomical data points in a single experiment, in contrast to present methods (e.g., cell-free extracts or tissue slices) which access only one or a few regions at a time. Aim 1 will develop and validate the ex vivo metabolic flux mapping assay against 13C fractional enrichment measured in cell-free extracts, and evaluate the accuracy of the ex vivo flux measurements against in vivo MRS time courses. A double-labeling approach to increase reliability and efficiency will also be evaluated. Aim 2 will develop automated metabolite quantification and analysis methods for the ex vivo 3D MRSI/MRI high density data sets for efficient and unbiased extraction of available information, enhanced data quality, and greatly increased throughput. Aim 3 will apply the ex vivo flux mapping assay to characterize and compare the acute effects of glutamate modulating drugs with a broad range of potential therapeutic effects, on regional rates of glutamate/GABA neurotransmission with receptor activated signaling (phosphoproteins).

描述(由申请人提供)：有证据表明大脑谷氨酸和GABA能功能改变，在一系列精神和神经疾病中都有报道。目前大多数神经精神疾病的治疗方法都是针对单胺系统，疗效有限。对这一事实的认识导致了开发通过替代机制起作用的新药的动力增加。目前，作为治疗靶点的氨基酸神经递质系统(谷氨酸/GABA/谷氨酰胺)受到高度关注，因此需要确定可靠的生物标志物分析方法。在体外，细胞培养和脑片制备往往无法预测人体或未麻醉动物对谷氨酸调节药物的体内反应。迫切需要对谷氨酸/GABA神经传递进行定量分析，以反映体内的生理状态，避免麻醉或死亡后的影响，并且可以更直接地翻译到人类身上。最近，一种新的体外三维原位磁共振波谱成像(MRSI)方法被引入，该方法从微波辐射安乐死啮齿类动物的脑中生成多种神经化学物质的高空间分辨率定量图，并保持神经化学水平和微结构。应用~(13)C标记的示踪剂，可以生成高空间分辨率的~(13)C标记氨基酸的2D和3D地图。经过适当验证，可以从数据集中提取反映神经元(谷氨酸和GABA能)和星形胶质细胞代谢以及神经递质循环的速率图。结合其他神经成像方法(例如，T1、T2扩散张量)，可以获得代谢、结构和连接性的多个端点的高信息量的定量测量。此外，还可以添加其他“组学”终点测量方法。由于所有的神经成像都是在同一个大脑和坐标空间有效地获得的，所提出的检测方法具有揭示谷氨酸/GABA能神经元和神经胶质通路改变的显著潜力，加速临床前药物评估和治疗反应。这种方法的发展将使研究人员有机会在一次实验中获得数千个精确定义的神经化学和解剖学数据点，而不是目前的方法(例如，无细胞提取物或组织切片)，这些方法一次只能访问一个或几个区域。目的1将建立和验证体外代谢通量图谱分析，对照在无细胞提取物中测量的13C部分浓缩，并评估体外通量测量对照体内MRS时间进程的准确性。还将对提高可靠性和效率的双重标签方法进行评估。AIM 2将为体外3D MRSI/MRI高密度数据集开发自动代谢物定量和分析方法，以高效和公正地提取可用信息，提高数据质量，并大幅提高吞吐量。目的3应用体外通量图分析方法，研究和比较谷氨酸调节药物和多种潜在治疗药物对谷氨酸/氨基丁酸神经递质和受体激活信号(磷蛋白)的局部传递速率的急性影响。