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: 8192458
• 负责人: KEVIN L BEHAR
• 金额: $ 46.85万
• 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/8192458
• 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). PUBLIC HEALTH RELEVANCE: Altered brain amino acid neurotransmitter function is found in a wide array of psychiatric and neurological disorders, and is the focus of intense efforts to determine their role in the underlying disorder and to develop new treatments. Recognition of this has lead to the search for new biological markers and assays that can be used to better visualize these neurochemical neurotransmitter systems and their response to treatment. The studies outlined in this proposal addresses this need and are of direct relevance to public health and consistent with the NIMH Strategic Plan by both helping to identify predictive biomarkers that can be used to better understand mental disorders and the search for new drug treatments.

描述（由申请人提供）：在广泛的精神和神经疾病中报道了脑谷氨酸能和gaba能功能改变的证据。目前大多数神经精神疾病的治疗针对单胺系统，疗效有限。认识到这一事实导致了通过替代机制开发新型药物的动力增加。现在人们强烈关注氨基酸神经递质系统（谷氨酸/GABA/谷氨酰胺）作为治疗靶点，这就需要确定可靠的生物标志物测定方法。体外细胞培养和脑切片制备通常不能预测人类或未麻醉动物对谷氨酸调节药物的体内反应。迫切需要能够反映体内生理状态，避免麻醉或死后影响，并能更直接地转化为人类的谷氨酸/GABA神经传递定量分析。最近，一种新型的离体三维原位磁共振光谱成像（MRSI）方法被引入，该方法可以从微波辐射安乐死的啮齿动物的大脑中生成高空间分辨率的大量神经化学物质的定量图，保留了神经化学物质的水平和微观结构。使用13C标记的示踪剂，可以生成13C标记的氨基酸的高空间分辨率2D和3D图。经过适当验证，可以从数据集中提取反映神经元（谷氨酸能和gaba能）和星形胶质细胞代谢以及神经递质循环的速率图。结合其他神经成像模式（如T1、T2弥散张量），可以获得代谢、结构、连通性等多个端点高信息量的定量测量。添加其他“组学”终点测量也是可能的。由于所有的神经成像都是在同一个大脑和坐标空间中有效获得的，因此该方法有可能揭示谷氨酸能/ gaba能神经元和胶质通路的改变，从而加快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，而目前的方法（例如，无细胞提取物或组织切片）一次只能访问一个或几个区域。目标1将开发和验证针对无细胞提取物中测量的13C分数富集的离体代谢通量制图分析，并评估离体通量测量相对于体内MRS时间过程的准确性。还将评估提高可靠性和效率的双重标签方法。目标2将为离体3D MRSI/MRI高密度数据集开发自动代谢物定量和分析方法，以有效和公正地提取可用信息，提高数据质量，并大大提高吞吐量。目的3将应用体外通量测图法来表征和比较谷氨酸调节药物的急性效应和广泛的潜在治疗效果，对谷氨酸/GABA神经传递与受体激活信号（磷酸化蛋白）的区域速率。