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）：在各种精神病和神经系统疾病中报道了脑谷氨酸能和GABA能作用改变的证据。当前关于神经精神疾病的治疗方法针对单胺系统，并且功效有限。对这一事实的认可导致增加了通过替代机制起作用的新型药物的动力。现在，人们对氨基酸神经递质系统（谷氨酸/GABA/谷氨酰胺）作为治疗靶标有强烈的关注，从而需要鉴定可靠的生物标志物分析。体外细胞培养和脑切片制剂通常无法预测人类或一han虫动物中对谷氨酸调节药物的体内反应。迫切需要对反映体内生理状态的谷氨酸/GABA神经传递的定量测定，避免麻醉或后验证后效应，并可以更直接地转化为人类。最近，引入了一种新型的Ex Vivo 3D原位磁共振光谱成像（MRSI）方法，该方法从微波辐照的啮齿动物的大脑中生成了许多神经化学物质的高空间分辨率定量图，从而确保了神经化学水平和微观结构。可以生成13C标记的示踪剂，可以生成13c标记氨基酸的高空间分辨率2D和3D地图。经过适当验证的速率图反映了神经元（谷氨酸能和GABA能）和星形胶质胶质代谢，以及神经递质循环可以从数据集中提取。结合其他神经成像模式（例如T1，T2扩散量），可以获得多个终点的高信息含量的定量测量，以实现代谢，结构和连接性。还可以增加其他“ - 组”终点测量值。在所有神经影像中有效地在同一大脑和协调空间中获得的所有神经成像，该拟议的测定具有巨大的潜力，可以揭示改变谷氨酸能/GABA能神经元和神经胶质途径的改变，从而加速了临床前药物评估和治疗反应。与当前的方法（例如，一次或几个区域仅访问一个或几个区域的当前方法相比，这种方法的开发将使研究人员有机会在单个实验中获得数千个精确定义的神经化学和解剖学数据点。 AIM 1将针对在无细胞提取物中测量的13C分数富集开发并验证离体代谢通量映射测定法，并评估对体内MRS时间课程的离体通量测量的准确性。还将评估提高可靠性和效率的双标签方法。 AIM 2将开发自动代谢物量化和分析方法，用于实体3D MRSI/MRI高密度数据集，以有效地提取可用信息，增强的数据质量并大大增加了吞吐量。 AIM 3将应用离体通量映射测定法来表征和比较谷氨酸调节药物的急性作用，并在谷氨酸/GABA神经传递的区域速率上与受体激活的信号传导（磷蛋白）（磷酸蛋白）的区域速率，对谷氨酸/GABA神经传递的区域速率进行比较。 公共卫生相关性：在各种精神病和神经系统疾病中发现了改变的大脑氨基酸神经递质功能，并且是确定其在潜在疾病中的作用并开发新疗法的强烈努力的重点。对此的认识已导致寻找新的生物学标记和测定法，这些标志物可更好地可视化这些神经化学神经递质系统及其对治疗的反应。该提案中概述的研究解决了这一需求，与公共卫生直接相关，并与NIMH战略计划保持一致，这两者都可以帮助识别可用于更好地了解精神障碍和寻找新药物治疗的预测生物标志物。