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

描述（由申请方提供）：在多种精神和神经系统疾病中报告了脑多巴胺能和GABA能功能改变的证据。目前大多数神经精神疾病的治疗靶向单胺系统，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。现在人们非常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，因此需要确定可靠的生物标志物测定。体外细胞培养和脑切片制备通常不能预测人体或未麻醉动物对谷氨酸调节药物的体内反应。有一个迫切需要的定量分析谷氨酸/GABA神经传递，反映在体内的生理状态，避免麻醉或死后的影响，并可以更直接地翻译给人类。最近，一种新的离体3D原位磁共振光谱成像（MRSI）的方法被引入，它产生高空间分辨率的定量地图的许多神经化学物质从大脑的啮齿动物安乐死的微波辐射，保留神经化学水平和微观结构。应用13 C标记的示踪剂，可以生成13 C标记的氨基酸的高空间分辨率2D和3D图谱。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。结合其他神经成像方式（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含量的定量测量。也可以添加其他“组学”终点测量。由于所有神经成像都在相同的大脑和坐标空间中有效获取，因此所提出的测定具有揭示改变的谷氨酸能/GABA能神经元和神经胶质通路的显著潜力，从而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个实验中获得数千个精确定义的神经化学和解剖数据点，与目前的方法（例如，无细胞提取物或组织切片），其一次仅进入一个或几个区域。目标1将开发和验证针对在无细胞提取物中测量的13 C部分富集的离体代谢通量图测定，并评价针对体内MRS时间过程的离体通量测量的准确性。还将评估一种提高可靠性和效率的双标签方法。目标2将开发用于离体3D MRSI/MRI高密度数据集的自动代谢物定量和分析方法，以有效和无偏地提取可用信息，提高数据质量，并大大提高通量。目的3将应用离体通量图测定来表征和比较具有广泛潜在治疗作用的谷氨酸调节药物对谷氨酸/GABA神经传递与受体激活信号传导（磷蛋白）的局部速率的急性作用。

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).