Simultaneous kinetic analyses of neuronal connectivities

 描述(申请人提供)：异常的神经元连接与许多神经病理和神经精神疾病/障碍有关。低效的发育连通性、错误的连通性或中断和退化的连通性可以解释正常神经元通信的丧失以及随后的精神或身体健康的损失。轴突引导蛋白表达的改变、神经炎症、神经元毒性和神经胶质细胞功能障碍可能与神经元连接不足有关。本申请中描述的工作的目标是开发一种仪器平台，该平台可以列举轴突的发展或丧失以及这些变化对分子和细胞修饰剂的响应的动力学。提出的基于微流生物传感器芯片的光栅耦合表面等离子激元共振(GCSPR)和光栅耦合表面等离子体激元耦合发射(GCSPCE)仪器将被开发和验证，用于量化大脑区域切片的冲孔之间的发育轴突以及先前建立的连接的丢失。最多可以在1cm2生物传感器金芯片上放置五个1-3 mm的冲头，芯片上预涂有细胞外基质蛋白和特定分子(引导蛋白)或特定位置的细胞(小胶质细胞)。轴突连接的缺失或丢失将通过SPR进行动态监测，突触前和突触后的抗原可以通过荧光标记的神经元抗原的SPCE进行检测。将确定特定大脑区域的几何位置，以便可以量化优先的区域交互作用。计划中的评估神经元电路的设备建立在SPR微阵列的丰富经验基础上，SPR微阵列可以表征单个细胞及其释放的产品的存在。所描述的工作将产生能够提供更完整和连贯的不同神经元连接的图像的仪器，以提供更全面的神经元通信的视图，这将揭示治疗干预的新机会。

 DESCRIPTION (provided by applicant): Aberrant neuronal connectivity is associated with a number of neuropathological and neuropsychiatric diseases/disorders. Inefficient developmental connectivity, misdirected connectivity, or disrupted and degenerated connectivity can account for losses of normal neuronal communication and subsequent losses of mental or physical health. Altered expression of axonal guidance proteins, neuroinflammation, neuronal toxicity and glial disturbances may be involved in inadequate neuronal connectivity. The goal of the work described in this application is to develop an instrument platform that can both enumerate development or loss of axons and the kinetics of these changes in response to molecular and cellular modifiers. The proposed grating coupled surface plasmon resonance (GCSPR) and grating coupled surface plasmon coupled emission (GCSPCE) instrumentation with a microflow biosensor chip will be developed and validated for quantification of developmental axonal outgrowths among punches of brain regional sections as well as loss of connections previously established. Up to five 1-3mm punches can be placed on the 1 cm2 biosensor gold chip precoated with extracellular matrix proteins and specific molecules (guidance proteins) or cells (microglia) spotted at specific locations. Lack or loss of axonal connections will be kinetically monitored by SPR and presynaptic and postsynaptic antigens can be assayed by SPCE with fluorochrome conjugated antibodies to neuronal antigens. The geometrical placement of specific brain regions will be identified so that preferential regional interactions can be quantified. The planned device to assess neuronal circuitry is built upon extensive experience with SPR microarrays that can characterize presence of single cells and their released products. The work described will produce instrumentation capable of providing a more complete and coherent picture of differential neuronal connectivities to provide a more comprehensive view of neuronal communications, which will reveal new opportunities for therapeutic interventions.