Microfluidic Systems to Address Networks of Neurons

描述（由申请人提供）：本研究的长期目标是开发一个微流控平台，以研究神经细胞在网络中的相互通讯，以及细胞之间的相互连接对神经细胞对物理创伤和化学毒素暴露的反应的影响。这项工作的一个关键方面将是开发和利用三维（3D）微流体系统来精确控制网络中单个细胞的试剂输送，并使用该系统进一步了解细胞间通信。体外细胞网络将被用于模拟更大的大脑网络中的复杂通信。特别的重点将放在理解突触前和突触后受体，模式的可塑性和筛选的药理学效力与多个细胞一次。由于无法同时刺激（或暴露于药物、毒素）和记录阵列或网络中的几个特定细胞，通常很难对神经网络中的药物、毒素或特定细胞的损伤进行详细研究。所提出的系统将为这一问题提供一个解决方案。我们期望这项工作将扩展现有的关于基于分子的细胞间通讯途径的知识，并为筛选涉及大脑的药物和毒素提供新的途径。该提案有4个具体目标。首先，将设计和制造一个集成系统，以允许线性细胞阵列进行非侵入性刺激和连续实时监测。其次，结合小孔径和交叉流的三维（3D）微流体装置将用于解决更复杂的二维（2D）细胞网络。第三，结合细胞活性荧光监测的微流控系统将以PC12和P19细胞网络为模型进行测试。第四，将监测具有功能突触的神经细胞培养。长期目标是使用该系统来检查物理和化学损伤对神经网络中特定细胞的影响。我们建议测试以下假设：1)神经元连接改变以补偿物理损伤后的神经元损失；2)神经元网络的活动影响暴露于毒素后的降解速度；3)谷氨酸神经元在暴露于毒素后多巴胺能神经元细胞损失中发挥积极作用；4)左旋多巴参与帕金森病细胞丢失的进程，可能参与神经保护剂对多巴胺细胞的作用机制。提出的研究的总体目标是建立微流体作为生物学的有价值的工具。

DESCRIPTION (provided by applicant): The long term goal of this research is to develop a microfluidic platform to investigate the intercommunication of nerve cells in networks and the influence of the interconnections between cells on the neuronal response to physical trauma and exposure to chemical toxins. A key aspect of this work will be to develop and utilize three-dimensional (3D) microfluidic systems to precisely control delivery of reagents to individual cells in a network and to use this system to further our understanding of cell-to-cell communication. Cell networks in vitro will be used to model complex communication in the larger network of the brain. Special emphasis will be placed on understanding pre- vs. postsynaptic receptors, plasticity in patterns and screening for pharmacological efficacy with multiple cells at once. It is normally difficult to carry out detailed studies of drugs, toxins or damage to specific cells in neural networks owing to an inability to simultaneously stimulate (or expose to drug, toxin) and record from several specific cells in an array or network. The system proposed will provide a solution to this problem. We expect the work will expand existing knowledge concerning molecule-based cell-to-cell communication pathways and provide new avenues for screening drugs and toxins involved in the brain. There are 4 specific aims for this proposal. First, an integrated system will be designed and fabricated to allow linear cell arrays to be non-invasively stimulated and continuously monitored in real-time. Second, a three-dimensional (3D) microfluidic device combining small apertures with cross flow will be used to address more complicated two-dimensional (2D) cell networks. Third, the microfluidic system combined with fluorescence monitoring of cell activity will be tested on PC12 and P19 cell networks as models. Fourth, neuronal cell cultures with functional synapses will be monitored. The long-term goal is to use this system to examine the effects of physical and chemical damage to specific cells in a neuronal network. We propose to test the hypotheses that 1) neuronal connectivity changes to compensate for neuronal loss following physical damage, 2) activity in neuronal networks affects the rate of degradation following exposure to toxins, 3) glutamate neurons play an active role in dopaminergic neuronal cell loss following exposure toxins, and 4) L-DOPA plays a role in the progression of cell loss in Parkinson's Disease and might be involved in the mechanism of action of neuroprotective agents on dopamine cells. The proposed research has the overarching goal of establishing microfluidics as a valuable tool for biology.