Microfluidic Systems to Address Networks of Neurons

描述(申请人提供)：这项研究的长期目标是开发一个微流体平台，以研究网络中神经细胞之间的相互联系，以及细胞之间的相互连接对神经元对物理创伤和暴露于化学毒素的反应的影响。这项工作的一个关键方面将是开发和利用三维微流控系统来精确控制试剂向网络中单个细胞的输送，并使用该系统来加深我们对细胞间通信的理解。体外细胞网络将被用来模拟更大的大脑网络中的复杂交流。重点将放在了解突触前和突触后受体、模式的可塑性以及同时用多个细胞筛选药理效果。通常很难对神经网络中的特定细胞进行药物、毒素或损害的详细研究，因为无法同时从阵列或网络中的几个特定细胞进行刺激(或接触药物、毒素)和记录。建议的系统将为这一问题提供解决方案。我们希望这项工作将扩大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。这项提议有四个具体目标。首先，将设计和制造一个集成系统，以允许对线性单元阵列进行非侵入性刺激和实时连续监测。第二，将小孔与横流相结合的三维微流控装置将被用于解决更复杂的二维细胞网络。第三，结合荧光监测细胞活性的微流控系统将在PC12和P19细胞网络上作为模型进行测试。第四，将对具有功能性突触的神经细胞培养进行监测。长期目标是使用这个系统来检查物理和化学损伤对神经元网络中特定细胞的影响。我们提出的假设是：1)神经元连接性改变以补偿物理损伤后的神经元丢失，2)神经元网络的活动影响暴露于毒素后的退化速度，3)谷氨酸神经元在暴露于毒素后的多巴胺能神经元丢失中发挥积极作用，以及4)L-多巴酚A在帕金森病细胞丢失的进展中起作用，并可能参与神经保护剂对多巴胺细胞的作用机制。这项拟议的研究的首要目标是将微流体作为生物学的一种有价值的工具。

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 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 four 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 microfluidic device combining small apertures with cross flow will be used to address more complicated two-dimensional 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.