Monitoring Of Neuronal Communications Using Nanometer-sized Electrochemical Sensors And Microfluidic Devices

The nervous system, by which all the organs and systems work together to maintain normal life activities in a direct or indirect regulation, is the main control system of living beings. The communication between cells in complex neural network relies on the release of neurotransmitter molecules via exocytosis from the terminal of axon into the synaptic cleft. Monitoring of vesicular exocytosis in real-time is not only beneficial to the understanding of normal physiological processes, but also for revealing the molecular mechanisms of human neurological disease and screening of drugs. However, because a single vesicle exocytosis rapidly (ms) occurred in the extremely tiny synaptic cleft (10?50nm), it is still a great challenge to real-time monitoring the synaptic neurotransmitter exocytosis dynamics from inside single synapse. Ultramicroelectrodes electrochemical detection provides a powerful tool to directly real-time monitoring vesicular release of neurotransmitter in single cell with high spatiotemporal resolution. However, there has been no means for direct monitoring of neurotransmitter exocytosis and its precise kinetics inside individual synapse. Therefore, it is important to develop novel extremely small electrochemical sensor capable of probing into single synapse.On the other hand, cells in vivo are in a complicated microenvironment. Precisely monitoring cells in vitro depends on the successful construction of cellular physiological microenvironment. Culturing nerve cells in traditional model (such as in flasks and dishes) always leads to change of their morphologies, structures as well as their specific functions. Therefore, in vitro mimicking the in vivo environment to construct more actual neural network is of great significance, to real-time monitor and obtain better understanding of the neuronal communications. Over the past decade, microfluidics has emerged as a technology with the potential to make significant impact on cell biology research, and showed the powerful capability in reconstructing cellular microenvironment in vitro.Based on the research background and relied on the unique advantage of nano electrode amperometric method and microfluidic technology in cell analysis, in this thesis, we focus on the study including fabrication of a novel finite conical nanoelectrode, monitoring of vesicular exocytosis in single synapse, patterning the neurons and constructing neural network on microfluidic device. The main work and conclusions are summarized as follows: We developed a facile and robust technique to routinely fabricate finite conical carbon fiber nanoelectrodes (CFNEs) based on flame etching carbon fiber combined with the pulled glass sub-micropipette or nanopipette. The CFNEs, with extremely small tips (less than100in tip diameter and less than1?m in shaft length), showed excellent electrochemical characteristics. For the first time, we successfully probed the nanoelectrode into single synapses formed between cultured superior cervical ganglion (SCG) sympathetic neurons and synapse between SCG sympathetic neuron and A7r5aortic smooth muscle cells (SMCs) and used to real-time monitor the kinetics of norepinephrine release inside synaptic cleft. Our results showed that secretary vesicles are concentratedly distributed at the position apposing the postsynaptic targets and complex exocytosis via flickering pore (kiss and run) is possibly a major mode of fusion in the synaptic cleft. Moreover, the electrochemical detection results showed direct evidences that the heterogeneities in both release probabilities and fuse model are possibly controlled by their targets. This electrochemical detection method has the potential to revolutionize the way we probe into single synapse by direct monitoring inside the real synapse composed of presynaptic membrane, synaptic cleft and postsynaptic membrane.Various microfluidic-based technologies including microcontact printing, microchannel-network by reversible bonding of PDMS with glass, and spatial confinement method by irreversible bonding, have been applied to study their capability in patterning cells and their potential in constructing neuronal communication respectively. The results showed that single axon could be successfully isolated and guided through special microgroove architecture made by the spatial confinement method. Furthermore, using gradient-generation microfluidic devices combined with culturing of SCG neurons and their target SMC in isolated channels, the neuronal growth, differentiation and axon guidence have been investigated. The results demonstrated that the development and axon guidance of SCG neuron could be well regulated and induced by SMC. Based on which synaptic connections and communications network of SCG-SMC have been successfully constructed, showing the potential of real-time monitoring the neuronal communications in an integrated microdevice.