Fast methods for analysis of neurotransmitters from single cells and monitoring their releases in central nervous system by capillary electrophoresis, fluorescence microscopy and luminescence imaging

摘要

Fast methods for detection of important neurotransmitters and their releases in central nervous system (CNS) were developed. Enzymatic assay combined with capillary electrophoresis was used to analyze glutamate from single neuron cells. The release of glutarmate from neurons was monitored by fluorescence imaging method. The release of adenosine triphosphate (ATP) in CNS was studied with sensitive luminescence imaging method;A new dual-enzyme assay method combined with capillary electrophoresis has been developed for determining the glutamate in single cells. The measured laser-induced fluorescence intensity of product NADH was related to the concentration of glutamate in each cell. The detection limit is 10--8 M level, I order lower than previous reports. The mass detection limit of a few attomoles is far superior to that of any other reports. The glutamate content in single human erythrocyte and rat brain neurons were determined and the results agreed well with literature values;A noninvasive detection scheme based on above assay combined with microscopy was developed to monitor the glutamate release in CNS. The detection limit with CCD imaging is down to 10--8M concentration level with reasonable response (∼30 s). The in vitro monitoring of glutamate release from cultured neurons demonstrated excellent spatial and temporal resolutions that is superior to prevalent electrophysiological or chromatographic methods;I A novel chemiluminescence method was developed to monitor the ATP release from astrocytes. The assay has linear response over 3 orders of magnitude. The detection limit of ATP is down to 10--8 M concentration levels at milliseconds exposure time scale with intensified charge coupled device (ICCD), which is superior to seconds of collection time with luminometer. The in vitro experiment of ATP release from astrocytes was successfully developed, the ATP release upon stimulation was recorded first time in real time scale. The data shows that stimulation triggered an extracellular ATP wave, which reveals the communications among cells along the signaling pathway. Furthermore, the intracellular calcium propagation underneath this ATP wave was successfully simultaneously imaged. The data shows the ATP signaling process is a dominant pathway for evoked calcium wave in glials, but ATP release itself is not calcium-dependent.