Development of in situ detection methods for substances in brain by using bioelements and bilayer lipid membranes

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For the design of in situ sensing systems for biologically important substances that participates in neuronal communication in acute mouse brain slices, the use of artificial and natural bilayer lipid membranes and biological elements has be investigated. A glass capillary-based enzyme electrode was designed to detect L-glutamate that is released under glucose-free and oxygen-deficient conditions (Ischemia) by using glutamate oxidase and a gall capillary having the ability of sampling a small volume of extracelluar solution. The sensor applied to acute mouse brain slices showed that release of L-glutamate is neuronal region-dependent and the amount of L-glutamate release increases in the order of CA1【approximately equal】CA3>DG. This order was in agreement with those obtained by differential imaging of L-glutamate fluxes using enzyme-immobilized membrane developed newly in this research project. A patch sensor containing NMDA receptors was developed for detecting GABA-induced release of glutamate in acute hippocampal slices. The results showed that in the CA1 region, glutamate release by GABA stimulation is induced via GABA_A receptor and in the CA3 region, release of glutamate was not observed due to inhibitory effect of GABA_B receptor. An excised patch membrane sensor for arachidonic acid was also developed and applied to detect L-glutamate-stimulated release of arachidonic acid. The results exhibited that release of arachidonic acid increases in the order of CA3>CA1【approximately equal】DG. Also, bilayer lipid membranes (BLMs) containing a single gramicidin channel was prepared by the tip-dip method. The BLMs exhibited current responses, corresponding to modulation of gramicidin monomer/dimer kinetics caused by the binding of analytes to membrane bound receptor sites in the BLMs. The BLMs were used for detecting antibodies.