Application of imaging technique for characterization of ionotropic glutamate receptor ligands in cultured neurons

Abstract

This article presents a non-invasive microphotometrical method for identification and the quantitative analysis of the compounds interacting with glutamate NMDA, AMPA, and KA-receptors. The method is based on the registration of ligand-gated and voltage-gated calcium channels activity using ratiometric fluorescent probe Fura-2. We have found conditions when repeated short-term (up to 30 s) applications of NMDA-, AMPA- and kainate-receptor agonists induce in most cells a calcium response almost identical to the first one. In the experiment we used the cell reaction to the first ligand application as a control, which significantly simplified the process of ligand titration. The optimal culture age was determined to enhance sensitivity of the method for each ligand. Certain rules of signal normalization were established to compare the results of different experiments and to obtain more precise values of activation and inhibition constants. It has been shown that the variability of calcium responses of individual neurons to the glutamate receptor agonists significantly depends on the degree and kinetics of their desensitization. The AMPA and KA-receptor ligands were characterized in the experiments performed in the presence of desensitization inhibitors cyclothiazide and concanavalin A, respectively. The inhibitors increased the amplitude of the response and converted the transient responses into the step ones. The method permits the detection of receptor ligands under study in an unknown sample, as well as determination of the activation and inhibition constants and the type of the inhibition. The method combines, high sensitivity, specificity and performance due to the possibility of analyzing individual activities of hundreds of cells simultaneously. The method can be used to obtain both the averaged response for all of the studied neurons and the responses of the selected cell populations or single neurons.