Image Analysis Application for Ionotropic Glutamate Receptor Ligands Characterization in Cultured Neurons

The non-invasive microphotometrical method for identification and quantitative analysis of the compounds interacting with glutamate NMDA-, AMPA-, and KA-receptors is presented in this paper. The method is based on the registration of the ligand-gated and voltage-gated calcium channels activity using two-wavelength fluorescent probe Fura-2. We found conditions under which repeated short-term (in up to 30 seconds) applications of NMDA-, AMPA and kainate receptor agonists induce calcium response in majority of the cells, which was almost identical to the first response. In the experiment we use the reaction of the cell to the first ligand application as a control that significantly simplifies the process of ligands titration. To increase the sensitivity of the method for each ligand the optimal age of the culture was determined. To compare the results in different experiments and to obtain more precise values of activation and inhibition constants, the certain rules of signal normalization were introduced. It is shown that the variability of calcium responses of individual neurons to the glutamate receptor agonists significantly depends on the degree and kinetics of desensitization. To characterize the AMPA and KA receptor ligands, the experiments were carried out in the presence of desensitization inhibitors, cyclothiazide and concanavalin-A, respectively. The inhibitors increase the amplitude of the response and turned impulse responses into gradual ones. The developed method allows all studied receptor ligands to be found in the unknown sample, and the constants of activation and inhibition and the type of inhibition to be determined. The method combines high speed, high sensitivity, specificity and performance due to the possibility to analyze simultaneously individual activity of hundreds cells. The method is applicable to obtain both averaged response over all investigated neurons and the response of selected cell populations or single neurons.