Measurement of Free Ca From Digital Images in Cultured Rat Brain Neurons

Recently it has been reported that the intracellular Ca^<2+> concentration of neurons can be obtained by analyzing suitably the intensity of the fluorescent light of 500nm emitting from the neurons loaded with fura-2 when they are excited by the light of 340,360 or 380nm. The present paper represents the measurement of free Ca^<2+> distribution in the cultured rat hippocampal neurons using the digital analysis system, ARGUS100/CA. The ratio images are obtained from the ratio of the brightness of the pixels in the picture obtained by the excited light of 340nm to that in the images obtained by the excited light of 360nm. In order to obtain the intracellular Ca^<2+> concentration from the ratio images, the corrected in vitro calibration curve is calculated by multiplying 0.85 to the values obtained from the intensity of F_<max> and F_<min>. This multiplication considers the effect of cytoplasmic microviscosity in the intracellular region of the neuron. The intracellular free Ca^<2+> concentration obtained is about 8×(10)^<-8>M in the resting state of the neurons. After application of the neural transmitter L-glutamate of (10)^<-4>M, the level of Ca^<2+> increases to about 6×(10)^<-7>M, These Ca^<2+> levels agree with the data reported by other groups.