A single optical fiber fluorometric device for measurement of intracellular Ca2+ concentration: its application to hippocampal neurons in vitro and in vivo.
We developed a new system to measure the intracellular Ca2+ concentration in the deep region of the central nervous system with a single optical fiber (300 microns in diameter), used for both excitation and detection of the fluorescence of previously loaded fura-2. With this system, a brain region loaded with fura-2 was illuminated by a rotating disc bearing three different interference filters of 340, 360 and 380 nm at a rate of 600 rpm. The emitted fluorescence was collected by the same fiber connected to a photomultiplier whose output was fed into a computer which regulates the timing of illumination and detection. The time course of the change in the fluorescence due to 340, 360 or 380 nm excitation was measured simultaneously at the maximum sampling rate of 10 points/s. Ratios of fluorescence intensities were obtained after the experiment. After confirming that this system was sensitive enough to detect the change of intracellular Ca2+ concentration in cultured hippocampal neurons and hippocampal slices during depolarization by high potassium medium (50 mM), we applied this system to anesthetized rats. In the hippocampus preloaded with fura-2, characteristic changes in fluorescence intensities ascribed to an increase in intracellular Ca2+ concentration were detected after asphyxia. The system is potentially useful for investigating the physiological and pathological roles of Ca2+ in the brain.
Kudo
Y
Y
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Akita
K
K
Nakamura
T
T
Ogura
A
A
Makino
T
T
Tamagawa
A
A
Ozaki
K
K
Miyakawa
A
A
D000758:Anesthesia; D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D005336:Fiber Optic Technology; D016257:Fura-2; D006624:Hippocampus / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism; D055100:Optical Fibers; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*
D002118:Calcium; D016257:Fura-2