A single optical fiber fluorometric device for measurement of intracellular Ca2+ concentration: Its application to hippocampal neurons in vitro and in vivo

doi:10.1016/0306-4522(92)90451-7

Neuroscience

Volume 50, Issue 3, October 1992, Pages 619-625

https://doi.org/10.1016/0306-4522(92)90451-7 Get rights and content

Abstract

We developed a new system to measure the intracellullar Ca²⁺ concentration in the deep region of the central nervous system with a single optical fiber (300 μm 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.

Reference (19)

GarthwaiteJ. et al.
Amino acid neurotoxicity: relationship to neuronal depolarization in rat cerebellar slices
Neuroscience
(1986)
GrynkiewiczG. et al.
A new generation of Ca²⁺ indicators with greatly improved fluorescence properties
J. biol. Chem.
(1985)
IijimaT. et al.
Variation in the pattern of [Ca²⁺]₁ change induced by acetylcholine in cultured hippocampal neurons
Brain Res.
(1990)
ItoM. et al.
Long term desensitization of quisqualate-specific glutamate receptors in Purkinje cells investigated with wedge recording from rat cerebellar slices
Neurosci. Res.
(1989)
KudoY. et al.
Monitoring of intracellular Ca²⁺ elevation in a single neural cell using fluorescence microscope/video camera system
Jap. J. Pharmac.
(1986)
KudoY. et al.
Cytosolic calcium elevation in hippocampal granule cell induced by perforant path stimulation andl-glutamate application
Brain Res.
(1987)
KudoY. et al.
A new device for monitoring concentration of intracellular Ca²⁺ in CNS preparations and its application to the frog's spinal cord
J. Neurosci. Meth.
(1989)
KudoY. et al.
A “macro” image analysis of fura-2 fluorescence to visualize the distribution of functional glutamate receptor subtypes in hippocampal slices
Neurosci. Res.
(1991)
OguraA. et al.
Non-NMDA receptor mediates cytoplasmic Ca²⁺ elevation in cultured hippocampal neurones
Neurosci. Res.
(1990)

There are more references available in the full text version of this article.

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A single optical fiber fluorometric device for measurement of intracellular Ca2+ concentration: Its application to hippocampal neurons in vitro and in vivo

Abstract

Neuroscience

J. biol. Chem.

Brain Res.

Neurosci. Res.

Jap. J. Pharmac.

Brain Res.

J. Neurosci. Meth.

Neurosci. Res.

Neurosci. Res.

A single optical fiber fluorometric device for measurement of intracellular Ca²⁺ concentration: Its application to hippocampal neurons in vitro and in vivo