sq-23377 and fura-red

Ca2+ signalling influences many processes in the adult and developing nervous system like exocytosis, synaptic plasticity, and growth cone motility. Optical techniques in combination with fluorescent Ca2+ indicators are the most frequently used methods to measure Ca2+ signalling in cells. In the present study, a new method for ratiometric confocal Ca2+ imaging was developed, and the usefulness of the system was tested with two different neuronal preparations. Developing Manduca sexta antennal lobe neurons were loaded with the Ca2+-sensitive dye Fura Red-AM, and the ratio of fluorescence excited at 457 and 488nm was measured with a confocal laser scanning microscope. During pupal stages 4-12, the antennal lobe neuropil is restructured which includes the ingrowth of olfactory receptor axons, dendritic outgrowth of antennal lobe neurons, and synaptogenesis. In antennal lobe neurons, application of the AChR agonist carbachol induced Ca2+ oscillations the amplitude and frequency of which changed during stages 4-9, while at the end of synaptogenesis, at stages 11 and 12, only single Ca2+ transients were elicited. The Ca2+ oscillations were blocked by D-tubocurarine and Cd2+, indicating that they were due to Ca2+ influx through voltage-gated Ca2+ channels, activated by nAChR-mediated membrane depolarization. To test whether single action potentials can induce Ca2+ transients detectable by Fura Red, individual leech Retzius neurons were injected iontophoretically with the Ca2+ indicator, and the membrane potential was recorded during Ca2+ imaging. Single action potentials induced transient increases in the Fura Red ratio measured in the axon, while trains of action potentials elicited Ca2+ transients that could also be recorded in the cell body and the nucleus. The results show that Fura Red can be used as a ratiometric Ca2+ indicator for confocal imaging.

Topics: Action Potentials; Algorithms; Animals; Benzofurans; Brain; Cadmium; Calcimycin; Calcium; Calcium Signaling; Calibration; Carbachol; Cell Nucleus; Cytoplasm; Data Interpretation, Statistical; Imidazoles; Ionomycin; Leeches; Manduca; Metamorphosis, Biological; Microscopy, Confocal; Microscopy, Fluorescence; Neurons; Photobleaching; Pupa; Tubocurarine

Ischemia produces striking electrophysiological abnormalities in blood-perfused hearts that may be caused, in part, by effects of ischemia on intracellular calcium. To test this hypothesis, intracellular Ca2+ concentration ([Ca2+]i) transients were recorded from the epicardial surface of blood- and saline-perfused rabbit hearts using the long-wavelength indicator Fura Red. Calcium transients were much larger than the movement artifact, representing up to 29% of the total signal. Switching the perfusate from saline to blood did not affect the time course of the transients or the apparent level of [Ca2+]i. Compartmentation of Fura Red fluorescence was estimated by exposure to Mn2+. The results were cytosol 60 +/- 3%, organelles 12 +/- 2%, and autofluorescence plus partly deesterified Fura Red 29 +/- 4%. [Ca2+]i transients were calibrated in situ by perfusion of the extracellular space with high-Ca2+ and Ca(2+)-free EGTA solutions. Peak systolic [Ca2+]i was 663 +/- 74 nM, and end-diastolic [Ca2+]i was 279 +/- 59 nm. Ischemia was produced by interruption of aortic perfusion for 2.5 min during pacing (150 beats/min). Ischemia produced broadening of the [Ca2+]i transient, along with beat-to-beat alternations in the peak systolic and end-diastolic level of [Ca2+]i (calcium transient alternans). [Ca2+]i transient alternans occurred in 82% of blood-perfused hearts vs. 43% of saline-perfused hearts. The discrepancy between large and small transients (mean alternans ratio) was larger in the blood-perfused hearts (0.23 +/- 0.04 vs. 0.07 +/- 0.03, P = 0.005). These observations are important because of the apparent relationship of [Ca2+]i transient alternans to electrical alternans and arrhythmias during ischemia.

Topics: Animals; Benzofurans; Calcium; Calibration; Female; Fluorescent Dyes; Heart; Imidazoles; Ionomycin; Kinetics; Male; Manganese; Myocardial Ischemia; Myocardium; Rabbits; Spectrometry, Fluorescence; Subcellular Fractions; Time Factors