Monitoring neuronal calcium signalling using a new method for ratiometric confocal calcium imaging

Abstract

Ca 2+ 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 Ca 2+ indicators are the most frequently used methods to measure Ca 2+ signalling in cells. In the present study, a new method for ratiometric confocal Ca 2+ 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 Ca 2+-sensitive dye Fura Red-AM, and the ratio of fluorescence excited at 457 and 488 nm 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 Ca 2+ 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 Ca 2+ transients were elicited. The Ca 2+ oscillations were blocked by d-tubocurarine and Cd 2+, indicating that they were due to Ca 2+ influx through voltage-gated Ca 2+ channels, activated by nAChR-mediated membrane depolarization. To test whether single action potentials can induce Ca 2+ transients detectable by Fura Red, individual leech Retzius neurons were injected iontophoretically with the Ca 2+ indicator, and the membrane potential was recorded during Ca 2+ imaging. Single action potentials induced transient increases in the Fura Red ratio measured in the axon, while trains of action potentials elicited Ca 2+ 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 Ca 2+ indicator for confocal imaging.