title: Calcium Imaging of AM Dyes Following Prolonged Incubation in Acute Neuronal Tissue.
abstract: Calcium-imaging is a sensitive method for monitoring calcium dynamics during neuronal activity. As intracellular calcium concentration is correlated to physiological and pathophysiological activity of neurons, calcium imaging with fluorescent indicators is one of the most commonly used techniques in neuroscience today. Current methodologies for loading calcium dyes into the tissue require prolonged incubation time (45-150 min), in addition to dissection and recovery time after the slicing procedure. This prolonged incubation curtails experimental time, as tissue is typically maintained for 6-8 hours after slicing. Using a recently introduced recovery chamber that extends the viability of acute brain slices to more than 24 hours, we tested the effectiveness of calcium AM staining following long incubation periods post cell loading and its impact on the functional properties of calcium signals in acute brain slices and wholemount retinae. We show that calcium dyes remain within cells and are fully functional >24 hours after loading. Moreover, the calcium dynamics recorded >24 hrs were similar to the calcium signals recorded in fresh tissue that was incubated for <4 hrs. These results indicate that long exposure of calcium AM dyes to the intracellular cytoplasm did not alter the intracellular calcium concentration, the functional range of the dye or viability of the neurons. This data extends our previous work showing that a custom recovery chamber can extend the viability of neuronal tissue, and reliable data for both electrophysiology and imaging can be obtained >24hrs after dissection. These methods will not only extend experimental time for those using acute neuronal tissue, but also may reduce the number of animals required to complete experimental goals.
authors.affiliation: Biomedical Engineering and Neuroscience group, The MARCS Institute, Western Sydney University, Penrith, NSW, Australia.
authors.forename: Morven
authors.initials: M
authors.lastname: Cameron
authors.affiliation: School of Medicine, Western Sydney University, Penrith, NSW, Australia.|Biomedical Engineering and Neuroscience group, The MARCS Institute, Western Sydney University, Penrith, NSW, Australia.
authors.forename: Orsolya
authors.initials: O
authors.lastname: Kékesi
authors.affiliation: School of Medicine, Western Sydney University, Penrith, NSW, Australia.|Biomedical Engineering and Neuroscience group, The MARCS Institute, Western Sydney University, Penrith, NSW, Australia.
authors.forename: John W
authors.initials: JW
authors.lastname: Morley
authors.affiliation: School of Medicine, Western Sydney University, Penrith, NSW, Australia.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Tapson
authors.affiliation: School of Medicine, Western Sydney University, Penrith, NSW, Australia.
authors.forename: Paul P
authors.initials: PP
authors.lastname: Breen
authors.affiliation: School of Medicine, Western Sydney University, Penrith, NSW, Australia.
authors.forename: André
authors.initials: A
authors.lastname: van Schaik
authors.affiliation: School of Medicine, Western Sydney University, Penrith, NSW, Australia.|Biomedical Engineering and Neuroscience group, The MARCS Institute, Western Sydney University, Penrith, NSW, Australia.
authors.forename: Yossi
authors.identifier: 0000-0001-8500-469X
authors.initials: Y
authors.lastname: Buskila
mesh_terms: D000818:Animals; D001921:Brain / Q000000981:diagnostic imaging / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D005456:Fluorescent Dyes; D051379:Mice; D057054:Molecular Imaging / Q000379:methods; D009457:Neuroglia / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D012160:Retina / Q000000981:diagnostic imaging / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D002118:Calcium