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.

Mitochondrial Dysfunction and Calcium Dysregulation in Leigh Syndrome Induced Pluripotent Stem Cell Derived Neurons

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Deleterious effects of calcium indicators within cells; an inconvenient truth

“…The ease with which Ca 2+ indicators can be loaded and monitored has led to their use in thousands of studies that have characterised the spatial and temporal properties of Ca 2+ signals in various cell types. Chemical Ca 2+ indicators are ideal in many ways; they have rapid Ca 2+ -binding kinetics, are brightly fluorescent, and are functional within cells for long periods of time [6]. Moreover, the output from these Ca 2+ indicators can usually be readily calibrated into Ca 2+ concentration [7].…”

SUR1-TRPM4 channel activation and phasic secretion of MMP-9 induced by tPA in brain endothelial cells

“…bEnd.3 cells grown on Fluorodish (WPI, Sarasota, FL) were activated with 20 ng/mL TNF for 3–16 hours. The cells were rinsed twice with a bath solution (HBSS containing 10 mM HEPES, pH 7.4) and then incubated with 5 μM Fluo-4 AM and 0.02% F-127 pluronic acid in the bath solution in the dark for 1 hour at room temperature [ 32 , 33 ]. The cells were washed twice with the bath solution and mounted in an inverted microscope.…”