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.
Highlights
IntroductionCalcium is one of the most common second messengers in the brain, playing a key role in a variety of intracellular physiological processes, ranging from cell proliferation [1] to synaptic plasticity [2] and cell death [3]
Calcium signalling in the central nervous systemCalcium is one of the most common second messengers in the brain, playing a key role in a variety of intracellular physiological processes, ranging from cell proliferation [1] to synaptic plasticity [2] and cell death [3]
This study aimed to demonstrate the length of time acute brain slices and excised retinae, loaded with calcium indicators, can be maintained to produce reliable and reproducible data about cell function
Summary
Calcium is one of the most common second messengers in the brain, playing a key role in a variety of intracellular physiological processes, ranging from cell proliferation [1] to synaptic plasticity [2] and cell death [3]. An intracellular increase in free calcium concentration modulates the activity of numerous proteins, including kinases, phosphatases, transcription factors and enzymes, which participate in many physiological and pathological processes (reviewed by [4]). Intercellular calcium signalling takes place in two ways: direct transmission through gap junctions–mainly in glial cells [6], or through transmitter gated channels (e.g. NMDA and voltage gated calcium channels)[5], in which neurotransmitter release from a presynaptic cell leads to a cascade of processes in the postsynaptic region, that increase the cytoplasmic calcium concentration. It has been shown that calcium signals fall into two main categories: calcium oscillations, and calcium waves, in which the calcium signal spreads via gap junctions from one astrocyte to another as an inter-glial communication signal [6]
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