Take an imaging chamber containing immobilized mouse colonic myenteric plexus tissue, which houses ganglionated plexuses composed of networks of enteric neurons and glial cells.
These cells are labeled with a calcium indicator that fluoresces upon calcium binding.
Next, place the chamber on a fluorescence microscope stage. Perfuse the chamber with a pre-warmed buffer containing inhibitors to suppress muscle contractions during imaging.
Using a microscope, identify healthy ganglionic regions that exhibit uniform fluorescence.
Acquire fluorescent images to measure baseline activity, providing a reference for intracellular calcium levels.
Perfuse the tissue with a receptor agonist to activate neuronal receptors, which triggers intracellular calcium influx and an increase in fluorescence intensity.
Neuronal activation indirectly stimulates surrounding glial cells, leading to calcium influx and a subsequent increase in fluorescence.
Capture time-lapse fluorescent images to measure changes in fluorescence intensity, which indicate calcium dynamics and signaling activity in both neurons and glial cells relative to baseline levels.
During the incubation, make a modified Kreb's buffer according to the instructions in the written portion of the protocol, and add 3 micromolar nicardipine and 1 micromolar scopolamine to inhibit muscle contractions during calcium 2 plus imaging at whole mount dissections. Position the recording chamber under the fluorescent microscope, and using a gravity flow perfusion system with multiple heated syringe reservoirs, establish a continuous perfusion rate of 2 to 3 milliliters per minute of 37 degrees Celsius Kreb's buffer. Make sure to prevent air bubble formation in both and the suction line connected to a vacuum trap. Bring the desired plexus into focus under brightfield illumination. Avoid overexposing the tissue, which may lead to photobleaching.
Examine the fluorophore loading within the ganglia and select healthy ganglia for imaging. Unhealthy damaged ganglia will exhibit autofluorescence or punctate morphology and should not be used for imaging. Once a ganglion is selected, divert the light path to the camera and obtain a live image with image acquisition software. Ensure that the ganglion is in focus and set the image acquisition rate and exposure times.
Image acquisition rates and times will vary depending on the events investigators wish to record. For most experiments, images are traditionally acquired at 0.5 to 1 hertz for glial cells, and up to 2 to 10 Hertz for neurons, because glial calcium transients are not as rapid as calcium transient neurons.
Start the recording and establish baseline physiological activity of the chosen ganglion in the absence of experimental stimuli for 30 seconds. Then apply pre-warmed drugs of interest, such as receptor agonists and antagonists, using the gravity flow perfusion system at a rate of 2 to 3 milliliters per minute according to an optimized protocol for your drug. Stop the recording and view the time lapse movie of the experiment.
Carefully select the regions of interest, or ROIs, using the appropriate image analysis software.
Finally, use appropriate imaging software to normalize and compare fluorescence intensity of regions of interest against its initial baseline value. Changes in normalized fluorescence are directly proportional to changes in calcium.
