Calcium Imaging of Enteric Neuron and Glia Activities in a Mouse Colonic Myenteric Plexus

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 

1.Prepare Modified Krebs buffer.

Make a modified Krebs buffer such that the final concentrations (in mM) of the components are as follows: 121 NaCl, 5.9 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 1.2 NaH₂PO₄, 10 HEPES, 21.2 NaHCO₃, 1 pyruvic acid and 8 glucose (pH adjusted to 7.4 with NaOH). Add 3 μM nicardipine and 1 μM scopolamine to inhibit muscle contractions during Ca²⁺ imaging and whole-mount dissections.

2. Imaging and Analysis

NOTE: Use at least a basic imaging rig with a fluorescent light source, microscope, a quality CCD camera and appropriate acquisition software. Vary the addition of other components depending on the light source and specific application. A filter wheel and shutter must be used with a traditional xenon arc light source. However, LED light sources and illumination systems do not require those components.

1. Position the recording chamber under the microscope and using a gravity flow perfusion system with multiple heated syringe reservoirs establish a continuous perfusion rate of 2–3 ml/min of 37 °C Krebs buffer. Make sure to prevent air bubble formation in both the input and suction line connected to a vacuum trap.
2. Bring the desired plexus into focus under bright field illumination. Avoid overexposing tissue, which may lead to photobleaching.
3. Examine the Fluo-4 loading within ganglia and select healthy ganglia for imaging. Unhealthy/damaged ganglia will exhibit autofluorescence or punctate morphology and should not be used for imaging.
4. Once ganglion is selected, divert light path to camera and obtain live image with image acquisition software. Ensure that ganglion is in focus and set image acquisition rate and exposure times.
   NOTE: Image acquisition rates and times will vary depending on the events investigators wish to record. For most experiments, images are traditionally acquired at 0.5-1 Hz for glial cells and up to 2-10 Hz for neurons because glial Ca²⁺ responses are not as rapid as Ca²⁺ transients in neurons.
5. Begin experiment and establish baseline activity for 30 sec.
6. Apply pre-warmed drugs of interest such as receptor agonists and antagonists using the gravity flow perfusion system at a rate of 2-3 ml/min. Follow application of agonists/antagonists by returning to a perfusion of normal buffer and allow a wash/recovery period of at least 10 min.
   NOTE: The drug application times will vary depending on the individual compound and experimental design. In general, a 20-30 sec application of agonist is sufficient to activate G-protein coupled receptors in neurons and glial cells. However, ligand-gated ion channels (such as nicotinic acetylcholine receptors) require application times of no more than 5-10 sec. Further duration exposures will cause ligand gated ion channels to rapidly desensitize. Antagonists should be applied for approximately 3-15 min to ensure complete blockade of receptor pathways. However, this is a gross generalization and investigators should always optimize any experimental drug in their particular paradigm.
7. Stop recording and view time-lapse movie of the experiment. Carefully select regions of interest (ROI’s) using the appropriate image analysis software.
8. Use software to normalize and compare ROI fluorescent intensity against its initial baseline fluorescent value. Changes in normalized fluorescence are directly proportional to changes in [Ca²⁺].
   1. Use a modification of a method described previously using ΔF/F = ((F1− F0)/F0)ROI − ((F1− F0)/F0)background, where F1 is the fluorescence at any given point and F0 is the baseline fluorescence, to improve the assessment accuracy. This modification aids in reducing noise from fluorescence changes in tissue preparations that exhibit movement of the muscle layer underlying the myenteric plexus.