In Situ Ca²⁺ Imaging of the Enteric Nervous System

NOTE: The following procedures involving tissue from laboratory animals are consistent with the AVMA Guidelines for the Euthanasia of Animals 2013 and were approved in advance by the Michigan State University IACUC.

1. Tissue Preparation

1. Anesthetize research animal in a chamber containing 2.5% isoflurane in oxygen or by placing 3-5 ml of liquid isoflurane onto an absorbent material on the floor of the chamber, ensuring that a physical barrier prevents animals from direct contact with the isoflurane. Test for the depth of anesthesia by pinching the footpad.
   NOTE: The depth of anesthesia is deemed appropriate when there is no withdrawal reflex of the hind limb. Once appropriately anesthetized, euthanize the mouse by cervical dislocation
2. Place the animal in a supine position and clean abdominal skin with 70% ethanol. Use forceps to pinch abdominal skin at midline and use surgical scissors to make a 6 cm medial incision along the linea alba to expose internal digestive organs.
3. Use blunt forceps to locate and expose the ileum inside the peritoneum. Cut the ileal/colon mesentery with scissors and begin unraveling the intestine.
4. Once the length of the intestine is adequately unraveled, cut the intestine distal to the stomach and proximal to the cecum for an ileum preparation. For a large intestine preparation, cut the colon distal to the cecum and proximal to the rectum.
5. Quickly remove the intestinal segment and place it in a beaker with DMEM/F12 media supplemented with 3 μM nicardipine hydrochloride and 1 μM scopolamine hydrochloride (hereafter referred to as “media”) on ice. The addition of these inhibitors facilitates microdissection and subsequent imaging by paralyzing the gut smooth muscle.
6. Cut segment of interest (e.g., jejunum, ileum, distal or proximal colon) based on established anatomical markers. Typically utilize tissue from the distal ileum or distal colon. However, use the same basic procedure to isolate, load and image myenteric neurons and glia in all intestinal regions.
7. Remove a small segment (4-6 cm) of desired intestine segment and place in a Sylgard–coated petri dish filled with chilled media.
8. Secure the proximal and distal ends of the intestinal segment with insect pins and open the intestinal tube by making a straight, lengthwise cut along the mesenteric border.
9. Pin tissue flat under light tension with mucosa side up and carefully dissect away mucosal layer using fine forceps (#5 and #5/45 work best) and very fine spring scissors.
   NOTE: The removal of the mucosa can be quite traumatic for the ENS if not done properly. For quality preparations, take care to limit abrupt removal of the mucosa by peeling or scraping. The best practice is to lift the mucosa and cut underneath with fine scissors.
10. Cut the tissue into smaller preparations (approximately 0.5 cm²) and pin into imaging dishes (4 corners with circular muscle layer facing up) placed on ice with fresh media.
11. Carefully dissect away circular muscle by teasing apart with fine tweezers to expose the myenteric plexus. Avoid excessive stretching.
12. Place imaging dish back on ice and change solution with fresh media.
13. Prepare 2 ml of enzyme mix per dish [Dispase 1 U/ml (4.48 mg/8 ml), Collagenase Type II 150 U/ml (5.45 mg/8 ml) in media].
14. Remove dishes from ice and add the enzyme mix from step 1.13.
15. Incubate dishes at RT for 15 min with 5% CO₂ / 95% air.
16. Wash tissue preparations with media 3 times and re-pin corners.

2. Loading Fluo-4 Dye

NOTE: Avoid photobleaching by working with limited light while handling fluorescent dyes and tissue loaded with indicator dyes.

1. Prepare 4 μM Fluo-4 loading solution.
   1. Add 1.5 ml media and 1.2 μl of a 250 mM probenecid stock to a 1.5 μl aliquot of 4 mM Fluo-4 stock. 4 mM Fluo-4 stock is prepared by adding 11.4 μl of pluronic F-127 (20% in DMSO; supplemented with 0.25% cremaphor-EL) to 50 μg of Fluo-4, AM.
2. Incubate preps in Fluo-4 loading solution for 45 min in a dark incubator at 37 °C.
3. Remove from incubator and wash preparations with media 3 times.
4. Exchange media for media containing 200 μM probenecid and incubate 15 min at 37 °C before imaging.
   NOTE: Probenecid is a drug that inhibits multidrug resistance transporters in neurons. Addition of this drug inhibits the ability of neurons to extrude dyes and enhances neuronal labeling. Bulk-loading of Ca²⁺ indicator dyes in the absence of probenecid produces mainly glial cell loading. The addition of probenecid allows for visualization of neuronal and glial responses.
5. Prepare Modified Krebs buffer.
   1. Make a modified Krebs buffer such that the final concentrations (in mM) of the components are as followed: 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.

3. 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 photo bleaching.
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 ²⁶ 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.