In Situ Ca²⁺ Imaging of the Enteric Nervous System

The overall goal of this procedure is to image the activity of enteric neurons and glia in live whole mount preparations of intestine using fluorescent calcium indicator dyes. This is accomplished by first excising a portion of intestine from the animal and placing it in a prewarm media. The second step is to open the intestine along the mesenteric border and pin it flat under light tension with a mucosal surface facing up.

Whole mount preparations of the myenteric plexus are prepared by microdissection and placed in imaging dishes. Next, the whole mount preparations are loaded with a fluorescent indicator dye flu oh four in an incubator. In the final steps, the loaded preparations are imaged using a fluorescent imaging microscope equipped with a high resolution camera controlled by image acquisition and analysis software to record baseline activity.

Then the drugs under investigation are added and calcium transients in neurons and glia are recorded. Ultimately in situ. Calcium imaging of the enteric nervous system is used to study how cellular activity in neurons and glia contributes to the regulation of physiological gut functions and enteric nervous system dysfunction in gut pathophysiology.

The implications of this technique extend toward establishing an understanding of the physiology and pathophysiology of functional bowel disorders and inflammatory bowel disease through the use of a simple and robust method to examine the complex interplay between neurons and enteric glia using NC two calcium imaging demonstrating this procedure will be David free to scientists from my laboratory. The following procedures involving tissue from laboratory animals are consistent with the A VMA guidelines for the euthanasia of animals 2013, and were approved in advance by the Michigan State University I-A-C-U-C After euthanizing the animal according to approved procedures, place the animal in a supine position and clean the skin over the abdomen with 70%ethanol. Use forceps to pinch the abdominal skin at midline and then use surgical scissors to make a six centimeter medial incision along the linear elbow to expose the internal digestive organs.

Next, use blunt forceps to locate and expose the colon inside the peritoneum. Cut the ileal colon mesentery with scissors and begin unraveling the intestine. Once the length of the intestine is adequately unraveled, cut the colon distal to the cecum and proximal to the rectum for a large intestine preparation, which will be shown in this video.

Then quickly remove the intestinal segment and place it in a beaker containing DM EMF 12 Media supplemented with three micromolar nicardipine hydrochloride and one micromolar S scopolamine hydrochloride on ice. The addition of these inhibitors facilitates microdissection and subsequent imaging by paralyzing the gut smooth muscle. Remove a small four to six centimeter segment of the desired intestine segment and place in a syl guard coated Petri dish filled with chilled supplemented media.

Then 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. Now pin the tissue flat under light tension with a mucosa side up and carefully dissect away the mucosal layer by lifting the mucosa with number five fine forceps and cutting underneath with very fine spring scissors. Cut the tissue into smaller preparations of approximately 0.5 centimeters square and place each piece into an imaging dish filled with supplemented media and place on ice.

Pin each preparation at four corners with a circular muscle layer facing up. Carefully dissect away the circular muscle by teasing it apart with fine tweezers. To expose the myenteric plexus, avoid excessive stretching, place the imaging dishes back on ice and replace the solution in each dish with fresh supplemented media.

Next, remove the dishes from ice and add two milliliters of enzyme mix to each dish and incubate at room temperature with 5%carbon dioxide and 95%air for 15 minutes. Finally, wash the tissue preparations with media three times and rein the corners while working under conditions of limited light to avoid photobleaching at 1.5 milliliters of supplemented media and 1.2 microliters of a 250 millimolar probit stock to a 1.5 microliter Eloqua of four millimolar Fluro four stock at approximately 1.5 milliliters of flu oh four loading solution to the imaging dishes and incubate in a dark incubator at 37 degrees Celsius for 45 minutes. After removing the dishes from the incubator, wash the preparations three times with media.

Then add fresh media containing 200 micromolar prop acid to enhance neural neuronal labeling and incubate as before for 15 minutes. During the incubation, make a modified Krebs buffer according to the instructions in the written portion of the protocol, and add three micromolar nicardipine and one micromolar scopolamine to inhibit muscle contractions. During calcium two plus imaging and 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 two to three milliliters per minute of 37 degrees Celsius Krebs buffer. Make sure to prevent air bubble formation in both the input and the suction line connected to a vacuum trap. Bring the desired plexus into focus under bright field illumination.

Avoid overexposing the tissue which may lead to photobleaching. Examine the flu oh four 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 one hertz for glial cells and up to two to 10 hertz.

For neurons, because glial calcium transient 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 prewarm drugs of interest such as receptor agonists and antagonists using the gravity flow perfusion system at a rate of two to three 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 fluorescent intensity of regions of interest against its initial baseline Value changes in normalized fluorescence are directly proportional to changes in calcium. The following three images demonstrate that enteric glia in the Guinea pig respond to a TP in situ.

Under basal conditions, a low level fluoro four fluorescence as outlined by the dashed line is visible. The arrows point to thick inter ganglionic fiber tracts upon stimulation with 100 micro mo per liter A TP glial cells, but not neurons rapidly increase fluoro four fluorescence indicating an increase in calcium. Note that the responding cells are small and surround the much larger neurons indicated by the dark spaces marked by asterisk.

This histogram shows that enteric glia respond to a TP in a dose dependent manner with one milli mole per liter eliciting maximal responses. This video shows a myenteric ganglion from the mouse distal colon loaded with a calcium two plus indicator dye flu oh four. The glial cell agonist a DP is added to the bath when indicated.

A DP elicits an increase in intracellular calcium two plus in enteric glia as observed by the transient elevation in flu oh four fluorescence. After watching this video, you should have a good understanding of how to accurately examine the complex interplay between neurons and empirically using calcium imaging. Characterizing the mechanisms and potential functional consequences of calcium responses in whole mount preparations requires precise dissections for ideal imaging quality.

The use of fluorescent labeling and pharmacological stimuli within this microscopy based technique enables improved assessment of these cells in their native multicellular environment.