This article describes an electrophysiological setup to study the afferent nerve signals from the mouse colon. The methodology involves monitoring nerve activity in response to colon distension.
Begin with an electrophysiology setup containing an immobilized mouse colon segment in a balanced electrolyte solution.
The colon has an exposed major pelvic ganglion with a pre-dissected colon afferent sensory nerve branch that transmits signals from the colon to the brain.
The colon is cannulated at both ends with three-way valves for saline infusion.
Fill the recording electrode and position it near the afferent nerve.
Apply mild suction to pull the nerve into the electrode, forming a tight seal.
Monitor the baseline nerve electrical signal and the colon's internal pressure.
Close the tap on the outlet cannula while continuously infusing the saline into the colon.
This causes a gradual rise in pressure inside the colon, resulting in colon distension and stimulating the afferent nerve.
Re-record the nerve signal and the colon pressure.
A gradual rise in the nerve signal with increased pressure confirms the afferent nerve stimulation.
Inspect a prepulled glass pipette under the dissecting microscope. Break the tip of the electrode with a pair of forceps, so that it is of a size compatible with the diameter of the nerve to be recorded. Following that, bevel the tip by placing it close to a lighter flare.
Individuals new to this methodology may also find it tricky to bevel the tip of the electrode compatible with the size of the nerve.
Next, place the beveled electrode in the electrode holder. Then, connect a 10-milliliter syringe to the side port of the holder for the application of negative or positive pressure to the electrode. After that, connect the holder to the head stage of the bioamplifier, and mount the head stage onto a manipulator.
Following this, move the electrode to the tissue bath, and fill the electrode with the Krebs solution by applying gentle suction, until it contacts the silver wire of the holder. Next, place the electrode tip close to the cut end of the nerve, and apply negative pressure to suck the nerve into the electrode. Then, apply more negative pressure so that about 1 millimeter of the nerve is pulled into the electrode, and forms a tight seal.
Now, turn on the bioamplifier, and set the filter to 300 to 3,000 hertz. Monitor the signal on the oscilloscope, and record the nerve signal and the intraluminal pressure signal in the Spike Data processing software. Next, apply ramp distension of the colon by closing the three-way tap on the outlet cannula while continuously infusing intraluminally. Monitor the intraluminal pressure until it reaches 60 millimeters mercury.
繁體中文
