Aug 19, 2022
Measuring tension and calcium imaging within cell subtypes of the colon in response to vagus nerve stimulation
- 1University of Nevada, Reno
Protocol Citation: Dante Heredia, Thomas Gould 2022. Measuring tension and calcium imaging within cell subtypes of the colon in response to vagus nerve stimulation. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9y9wzg3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 19, 2022
Last Modified: August 19, 2022
Protocol Integer ID: 68870
Abstract
Protocol for measuring tension and activity in distinct cell types of the colon in response to stimulation of the vagus nerve in an ex vivo preparation lacking the afferent brainstem targets of the vagus nerve.
Guidelines
Nothing more than in steps.
Materials
Same as in dx.doi.org/10/17504/protocols.io.82fhybn
Safety warnings
Not applicable
The entire gut, including esophagus with vagus nerve trunks attached, and stomach and intestines and mesentery, is dissected and placed in a dish with oxygenated Krebs-Ringers solution. The small intestine, as well as pancreas and spleen, are removed carefully. The colon is pinned and gently flushed of its contents. The right vagus nerve trunk is gently dissected away from the esophagus. The tissue is perfused with 35-degree, oxygenated Krebs-Ringers for the duration of the experiment.
For tension, the colon is then drawn over a 1.5-mm diameter fire-polished capillary tube, whose length exceeds that of the colon. An artificial pellet is mounted to the capillary glass and the colon is positioned with the pellet in the middle. The capillary glass is then fixed to the bottom of the organ bath by the ends protruding from each colonic opening. Suture silk is used to connect three force transducers (model TST125C; Biopac Systems, Santa Barbara, CA) to the proximal, transverse and distal segments of the colon. Resting tension is initially set at 8 mN and monitored using an MP100 interface and recorded on a PC running Acqknowledge software 3.2.6 (Biopac Systems). A suction electrode connected to a 10ml syringe and tubing is then placed over the right vagus nerve trunk and upward pressure is applied to suck in the nerve. Stimulation is then performed with a variety of pulse duration durations and frequencies.
For calcium imaging, either Kit-GCaMP6f, ChAT-GCaMP6f, or nNOS-GCaM6f mice are used. In this dissection, the middle colon only is slit from the ventral midline, and pinned with gentle tension. This is the area of the colon that is imaged. A suction electrode connected to a 10ml syringe and tubing is then placed over the right vagus nerve trunk and upward pressure is applied to suck in the nerve. Stimulation is then performed with a variety of pulse duration durations and frequencies, and calcium responses are imaged.