May 22, 2020
Optogenetically stimulating enteric neurons in the murine large intestine.
- 1Department of Physiology and Cell Biology, University of Nevada Reno School of Medicine
Protocol Citation: Dante Heredia, Thomas Gould 2020. Optogenetically stimulating enteric neurons in the murine large intestine.. protocols.io https://dx.doi.org/10.17504/protocols.io.bgr9jv96
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 in our group and it is working. We are moving to a TTL-coupled laser-emitting diode system to control timing of laser pulses for nNOS-ChR2 mice, similar to the timed LED pulses that are sufficient to drive contractions in the colon of ChAT-ChR2 mice.
Created: May 22, 2020
Last Modified: May 22, 2020
Protocol Integer ID: 37409
Abstract
Protocol for optogenetically stimulating neuronal subtypes in murine colonic myenteric plexus.
Guidelines
This protocol applies to transgenic animals expressing the optogenetic protein channelrhodopsin-2 in colonic myenteric neurons.
Dissection and experiments are done in the dark under infrared illumination. A ventral midline incision is made and the whole colon is carefully excised into a Sylguard lined dissection dish containing oxygenated Krebs-ringer solution.
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).
After spontaneous colonic migrating motor complexes (CMMCs) are detected, blue light is shined continuously for 20s by a hand-held laser (450 nm Sapphire Galaxy 3; ZBolt; Happy Valley, OR) mounted on a clamp stand, positioned 30 cm above the preparation, or by a combination of 4 pairs of LEDs placed on either side of the colon, connected to a wire driven by an SD9 stimulator at 5Hz (20ms pulsewidth) for 5, 20 or 60 seconds.
After several control light stimulations, drugs are perfused and light stimulations are obtained again.