TITLE: Chemical colorectal stimuli for GCaMP6f characterization 
AUTHORS: Bin Feng

[DESCRIPTION]
By adopting our developed high-throughput optical recording system, a large scale of dorsal root ganglia (DRG) neurons were recorded and identified from a whole DRG. Based on this custom-built platform, we functionally characterize numerous chemosensitive colorectal DRG neurons and plot their topological distribution on the DRG. This protocol includes the steps for tissue preparation (colorectum, pelvic nerve, lumbar splanchnic nerve, thoracolumbar, and lumbosacral DRG), optical signal acquisition (GCaMP signals), and processing. Following this protocol, responses of colorectal DRG neurons to chemical stimuli were compared between zymosan-treated and control groups.

[STEPS]
SECTION: Tissue preparation for GCaMP recording
1. Mice (VGU/GCaMP) 8-14 weeks of age were deeply anesthetized by intraperitoneal and intramuscular injection of a 0.4 mL cocktail of ketamine (120 mg/kg) and xylazine (10 mg/kg).
SECTION: Tissue preparation for GCaMP recording
2. Mice were then euthanized by perfusion from the left ventricle with modified ice-cold Krebs solution.

Krebs Solution Composition:
- 6L DI Water
- 2.15g Sodium phosphate dihydrate monobasic (NaH2PO4-2H2O)
- 1.35g Potassium Chloride (KCl)
- 12.0g Sodium Bicarbonate (NaHCO3)
- 43.15g Sodium Chloride (NaCl)
- 12mL Calcium Chloride (CaCl2, 1.0M)
- 12mL Magnesium Sulfate (MgSO4, 480mM)
- 12g D-glucose
- pH 7.2-7.3
- Osmolarity 290-320
SECTION: Tissue preparation for GCaMP recording
3. A dorsal laminectomy was performed to expose the thoracic and lumbar spinal cord.
SECTION: Tissue preparation for GCaMP recording
4. The colorectum with attached pelvic and lumbar splanchnic nerves and DRGs were carefully dissected via blunt dissection.
SECTION: Tissue preparation for GCaMP recording
5. The tissue was then transferred to a recording chamber perfused with 32-34 degree Krebs solution (same composition as step 2) bubbled with carbogen (95% O2, 5% CO2).
SECTION: GCaMP signals recording
6. Turn on the light source and set up the microscope settings. An upright microscope platform was used to capture one whole DRG with a water immersed 10x objective using a halogen epi-illumination light source.
SECTION: GCaMP signals recording
7. Turn on the camera. The video was captured using a high-speed sCMOS camera. The resolution of video is 1920x1920. The sampling speed is 65 frames per second. Spatial resolution is 1.6 micrometer.
SECTION: GCaMP signals recording
8. Open the softwares that are used for the experiment. The software ImageJ was used to capture the video. A Matlab GUI software was used to control the custom-built system to distend the colorectum for mechanical stimulation.
SECTION: GCaMP signals recording
9. Distend the colorectum. The cannulated colorectum was distended by four ascending hydrostatic pressures (15, 30, 45, 60 mmHg). Each pressure lasts for 5 sec.
SECTION: GCaMP signals recording
10. Stimulate colorectum by shear flow. The colorectum was then flushed by a shear flow (20-30 mL/min) lasting for 23 sec.
SECTION: GCaMP signals recording
11. Introduce the acidic hypertonic solution (AHS) to the interior colon and focus on a single DRG segment, recording neural activity initially (0 minutes) and then once every 5 minutes up to 15 minutes.

AHS is composed of Krebs solution with:
- Additional sodium phosphate dihydrate monobasic until pH is approximately 6.0 (i.e. Acidic Krebs).
- Addition of D-Mannitol until osmolarity is approximately 800.
SECTION: GCaMP signals recording
12. Distend the colorectum and stimulate by shear flow.
SECTION: GCaMP signals recording
13. Remove all AHS from the interior colon and flush with water every 2 minutes for 10 minutes.
SECTION: GCaMP signals recording
14. Introduce the chemical cocktail IS to the interior colon and focus on a single DRG segment, recording neural activity initially (0 minutes) and then once every 5 minutes up to 15 minutes.
SECTION: GCaMP signals recording
15. Distend the colorectum and stimulate by shear flow.
SECTION: Image analysis and functional characterization of mechanosensitive neurons
16. Align the image stack using ImageJ to remove the spatial movement of the DRG.
SECTION: Image analysis and functional characterization of mechanosensitive neurons
17. Extract the potential active neurons based on intensity fluctuation across different frames.
SECTION: Image analysis and functional characterization of mechanosensitive neurons
18. Locate the neurons using marker-based watershed segmentation algorithm.
SECTION: Image analysis and functional characterization of mechanosensitive neurons
19. Active neurons are classified into four classes based on their response profiles. Low-threshold (LT) muscular neurons are activated by all pressures. High-threshold (HT) muscular neurons are evoked by pressures no lower than 30 mmHg. Mucosal neurons are exclusively activated by shear flow, and muscular-mucosal type is responsive to both distension and shear flow.
SECTION: Image analysis and functional characterization of mechanosensitive neurons
20. The topological distribution of responsive neurons is plot using Matlab.