Carbachol

The water-soluble tetrazolium-1 (WST-1) assay kit (Roche Diagnostics GmbH, Germany) was used to determine the viability of LIM-2405 and HT-29 cells. WST-1 is cleaved to form formazan dye via a complex cellular interaction at the cell surface. This interaction is contingent on the viable cells’ production of glycolytic nicotinamide adenine dinucleotide phosphate (NADPH). Hence, the amount of formed formazan dye correlates with the number of viable cells in the culture. LIM-2405 and HT-29 cells were seeded and cultured at 1 × 10⁴ cells per well in 96-well plates for 24 h (hrs). Cells were then treated with various concentrations of the general muscarinic receptor blocker, atropine (Sigma-Aldrich, Australia) for 1–48 h, selective M3R blocker, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) (Abcam, Australia), cholinergic agonist, carbachol (Abcam, Australia) and acetylcholinesterase inhibitor, donepezil (Abcam, Australia) for 8 h. All treatments were performed in triplicates, and three independent experiments were conducted. WST-1 reagent (10 µL) was added to each well and incubated at 37 °C for 1 h. Cellular proliferation at the absorbance of 450 nm was measured using a microplate reader (Varioskan Flash, Thermo Scientific, Australia).

Acute intact DRG preparations were prepared from GFAP-Cre::GCaMP6f mice. Briefly, vertebras and dura mater were removed and lumbar L4 and L5 DRGs were exposed and immediately covered with ice cold (slushy) incubation ACSF solution (95 mM NaCl, 1.9 mM KCl, 1.2 mM KH₂PO₄, 0.5 mM CaCl₂, 7 mM MgSO₄, 26 mM NaHCO₃, 15 mM glucose, 50 mM sucrose) and bubbled with 95% O₂ and 5% CO₂. DRGs were incubated for 30 min at 35°C in the incubation solution and then left to recover for 1 h 30 min at room temperature. A single DRG was placed in the recording chamber of a custom-built 2-photon laser-scanning microscope with a 20x water immersion objective (x20/0.95w XLMPlanFluor, Olympus). GCaMP6f was excited at 920 nm with a Ti:Sapphire laser (Mai Tai HP; Spectra-Physics). DRGs were continuously superfused at a rate of 4 ml/min with recording solution (127 mM NaCl, 1.9 mM KCl, 1.2 mM KH₂PO₄, 2.4 mM CaCl₂, 1.3 mM MgSO₄, 26 mM NaHCO₃, 15 mM glucose) and bubbled with 95% O2–5% CO2. To evoke intracellular Ca²⁺ elevations in DRG GCaMP6f-expressing cells, a cocktail of agonists to endogenous G_q GPCRs containing 50 μM DHPG (Abcam), 10 μM histamine (Sigma Aldrich), 10 μM carbachol (Abcam), and 50 μM ATP (Sigma Aldrich) was bath applied for 30 sec.