Devazepide

Each mouse was anaesthetized with ketamine (100 mg kg⁻¹) and xylazine (10 mg kg⁻¹). The mice were tracheotomized, and the nodose ganglion was exposed for imaging exactly as previously described^{4 (link)}.
For CCKAR blocker experiments, devazepide (Sigma) was dissolved in DMSO and diluted to a final dose of 4 mg kg⁻¹ in saline^{11 (link)}. For glutamate receptor blocker experiments, a mixture of metabotropic glutamate receptor antagonist AP3 (2 mg kg⁻¹) and ionotropic glutamate receptor antagonist kynurenic acid (300 μg kg⁻¹) was used. CCKAR and glutamate receptor blockers were delivered both into the intestines and abdominal cavity^{11 (link)}; after a 5 min incubation period, the imaging session was started. For CCK application, the intestines, still attached to the anaesthesized mouse, were partly placed on a 25 mm petri dish to allow delivery (60 s) and washout (>180 s) of the stimuli (1 μg ml⁻¹ CCK peptide; Bachem 4033101).
Note that for nodose imaging experiments using sugar, glucose stimuli consisted of 10 s pulses since stimulating with high concentration (>250 mM) for long pulses (60 s or more) strongly activates nutrient-independent vagal responses^{4 (link),22 (link),58 (link)}, severely masking sugar/nutrient-evoked responses.

The following neurotransmitter/neuropeptide receptor blockers were used: CCK-A receptor antagonist devazepide (2 mg kg^–1 in 5% DMSO PBS; Sigma)^{14 (link),53 (link)}, cocktail of the ionotropic glutamate receptor antagonist KA (150 μg kg^–1 in PBS, stock made in 1 M NaOH then diluted, pH 7.4; Sigma)^{14 (link),54 (link)} and the metabotropic glutamate receptor antagonist AP3 (1 mg kg^–1 in PBS, stock made in 1 M NaOH diluted, pH 7.4; Sigma)^{14 (link),55 (link)} and non-selective P2-purinoreceptor antagonist PPADs (25 mg kg^–1 in PBS; Sigma)^{32 (link),56 (link)–58 (link)}. Following recording of a preinhibitor response, one inhibitor was delivered over 1 min (devazepide and PPADs were delivered at 10 μl g^–1; the KA/AP3 cocktail was delivered at 20 μl g^–1). Infusion of the selected sugar ligand was repeated for postinhibitor recording after an incubation period of 5–8 min for devazepide and 3–5 min for KA/AP3 and PPADs.

Antagonists were delivered 30 min before optical activation of LPB^CCK neurons. For blocking glutamatergic neurotransmission, we firstly connected guiding cannula with a Hamilton syringe via a polyethylene tube. Then we infused 0.25 μl mixed working solution containing CNQX disodium salt hydrate (0.015 μg; Sigma-Aldrich), a glutamate AMPA receptor antagonist, and AP5 (0.03 μg; Sigma-Aldrich), a NMDA receptor antagonist, into the PVN with a manual microinfusion pump (RWD, 68606) over 5 min. For blocking CCKergic neurotransmission, 0.25 μl mixed solution containing Devazepide (0.0625 μg; Sigma-Aldrich), a Cckar receptor antagonist, and L-365,260 (0.0625 μg; Sigma-Aldrich), a Cckbr receptor antagonist, was infused into the PVN over a period of 5 min. To prevent backflow of fluid, the fluid-delivery cannula was left for 10 min after infusion.