*4.2. Preparation of Brainstem Slices*

Brains were removed and placed in an ice-cold oxygenated solution of the following composition (in mM): sucrose 250, KCl 1.5, NaH2PO4 2.23, MgCl2 5, CaCl2 0.5, NaHCO3 26, glucose 10 (pH 7.4) and bubbled with carbogen (95% O2 and 5% CO2). The brainstem was isolated and transferred to the cutting chamber of Vibratome-1000+ slicer (Leica Microsystems, Wetzlar, Germany) where horizontal brainstem slices (250–300 μm thickness) containing the NTS were prepared. The slices were then transferred to a storage chamber and incubated at 30 ◦C for ~60 min in an oxygenated artificial cerebrospinal fluid (ACSF) of the following composition (in mM): NaCl 125, KCl 1.5, NaH2PO4 2.23, MgCl2 1, CaCl2 2, NaHCO3 26, glucose 10 (pH 7.4). Slices were then stored in the identical oxygenated ACSF at 24 ◦C for up to 8 h.

#### *4.3. Electrophysiological Patch-Clamp Recordings*

For patch-clamp recordings, brainstem slices were placed in the recording chamber and perfused with oxygenated ACSF at a rate of 1 mL/min using a 2232 Microperpex S peristaltic pump (LK.B, Upsalla, Sweden). Slices were secured using a nylon mesh attached to platinum ring insert. Whole-cell recordings were conducted at room temperature. The patch electrode solution contained (in mM): K-gluconate 140, NaCl 1, MgCl2 2, Mg-ATP 2, Na-GTP 0.3, HEPES 10, KOH 0.42 (pH 7.4). Membrane voltages were not corrected for the liquid junction potential which was calculated using software available through pCamp: VLJ = 16.2 mV. The electrophysiological data were recorded using MultiClamp-700B patch-clamp amplifier (Molecular Devices, Sunyvale, CA, USA). The seal resistance was >2 GΩ. The access resistance was <30 MΩ and was not compensated. Patches with access resistances >30 MΩ were corrected by applying additional negative suction or discarded. Input resistance and series resistance were measured every five minutes and cells showing greater that 20% change in series resistance were not included in analysis. Data were sampled at 10–20 kHz and filtered at 5 kHz. The final drug concentration in the bath was calculated based on the known concentration of the stock solution and adjustable rates of pumps [70]. A picospritzer (Parker Hannifin Instrumentation, Cleveland, OH, USA) was used for agonist applications via pipettes (4–7 MΩ) identical to those used for patch clamp recordings. Agonist application was standardized by positioning the tip of the application pipette 15 μm from the recorded neuron. This distance was calibrated and marked on the TV monitor and used for visualization of neurons while patching. Off-line data analysis was done with the program Clampfit 9 (Molecular Devices, Sunnyvale, CA, USA). To obtain evoked EPSCs, a Grass Stimulator (S88) with stimulus isolation unit with constant current output (Grass Instrument, Quincy, MA, USA) was used to stimulate a concentric bipolar electrode (Rhodes Medical Instruments, Tujunga, CA, USA) placed on the solitary tract. The following parameters of electrical stimulation were used: stimulus duration, 50–400 μs; stimulus intensity,100–500 μA; inter-stimulus interval, 60 s.
