Axon clampfit

Functional data were analyzed using Axon Clampfit (Molecular Devices), GraphPad Prism 8 (GraphPad Software Inc.), or Excel (Microsoft Corp., Redmond, WA, USA) and presented as mean ± standard error (SE) for the specified number of samples (N). Statistical significance was determined using two-tailed t-tests, one-way ANOVA, or two-way ANOVA as appropriate. P-value ≤0.05 was considered statistically significant.

At least four weeks after viral injections, slices were prepared as previously reported using coronal brainstem slices in aCSF of a composition matching previous reports^{35 (link),48 (link)}. Coronal slices (300 µm) containing the DVC were prepared and transferred to a holding chamber oxygenated (95%O₂/5%CO₂) at 32–34 °C. On cell or whole-cell patch-clamp recordings were performed using glass pipettes (2–5 MΩ). Internal recording solution composition matched previous reports^{35 (link),48 (link)}. Action potentials were recorded at resting membrane potential in current-clamp mode. DMV cells were voltage-clamped at a holding potential of 0 mV to record IPSCs. Kynurenic acid (KYN; 1 mM) was added to perfusate for recordings in the DMV to block synaptic currents mediated by ionotropic glutamate receptors and isolate GABAergic currents; bicuculline methiodide (BIC; 30 µM) was used to block GABA_A receptor-mediated currents.
All recordings were low-pass filtered at 3 kHz and acquired digitally at 20 kHz using pClamp acquisition software (Axon Clampfit; Molecular Devices, San Jose, CA). Mini-analysis (Synaptosoft, Decatur, GA) was used to measure action potential firing and IPSC frequency and amplitude.