*4.5. Total RNA Extraction and RT-PCR*

Total RNA was extracted by Trizol reagent (Invitrogen Co., Carlsbad, CA, USA) from nucleus of the solitary tract (NTS) and dorsal root ganglion (DRG). cDNAs were prepared by reverse transcription. PCR was performed using a standard approach and PCR green master mix (Promega Corporation, Madison, WI, USA). The PCR products were electrophoresed in 1.5% agarose gel with ethidium bromide in Tris/Borate/EDTA buffer. The gel was scanned using Versa Doc imaging system (Bio-Rad, Hercules, CA, USA) and the blot band density was quantified by Quantity One (Bio-Rad, Hercules, CA, USA).

#### *4.6. Data Analysis*

All data are shown as means ± SEM. Significance is tested using unpaired Student's *t*-test, and the data were considered significant at *p* < 0.05. For analysis of synaptic currents, Kolmogorov-Smirnov (KS) test was used to compare the cumulative probability plots for inter-event intervals and amplitude between various treatment groups. Data are represented as means ± SE and expressed as percentage of control, which is scaled to 100%.

The spontaneous/miniature postsynaptic currents (s/mPSCs) were analyzed off-line using MiniAnalysis 6.0.3 (Synaptosoft Inc., Fort Lee, NJ, USA) and the threshold for event detection (usually 10 pA) was at least three times baseline noise levels.

All the chemicals used in this study were obtained from Sigma (St. Louis, MO, USA).

**Author Contributions:** Conceptualization, L.S.P. and L.F.; methodology, V.V.U. and L.F.; formal analysis, L.F. and V.V.U; investigation, L.F.; resources, L.S.P.; data curation, L.F.; writing—original draft preparation, L.F. and V.V.U., L.S.P.; writing—review and editing, L.F., V.V.U. and L.S.P.; project administration, L.S.P.; funding acquisition, L.S.P.

**Funding:** This work was supported with a grant from National Institutes of Health (DA028017 to L.S.P).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
