*2.1. Presynaptic Expression of TRPA1 in the Caudal NTS*

The existing literature indicates that sensory TRP channels are predominantly expressed in peripheral neurons and their expression in central neurons is limited [38,39]. To determine whether this rule applies to TRPA1 in the NTS, we used RT-PCR and immunohistochemistry. The TRPA1 mRNA was not detected in caudal NTS neurons while its presence was clearly detected in DRG neurons that served as a positive control (Figure 1A). Immunofluorescent staining then revealed that TRPA1 in the caudal NTS are selectively expressed only in nerve fibers i.e., pre-synaptically (Figure 1B,C), which is consistent with the expression of TRPA1 in the solitary tract fibers, but not the somatic expression in the second order caudal NTS neurons. However, these results alone do not rule out the expression of TRPA1 in non-solitary tract terminals and will be further confirmed in conjunction with our electrophysiological data (see Figures 2–4).

**Figure 1.** The lack of expression of TRPA1 in the nucleus of the solitary tract (NTS). (**A**) Expression of TRPA1 mRNA in sensory neurons but not in the NTS. (**B**,**C**) Immunostaining of TRPA1 (green), DAPI (blue) in the NTS. The TRPA1 staining was detected abundantly on the solitary tract, but not within the NTS. DRG: dorsal root ganglion.
