**3. Discussion**

In this study, the molecular biological and electrophysiological techniques were used to demonstrate TRPA1-mediated modulation of synaptic transmission at the NTS and the expression of TRPA1 in caudal NTS is strictly presynaptic. Together with the existing literature, these results support the exclusive expression of TRPA1 in the first order sensory neurons and indicate that the expression of TRPA1 in the caudal NTS is restricted to the primary afferent solitary tract terminals [30,35,36,47]. Activation of presynaptic TRPA1 by selective agonists was detected by its facilitating and modulatory effects on spontaneous and evoked glutamatergic synaptic transmission, respectively, recorded electrophysiologically in acute horizontal brainstem slices; while the specificity of TRPA1 in triggering synaptic glutamate release was confirmed in TRPA1 knockout mice. The effects of TRPA1 activation on glutamatergic synaptic transmission in the caudal NTS are consistent with those observed previously in other central sensory nuclei: the substantia gelatinosa and the caudal spinal trigeminal nucleus [30,48–53]. Agonists of TRPA1 (AITC) and TRPV1 (capsaicin; i.e., CAP) caused paired pulse depression. However, AITC did not depress evoked EPSC amplitude, while capsaicin did depress the evoked EPSC amplitude as described previously [45]. This observation is curious because

TRPA1 and TRPV1 channels are often co-expressed in the same subset of neurons [30] and their effects on synaptic neurotransmitter release are expected to be comparable. Differences in the sensitivity of presynaptic terminals to AITC and capsaicin, as well as the exact channel distribution within presynaptic terminals may be responsible for the apparent dichotomy of TRPA1- and TRPV1-mediated evoked presynaptic responses, respectively.

The functional role of TRPA1 in sensory signaling is suggested by the expression of these channels in subsets of primary sensory neurons in the trigeminal, jugular, geniculate, nodose and dorsal root ganglia [43,54–56]. The TRPA1 channels act as primary sensors for thermal (cold), mechanical and inflammatory noxious stimuli generated by environmental and endogenous agents [30–32,43,57–62]. Because TRPA1 channels are critical for transmission of visceral and inflammatory pain [21–27], the expression of functional presynaptic TRPA1 in primary afferents within the caudal NTS suggests a potential for TRPA1-dependent central component of nociception and its sensitization by TRPA1 agents at the level of the caudal brainstem.

Visceral pain is a common symptom of functional gastrointestinal disorder such as irritable bowel syndrome, ulcerative colitis and dyspepsia. As visceral structures are highly sensitive to distention, ischemia and inflammation, the features of chronic visceral pain are inflammatory and mechanical hyperalgesia and allodynia. Adopting strategies to reduce inflammatory and mechanosensory transduction may be particularly useful in relieving visceral pain [63–66]. The role of TRPA1 in gastrointestinal inflammatory disorders is becoming increasingly important as TRPA1 up-regulation has been confirmed in several disease model systems. Thus, TRPA1 may represent a useful target in treatments of chronic visceral pain. In fact, several TRPA1 antagonists have already entered clinical trials including one in phase II clinical trial [66].

A potential role of protein kinase C (PKC) in modulating TRPA1-mediated synaptic transmission was examined using PDBu, a PKC activator. The PKC-mediated phosphorylation plays an important role in modulation of synaptic neurotransmission [40,43–45,67,68]. For example, PDBu substantially increased the amplitude of TRPV1-mediated currents but had no effect on TRPA1-mediated currents in dorsal root ganglia neurons [41,69]. The direct PDBu-mediated facilitation of the frequency of synaptic events was observed in all neurons tested in this study, even in those cases where AITC failed to facilitate synaptic release. However, the effects of AITC and PDBu were simply additive (i.e., not synergistic) suggesting that AITC and PDBu likely employ independent pathways and thus, the TRPA1-dependent machinery is unlikely to involve PKC-mediated phosphorylation. This finding may reflect the nature of agonists used. AITC and NMM activate TRPA1 by covalent modification of cysteine residues. As a result, the affinity of these ligands for TRPA1 binding site(s) may not be altered by phosphorylation. In that event, the TRPA1 activity may still be susceptible to modulation by non-covalent modifying agonists and physical inputs, such as thermal (cold) and mechanical stimuli.

Taken together, the results of this study provide the first evidence of the TRPA1-dependent modulation of the primary afferent inputs to the caudal NTS and suggest that second order caudal NTS neurons serve as a TRPA1-dependent interface for visceral noxious-innocuous integration at the level of the caudal brainstem. Thus, TRPA1 may represent a useful target in treatments of chronic visceral and inflammatory pain.

## **4. Materials and Methods**

#### *4.1. Animals*

Young adult male Sprague-Dawley rats (P30-50) and TRPA1 knockout mice (Jackson Laboratories, Bar Harbor, ME, USA) were used in accordance with the Guide for the Care and Use of Laboratory Animals (NIH 865-23, Bethesda, MD, USA), and was approved by the Animal Care and Use Committee of Southern Illinois University (A3209-01; Approval date: 7/25/2013).
