*2.2. Modulation of Synaptic Transmission by AITC in the Caudal NTS*

To determine the functional characteristics of TRPA1 expressed in pre-synaptic terminals that synapse onto caudal NTS neurons, we used acute horizontal brainstem slices and patch-clamp electrophysiology. In voltage-clamp experiments, visualized caudal NTS neurons were held at −60 mV and synaptic currents were recorded at various experimental conditions. The expression of functional TRPA1 was confirmed using TRPA1 agonist, allyl isothiocyanate (i.e., AITC). Focal pressure puffs of AITC applied to recording caudal NTS neurons robustly increased the frequency of spontaneous excitatory synaptic currents (sEPSCs) (data not shown). To determine the effects of AITC on miniature excitatory synaptic activity (mEPSCs), patch-clamp recordings were conducted in the presence of 1 μM tetrodotoxin (TTX) to inhibit voltage-gated Na<sup>+</sup> channels and prevent action potential-dependent synaptic events. In these experiments, application of AITC (200 μM) significantly increased the frequency of mEPSCs in a pulse duration-/concentration-dependent manner (Figure 2).

**Figure 2.** Modulation of synaptic transmission in the NTS by allyl isothiocyanate (AITC). (**A**) Application of AITC (200 μM) increases the frequency of miniature excitatory synaptic activity (mEPSCs) in a reversible manner. The synaptic events are shown in higher time resolution below. (**B**) Cumulative probability plot showing decreased inter-event intervals representing increased frequency of mEPSCs (*p* < 0.0001, KS test). (**C**) The increase in frequency is not accompanied by a change in the amplitude. (**D**) Summary graph showing AITC-mediated increases in the frequency of mEPSCs in a dose-dependent manner (\* *p* < 0.05). Furthermore, the increase in AITC -induced synaptic events are blocked by 300 μM HC030031 (*n* = 5, \* *p* < 0.05). The asterisk (\*) represents *p* < 0.05 as compared to control.

The increase in mEPSC frequency is expressed as a percentage of control (i.e., no drugs applied): 500 ms, 230.25 ± 20.41% (*n* = 8, *p* < 0.05): 2 s, 450.63 ± 22.74% (*n* = 13, *p* < 0.05); 30 s, 610.79 ± 30.81% (*n* = 9, *p* < 0.05) (Figure 2A,B,D). The means mEPSC amplitude did not significantly change (control, 19.53 ± 2.53 pA, *n* = 5; after AITC, 21.2 ± 3.09 pA, *n* = 12) (Figure 2C). The increase in the mEPSC

frequency, but not the amplitude suggests that AITC acts only pre-synaptically, which is consistent with the pre-synaptic expression of TRPA1 (Figure 1). These potentiating effects of AITC were observed in ~40% (*n* = 143/358) of tested caudal NTS neurons. These results suggest that only neurons that received primary sensory afferent input responded by increasing the frequency of mEPSCs following AITC application.

mEPSCs were completely blocked by 6,7-Dinitroquinoxaline-2,3-dione (i.e., DNQX; 16 μM), a selective antagonist of AMPA receptors (Figure 2D). The involvement of TRPA1 was confirmed using HC030031 (i.e., HC), a TRPA1 selective antagonist. HC (300 μM) abolished the effects of AITC on mEPSC frequency without affecting the background synaptic activity (Figure 2D).

Similar results were obtained in experiments where two other TRPA1 agonists (i.e., N-methylmaleimide (i.e., NMM), an oxidizing agent that forms a covalent bond with TRPA1 and methylglyoxal (i.e., MG), a reactive molecule and an endogenous TRPA1 agonist, produced during hyperglycemia) were used. Pressure puffs of NMM or MG increased the frequency, but not amplitude of mEPSCs in caudal NTS neurons (Figures 3 and 4): 100 μM NMM (421.61 ± 24.09%, *n* = 8, *p* < 0.05; Figure 3B,D) and 50 μM MG (334.76 ± 30.62% *n* = 7, *p* < 0.05; Figure 4B,D).

By contrast, the somatodendritic responsiveness between NTS neurons to AITC, NMM and MG has not been detected in this study. Together with the molecular biological results (Figure 1), these data (Figures 2–4) support a strictly pre-synaptic expression of TRPA1 either in the primary afferent (solitary) terminals and/or pre-synaptic glutamatergic terminals of second- or higher-order NTS neurons.

**Figure 3.** Modulation of synaptic transmission in the NTS by N-methylmaleimide (NMM). (**A**) Application of NMM (100 μM) increases the frequency of mEPSCs in a reversible manner. The synaptic events are shown in higher time resolution below. (**B**) Cumulative probability plot showing decreased inter-event intervals representing increased frequency of mEPSCs (*p* < 0.0001, KS test). (**C**) The increase in frequency is not accompanied by a change in the amplitude. (**D**) Summary graphs showing that the NMM-mediated increase in mEPSCs (*n* = 8, \* *p* < 0.05). The asterisk (\*) represents *p* < 0.05 as compared to control.

**Figure 4.** Modulation of synaptic transmission in the NTS by methylglyoxal (MG). (**A**) Application of MG (50 μM) increases the frequency of mEPSCs in a reversible manner. The synaptic events are shown in higher time resolution below. (**B**) Cumulative probability plot showing decreased interevent intervals representing increased frequency of mEPSCs (*p* < 0.0001, KS test). (**C**) The increase in frequency is not accompanied by a change in the amplitude. (**D**) Summary graphs showing that MG-mediated increase in mEPSCs (*n* = 7, \* *p* < 0.05). The asterisk (\*) represents *p* < 0.05 as compared to control.
