*2.3. Changes in mEPSCs in Response to Continuous and Repeated Application of AITC*

TRPA1 agonists AITC, NMM and MG have been shown to activate the channel by covalent modification of cysteine and lysine residues [34,40]. Although covalent modification is expected to be an irreversible process, within the time course of electrophysiological experiments, NMM and AITC activate TRPA1 in a reversible manner [34,41]. We found that brief (2–10 s) puffs of AITC (200 μM), NMM (100 μM) or MG (50 μM) induced responses that were readily reversible (Figures 2–4) and a continuous application of AITC decreased the frequency of mEPSCs over time (Figure 2A).

In a separate experiment, changes in mEPSC frequency were analyzed with continuous application of AITC (Figure 5A,B). The AITC-mediated facilitation of mEPSCs showed a gradual decrease with time (Figure 5B). A persistent depolarization of presynaptic terminal and/or Ca2+-dependent decrease in TRPA1 activity and/or desensitization of TRPA1 may be responsible for the observed run-down of synaptic activity.

To determine whether AITC causes tachyphylaxis, AITC (2 s duration) was repeatedly applied to the recorded caudal NTS neurons every 10 s separated by a washout. Successive applications of AITC (200 μM) gradually decreased the mEPSC frequency (first application, 657.53 ± 135.2%, *n* = 5; second application, 487.64 ± 122.57%, *n* = 5; third application, 374.85 ± 73.50% of control, *n* = 6) (Figure 5A,C,D). These data support that TRPA1-mediated transmitter release is decreased with repeated application of AITC. Thus, in all experiments with multiple applications of agonists, at least 3 min of washout was given after each agonist application to avoid desensitization.

**Figure 5.** The tachyphylaxis of TRPA1. (**A**) AITC (200 μM) causes enhancement of mEPSCs progressively decreases with repeated AITC application. Synaptic currents are shown in a higher time resolution below. (**B**) Summary graph representing a progressive decrease in the number of mEPSC events immediately following AITC application. (**C**) Cumulative probability plots showing decreased mEPSC frequency as indicated by a progressive increase in the inter-event intervals with each subsequent AITC application. (**D**) Summary graph representing a progressive decline in mEPSC frequency with repeated AITC application. A significant decrease in frequency is observed only between the first and third AITC application (first application, *n* = 5; third application, *n* = 5, *p* < 0.05) (first application, 357.53 ± 135.2%, *n* = 5; second application, 271.64 ± 122.57%, *n* = 5; third application, 203.85 ± 73.50% of control, *n* = 6). The asterisk (\*) represents *p* < 0.05 as compared to control.
