*2.4. The Lack of Sensitization of Presynaptic TRPA1 by PDBu in the Caudal NTS*

Application of phorbol-12,13-dibutyrate (PDBu, 1–5 μM, a PKC activator) alone induced a dose-dependent increase in the frequency, but not amplitude of mEPSCs in caudal NTS neurons (Figure 6A,B,D). The effects of PDBu on mEPSC frequency were significant: 1 μM, 117.84 ± 19.66%, *n* = 6; 3 μM, 195.76 ± 22.15%, *n* = 5, *p* < 0.01; 5 μM, 295.13 ± 28.57%, *n* = 6, *p* < 0.01 (Figure 6D), whereas the mEPSC amplitudes remained unaltered (Figure 6C), supporting a presynaptic mechanism of action of PDBu [42–45].

Following application of PDBu (3 μM) to the ACSF, AITC increased the mEPSC frequency, but not amplitude (AITC alone, 298.51 ± 21.86%; AITC + PDBu, 358.69 ± 42.58% (*n* = 9, *p* > 0.05)) (Figure 7A,B,D). The effects of PDBu and AITC on mEPSC frequency appear to be additive, not synergistic suggesting that PDBu and AITC employ two different and independent mechanisms of action in presyanptic terminals of caudal NTS neurons.

**Figure 6.** Modulation of synaptic transmission in the NTS by phorbol-12,13-dibutyrate (PDBu). (**A**) PDBu increases the frequency of mEPSCs in a reversible manner. Synaptic currents are shown in higher time resolution below. (**B**,**C**) Cumulative probability graphs showing enhanced frequency of mEPSCs (*p* < 0.001,KS test) in response to PDBu without change in their amplitudes. (**D**) Summary graphs showing that PDBu-mediated increase in mEPSCs (1 μM, 117.84 ± 19.66%, *n* = 6; 3 μM, 195.76 ± 30.15%, *n* = 5, \* *p* < 0.01; 5 μM, 295.13 ± 38.57%, *n* = 6, \* *p* < 0.01). The asterisk (\*) represents *p* < 0.05 as compared to control.

**Figure 7.** Interaction between AITC and PDBu in the NTS. (**A**) Application of AITC (200 μM) increases the frequency of mEPSCs and this action is enhanced by incubation in 3 μM PDBu. Synaptic events are shown at a higher time resolution below. (**B**) The cumulative probability plots show an AITC-mediated increase in the frequency of synaptic events. These effects are further significantly enhanced by PDBu. (**C**) The increase in frequency of events is not accompanied by a change in the amplitude. (**D**) Summary graphs showing AITC-induced an increase in mEPSC frequency (*n* = 9) and potentiation by PDBu (*n* = 8). The asterisk (\*) represents *p* < 0.05 as compared to control.
