1. Activation of TRPA1 produced inward currents in the ACC
First, using whole-cell patch-clamp recordings from brain coronal slices, we examined if pharmacological activations of TRPA1 by CA could change the baseline currents in pyramidal neurons of layer II/III in the ACC of adult mice (Fig. 1A, B). Holding membrane potentials at -70 mV, we recorded sEPSCs. Three minutes after the stable recording, CA (300 mM) was applied for 1 minute in the bath solution 21. The application of CA led to inward currents (CA: -4.5 ± 0.3 pA, n = 7 neurons/7 mice, Fig. 1C). Subsequently, the CA-induced inward currents were inhibited in the presence of a TRPA1 antagonist, HC030031 (HC, 50 mM) (CA+HC; -0.74 ± 0.2 pA, n = 7 neurons/7 mice, Fig. 1D). Therefore, the acute application of CA-induced inward currents was mediated by TRPA1 (paired t-test, P<0.05, Fig. 1E). These results suggest that the acute generation of the inward currents were mediated by activations of TRPA1.
2. Activation of TRPA1 did not affect synaptic transmissions
Next, we tested if the stimulation of TRPA1 by CA could alter synaptic transmissions on both spontaneous excitatory and inhibitory synapses in the ACC (Fig. 2). Since sEPSCs were completely abolished by the AMPA/GluK antagonist (CNQX) sEPSCs must be mediated via AMPA/GluK receptors 22. We analyzed the electrophysiological property of sEPSCs before and after application of CA (n = 7 neurons/7 mice). The average amplitudes of sEPSCs were not changed between CA- and CA+ groups (paired t-test, P>0.05, Fig. 2B). The average frequency of sEPSCs was also unaffected between the two groups (paired t-test, P>0.05, Fig. 2C). In addition, we analyzed the electrophysiological property of sEPSCs including the rise time, decay time and area of the current curve. There was no difference in any of the parameters between the two groups (paired t-test, P>0.05, Fig. 2D-F).
We also analyzed if CA could alter the electrophysiological property of sIPSCs. Since the sIPSCs were completely abolished by the GABAA receptor antagonist (picrotoxin), sIPSCs were mediated through GABAA receptors 22. We analyzed the property of sIPSCs before and after CA (n = 6 neurons/6 mice, Fig 2G). The average amplitude of sIPSCs remained unchanged between the groups (paired t-test, P>0.05, Fig. 2H). The average frequency of sIPSCs was not altered between the groups (paired t-test, P>0.05, Fig. 2H). Furthermore, we analyzed rise time, decay time, and area of sIPSCs. Rise time, decay time and area were not altered (paired t-test, P>0.05, Fig. 2H-L). These results indicate that CA-induced activation of TRPA1 may not be mediated through neither excitatory nor inhibitory synaptic transmission in the layer II/III pyramidal neurons from the ACC.
3. Effect of TRPA1 on the production of APs and RMPs.
In order to analyze the roles of TRPA1 on neural activity, we examined if stimulating TRPA1 by CA could change the production of APs and RMPs (Fig. 3). The APs were generated by current injections (300 ms duration, from 0 to 500 pA, +20 pA steps, Fig. 3A). The frequency and shape of APs as well as RMPs were compared between the CA- and CA+ groups (n = 9 neurons/9 mice, Fig 3). The activation of TRPA1 changed generations and shapes of APs at injections of high current intensities of 400 pA (Fig. 3A-D). The activation of TRPA1 by CA decreased the frequency of APs (Two-way-repeated measure ANOVA, P<0.05, Fig. 3E). Conversely, there was no significant difference observed when comparing the stimulated (CA+) TRPA1 RMP to the non-stimulated (CA-) TRPA1 RMP (Fig. 3F).
Next, we analyzed whether CA stimulation could change the shapes of APs. We examined the first and last APs generated at high current injections (400 pA). The average threshold of the last APs was raised after CA stimulation, when compared to the non-stimulated TRPA1 (paired t-test, P<0.05, Fig. 3G). Furthermore, the average peak amplitude of the last AP was suppressed following CA stimulation (Last AP: CA-: 65.1 ± 2.3 mV; CA+: 54.5 ± 3.5 mV, paired t-test, P<0.05, Fig. 3H). The average rise time of last AP was delayed after CA stimulation (Last AP: CA-: 7.6 ± 0.1 ms; CA+: 8.1 ± 0.2 ms, paired t-test, P<0.05, Fig. 3I). Additionally, the average decay time of the last AP was delayed (Last AP: CA-: 2.8 ± 0.2 ms; CA+: 3.2 ± 0.2 ms, paired t-test, P<0.05, Fig. 3J). However, there was no difference in the average area of APs when comparing the stimulated CA+ TRPA1 to the non-stimulated CA- TRPA1 (paired t-test, P>0.05, Fig. 3L). The average halfwidth of last APs was wider after CA stimulation (paired t-test, P<0.05, Fig. 3K). These results suggest that stimulation of TRPA1 influences the production of APs and their electrophysiological property in the ACC.
4. Roles of TRPA1 and KATP channels on N2-induced acute hypoxia
Finally, we studied the functional role of TRPA1 in the ACC (Fig. 4). Hypoxia is a condition in which O2 is not available in sufficient amounts at the tissue level to maintain adequate homeostasis 23. To produce acute hypoxia, we replaced 95% O2 gas with 95% N2 in the bath solution 24. 95% N2 gas produced biphasic effects in the ACC (Fig. 4A). In the early phase, N2 produced inward currents (-18.4 ± 4.7 pA, 16 neurons/15 mice). In the late phase, N2 caused outward currents (30.3 ± 11.1 pA, Fig. 4A). In the presence of a TRPA1 antagonist (HC030031), we replaced O2 with N2 and found that HC030031 inhibited inward currents in the early phase (-7.9 ± 1.2 pA, one-way ANOVA, P<0.05 compared with N2, n = 15 neurons/12 mice), but had no effect on the late phase response (8.9 ± 6.3 pA, Fig. 4B, D). Furthermore, we explored the mechanism of outward currents in the late phase (Fig. 4C). Since O2 helps the generation of ATP, it is possible that KATP channels may be related to the outward currents 24. A KATP channel blocker, Glibenclamide (10 mM) completely blocked outward current in the late phase (-13.0 ± 5.5 pA, one-way ANOVA, P<0.05 compared with N2, n = 9 neurons/8 mice, Fig 4C, E).
These results suggest that TRPA1 plays a role in N2-induced inward response in the early phase and that the KATP channel is critical for the outward currents in the late phase under acute hypoxia conditions (Fig. 5).