Distinct mechanosensitive activation of trigeminal nerve terminals in male and female mouse meninges
To assess possible involvement of two types of mechanosensitive channels in the activity of meningeal TG nerve fibres in male and female mice, first 50 mM KCl was applied for 10 min to measure overall excitability of the preparation. Then, after washout of KCl, sequentially 5 μM Yoda1 (as Piezo1 agonist), 50 μM PregS (as TRPM3 agonist) and 1 μM capsaicin (as agonist of TRPV1 receptors, typically expressed in nociceptors) were tested in the same preparation. Examples of the firing activity of the nerve fibres before and after application of Yoda1 and PregS in male and female meningeal hemiskull preparations are shown in Fig. 1 A, B. Application of Yoda1 led to a moderate induction of persistent firing - compared to the spike activity during the 10 min control recording period directly prior to drug application (Fig. 1A and C) - with no difference between males (from 346.9 ± 73.9 prior to 1367 ± 297.3 during Yoda1) and females (from 370.2 ± 97.5 prior to 1507 ± 338.4 during Yoda1; p = 0.76, Mann–Whitney U test). In contrast, PregS application led to profoundly increased firing in females, namely from 515.1 ± 143.1 to 5084 ± 815.4 (n = 7, p = 0.01, Wilcoxon signed-rank test), while in males the observed increase in firing during PregS (from 343.2 ± 61.31 prior to 1503 ± 112.9) was not significant (n = 5, p = 0.06, Wilcoxon signed-rank test; Fig. 1 B-D). Thus, in females, PregS induced a much stronger increase in spiking activity than in males (p = 0.0025, Mann–Whitney U test; Fig. 1 C, D). The amplitude of action potentials in basal conditions did not show sex difference (7.6 ± 0.5 a.u. in females; n = 7 vs. 6.6 ± 0.4 a.u. in males; n = 5. p = 0.14; Mann–Whitney U test). However, there was a sex difference in amplitude of action potentials during PregS application which were larger in females (13.8 ± 1.2 a.u. in females; n = 7 vs. 8.9 ± 0.4 a.u. in males; n = 5, p = 0.01; Mann–Whitney U test).
Lack of interaction of Piezo1 and TRPM3 channels during sequential activation
The presence of two types of mechanosensitive channels in meningeal afferents, as detected during their sequential activation by Piezo1 and TRPM3 agonists, raises the issue whether or not the two channels interact, and if so, whether they promote or depress effects of the other. A functional calcium-dependent interaction has been presented for Piezo and TRPV1 channels [11]. As Piezo1 and TRPM3 mechanosensitive channels are both calcium-permeable [11, 27], one can envisage that they may also be able to provide sensitisation when activated after each other. To test for a possible interaction between Piezo1 and TRPM3 channels, the sequence of agonist application was swapped in female mouse meningeal preparations (Fig. 2 A, B). Spiking activity was calculated as the ratio between the number of spikes during the 10 min with the compound present, and the number of spikes during the 10-min period directly before compound application (i.e. control), the latter taken as 100%. When PregS was applied after Yoda1, firing induced by activation of TRPM3 channels by PregS increased to 1917 ± 1037% from the control level (n = 7; Fig. 2 C). Applying PregS before Yoda1 increased firing induced by activation of TRPM3 channels by PregS to 2339 ± 1221% from the control level (n = 5; Fig. 2 C), which was similar (p = 0.63, Mann–Whitney U test) to the alternate order of application. When Yoda1 was applied before PregS, the firing induced by activation of Piezo1 channels by Yoda1 increased to 415 ± 129.5% (n = 7; Fig. 2 D), while it increased to 466.8 ± 124.8% (n = 5; Fig. 2 D) when Yoda1 was applied after PregS. Thus, also for Yoda1 the spike frequencies were similar (p = 0.53, Mann–Whitney U test) regardless of the order of application of the agonists. This indicates a lack of interaction (including sensitization) between TRPM3 and Piezo1 channels when sequentially activated in female mouse meninges.
Profound activation of trigeminal nerve terminals by CIM0216 in females
To confirm our finding that TRPM3 channels are expressed in meningeal afferents, we tested the potent, selective synthetic TRPM3 agonist, CIM0216 [27]. Following the KCl and Yoda1 application we now applied 5 μM CIM0216 instead of PregS. Figure 3 A, B show example traces of spiking activity in meningeal TG nerve fibres from male and female mice in the control condition, during application of 5 μM CIM0216, and after 1 μM capsaicin. Figure 3 C, D shows that 5 μM CIM0216 raised the number of spikes during a 2-min window from 115.8 ± 47.2 during the control condition to 638.3 ± 52.5 in males (n = 6, p = 0.03, Wilcoxon signed-rank test) and from 127.0 ± 50.6 to 1839.0 ± 259.4 in females (n = 6, p = 0.03, Wilcoxon signed-rank test). Hence, like PregS, CIM0216 induced profound firing activity in TG nerve terminals, especially in female mice (p = 0.0022, Mann–Whitney U test; Fig. 3 C, D). Note that following CIM0216 application, the response to capsaicin was strongly impaired (Fig. 3 A-C) in both males and females, as compared to that observed following PregS application (Fig. 1 C).
Selective sex-dependence of TRPM3-mediated nociceptive firing with no sex difference in general excitability, Piezo1-mediated activity or capsaicin sensitivity
One key question is whether the observed sex difference in TG nociceptive firing is exclusively seen with TRPM3 channel activation or whether it is also observed for other nociceptive receptors and/or may relate to non-specific differences in neuronal excitability. To this end, we directly compared the general excitability features (as assessed with KCl application), effects of Piezo1 receptors (as assessed by Yoda1 application), and the capsaicin-induced firing of TG nerve terminals from the experiments from males vs. females. Figure 4 A, B shows the contribution of sex to the overall level of nociceptive neuronal firing by comparing the level of baseline firing to that after application of 50 mM KCl, for both sexes. Notably, the mean number of spikes during a 10-min window at baseline was similar (p = 0.78, Mann–Whitney U test) between males and females, i.e. 492.5 ± 158.9 (n = 11) and 472.5 ± 120.2 (n = 13), respectively. Following KCl application, the mean number of spikes within short burst of activity during the 30-s active phase was also similar (p = 0.29, Mann–Whitney U test) for both sexes, i.e. 241.4 ± 38.6 (n = 11) in males and 295.0 ± 37.5 (n = 13) in females. For the effect of Yoda1, the combined data (expressed as the mean number of spikes per 10 min) from all experiments during Yoda1 application revealed no sex difference for mechanosensitive Piezo1 channels (Fig. 4 C), with the mean number of spikes in males (1367.0 ± 297.3 (n = 11)) and females (1507.0 ± 338.4 (n = 13)) being similar (p = 0.94, Mann–Whitney U test). In addition, the mean number of spikes within the 2-min active phase of application of 1 μM capsaicin in males (166.8 ± 30 (n = 5)) and females (200.4 ± 32.9 (n = 7)) was also similar (p = 0.53, Mann–Whitney U test; Fig. 4 D). Notice that in the last comparison, data with 1 μM capsaicin were taken only from experiments with prior PregS application, as application of 5 μM CIM0216 had an impact on subsequent compound applications. Therefore, a lack of a sex difference for Piezo1 and TRPV1 channels indicates a specific role of sex in the regulation of mechanosensitive TRPM3 channels.
Cluster analysis of nociceptive spiking activity induced by Yoda1, PregS and capsaicin
To assess whether the activation of mechanosensitive Piezo1 and TRPM3 channels varies for single nerve fibres or small groups of fibres (clusters), we used a clustering approach [13]. Figure 5 A shows examples of multiple-spike clusters of experiments from female and male mice under control conditions and during 50 μM PregS application, whereby each dot represents one spike and different colours indicate different spike clusters. The data reveal that, especially in females, the TRPM3 channel agonist PregS increased the number of spikes in each of the identified clusters and additionally ‘wakes up’ previously silent clusters that consisted of spikes with a relatively large positive and negative amplitude. One advantage of the clustering approach is that it is able to shed light on whether different nociceptors are co-expressed within the same fibre, i.e. one cluster. Figure 5 Ba-d and Ca-d show example recordings illustrating the activity of selected clusters, indicating that different combinations of the two mechanosensitive Piezo1 and TRPM3 channels may occur within one cluster (fibre), as well as revealing whether a fibre responds to activation of classical nociceptors by capsaicin or not. Our functional data indicate that the latter are typically expressed in fibres with small- and prolonged-amplitude of spikes (Fig. 5 Ba,c and Ca,c), a signature of C-fibres [28] that are typically responsive to capsaicin [13]. Based on the joint responsivity to Yoda1 and PregS, Piezo1 channels are co-expressed with TRPM3 in capsaicin-sensitive (Fig. 5 Ba and Ca) as well as capsaicin-insensitive fibres (Fig. 5 Bb and Cb). Notably, TRPM3 channels could be activated in most (98% in females vs. 80% in males) fibres (including fibres that were insensitive to Yoda1 and/or capsaicin, see Fig. 5 Bb-d or Cb-d). Notably, the application of PregS caused a massive and repetitive escalation of spike frequency in clusters that had been silent throughout the whole application in females (Fig. 5 Bd), while in males (Fig. 5 Cd) in the same type of clusters PregS induced short peaks in firing rate. Thus, the different shapes of recorded spikes correlated to different types of activated fibres, based on the distinct responses to Yoda1, PregS, and capsaicin. Figure 5 D and E summarizes the neurochemical response profile of different meningeal fibres (represented by the distinct spike clusters) in females and males. Interestingly, in females, up to 44% clusters (63 out of 143) were activated by PregS and Yoda1 (23%) or by PregS and Yoda1 as well as capsaicin (21%), whereas in males the number of such ‘supermechanosensitive’ fibres was only 24% (17 out of 70; including PregS and Yoda1 (7%); PregS, Yoda1 and capsaicin (17%)). In contrast, the profile of ‘only-PregS-sensitive’ (46% in females vs. 44% in males) or all capsaicin-sensitive (30% in females vs. 33% in males) fibres did not show sex-dependence. In summary, spike cluster analysis revealed a prevailing functional activity of mechanosensitive TRPM3 channels in individual meningeal nerve fibres in females and, typical for this sex, massive and repetitive escalation of spike frequency in all clusters following pharmacological activation of these channels.
Spectral analysis of the nociceptive spiking activity induced by TRPM3 activation
Finally, we characterized the temporal patterns of spike activity in meningeal nerves induced by the TRPM3 channel agonists. Our spectral analysis showed that application of either CIM0216 or PregS remarkably increased nociceptive firing in the range of very brief interspike intervals (ISI), especially in females (examples of original traces in Fig. 6 A and B and averaged data in Fig. 6 C and D). Firing corresponding to the spiking activity in δ-range (1–4 Hz) was prevalent in the majority of fibres during control conditions with no difference between males (88 ± 5.9% of fibres, n = 6) and females (87.7 ± 5.6% of fibres, n = 6). In contrast, after application of 5 μM CIM0216, we observed additional spiking activity in the θ- and α-ranges (4–8 Hz and 8–13 Hz, respectively) in both sexes with a predominance in females, as well as β-range of spiking activity (13–20 Hz) only in females (Fig. 6 E, H). One can envisage that high-frequency firing induced by TRPM3 agonists is supposed to provide the temporal summation of nociceptive signalling at the level of second order nociceptive neurons in the brainstem [13, 25], thus facilitating nociceptive traffic to higher pain centers in the brain.