Piezo2 Expression in Mechanosensitive Dental Primary Afferent Neurons

Abstract

Mechanosensitive ion channels have been suggested to be expressed in dental primary afferent (DPA) neurons to transduce the movement of dentinal fluid since the proposal of hydrodynamic theory. Piezo2, a mechanosensitive, rapidly inactivating (RI) ion channel, has been recently identified in dorsal root ganglion (DRG) neurons to mediate tactile transduction. Here, we examined the expression of Piezo2 in DPA neurons by in situ hybridization, single-cell reverse transcriptase polymerase chain reaction, and whole-cell patch-clamp recordings. DPA neurons with Piezo2 messenger RNA (mRNA) or Piezo2-like currents were further characterized based on their neurochemical and electrophysiological properties. Piezo2 mRNA was found mostly in medium- to large-sized DPA neurons, with the majority of these neurons also positive for Nav1.8, CGRP, and NF200, whereas only a minor population was positive for IB₄ and peripherin. Whole-cell patch-clamp recordings revealed Piezo2-like, RI currents evoked by mechanical stimulation in a subpopulation of DPA neurons. RI currents were pharmacologically blocked by ruthenium red, a compound known to block Piezo2, and were also reduced by small interfering RNA-mediated Piezo2 knockdown. Piezo2-like currents were observed almost exclusively in IB₄-negative DPA neurons, with the current amplitude larger in capsaicin-insensitive DPA neurons than the capsaicin-sensitive population. Our findings show that subpopulation of DPA neurons is indeed mechanically sensitive. Within this subpopulation of mechanosensitive DPA neurons, we have identified the Piezo2 ion channel as a potential transducer for mechanical stimuli, contributing to RI inward currents. Piezo2-positive DPA neurons were characterized as medium- to large-sized neurons with myelinated A-fibers, containing nociceptive peptidergic neurotransmitters.

Keywords

neuroscience/neurobiology sensory neurons pulp biology mechanotransduction molecular biology electrophysiology

Introduction

Dental pain is elicited by various stimuli, such as noxious heat/cold or light air puff (Cook et al. 1997; Chung et al. 2013). Interestingly, not only noxious stimuli but also more subtle stimuli such as air puff or dentin probing onto teeth induce pain known as dental hypersensitivity. Dental hypersensitivity is classically explained by hydrodynamic theory, which hypothesizes the presence of mechanotransducers in the tooth pulp (Brännström and Åström 1964). However, the presence of mechanotransducers is yet to be identified in dental primary afferent (DPA) neurons.

Among the mechanotransducers identified to date, Piezo2 is the first to be identified in dorsal root ganglion (DRG) neurons. Piezo2-expressing neurons display a rapidly inactivating (RI) current in response to mechanical stimuli (Coste et al. 2010; Ikeda et al. 2014). Recent studies have shown that Piezo2 is preferentially found in medium- to large-sized A-fibers and is crucial for light touch sensation (Bron et al. 2014; Ikeda et al. 2014; Ranade et al. 2014). As dentinal tubules and the outermost pulp are mostly innervated by pulpal A-fibers (Jyväsjärvi and Kniffki 1987), Piezo2 may be expressed in such DPA neurons to mediate mechanotransduction.

In this study, we hypothesized that a subpopulation of DPA neurons may show mechanosensitive properties via expression of Piezo2. We therefore investigated the neurochemical profile of Piezo2 messenger RNA (mRNA)–positive DPA neurons and monitored the mechanical responsiveness of DPA neurons related to Piezo2 activation. We found that DPA neurons do indeed express Piezo2 and that these neurons are functionally distinct from conventional nociceptors.

Materials and Methods

Animals

All surgical and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Seoul National University. Male Sprague-Dawley rats (Orient Bio Inc.) (n = 63, 200–250 g) and male C57BL/6 mice (DBL Co.) (n = 3, 20–25 g) both at 8 to 10 wk of age were used for the experiments. The study conformed to ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for preclinical studies.

Primary Culture of Rat Trigeminal Ganglion Neurons after Retrograde Labeling of DPA Neurons

Rat DPA neurons were retrograde labeled by filling fluorescent tracer DiI into upper molars (Park et al. 2006). Two weeks later, trigeminal ganglions (TGs) were harvested and digested in collagenase and trypsin to obtain dissociated TG neurons. The separated cells were placed on poly-D-lysine–coated glass coverslips and were maintained at 37°C in a 5% CO₂ incubator. Individual DiI-labeled DPA neurons were observed under green fluorescent illumination.

In Situ Hybridization after Retrograde Labeling of Mouse DPA Neurons

Mouse DPA neurons were retrograde labeled by filling fluorescent tracer FluoroGold (FG) into an upper molar. Two weeks later, the mice were perfusion fixed with 4% paraformaldehyde in 0.1% diethylpyrocarbonate (DEPC)-treated phosphate-buffered saline (PBS). Serial frozen transverse sections were mounted on slide glasses for further processing. For fluorescent in situ hybridization, Mm-Piezo2 probe and RNAscope Multiplex Fluorescent Reagents Kit (ACD) were used. The mounted sections were examined by fluorescence microscopy.

Single-Cell Reverse Transcriptase Polymerase Chain Reaction

DiI-labeled DPA neurons were examined for isolectin B4 (IB₄) positivity prior to collection. The cells were collected with patch pipette and put into a polymerase chain reaction (PCR) tube containing reverse transcription agents for complementary DNA (cDNA) synthesis. All PCR amplifications were performed with nested primers (Appendix Table 1).

Whole-Cell Patch-Clamp Recordings

The pipette solution for voltage clamp recordings contained (in mM) 125 CsCl, 1 MgCl₂, 4.8 CaCl₂, 0.4 NaGTP, 4 MgATP, 1 MgCl₂, 10 HEPES, and 10 EGTA, adjusted to pH 7.3 with CsOH. The external solution contained (in mM) 132 NaCl, 3 KCl, 1 MgCl₂, 2.5 CaCl₂, 10 HEPES, and 10 D-glucose, adjusted to pH 7.3 with NaOH. Inward currents were recorded at a holding potential of −60 mV. In total, 30 μM Ruthenium red or 500 nM capsaicin was used for voltage clamp analysis.

Mechanical Stimulation and Analysis

Patch pipette with a 3- to 4-µm diameter tip was computer operated in 0.4- to 1-µm steps in an approaching motion via a piezoelectric device. The inactivation kinetics of the mechanically sensitive currents were fitted to exponentials using the Chebyschev nonlinear least squares fitting procedure.

Piezo2 Knockdown and Quantitative PCR

Primary cultured TG neurons were transfected with 10 nM Piezo2 or negative small interfering RNA (siRNA) (Ambion) 1 h after plating. Thirty-six hours after transfection, the cells were used for whole-cell recording or RNA extraction to evaluate Piezo2 knockdown. For quantitative PCR (qPCR), each sample containing 10 ng cDNA was run in triplicates. ΔΔCT values were analyzed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference gene.

Statistical Analysis

All data are presented as mean ± SEM unless stated otherwise except the fitting procedure for inactivation kinetics analysis. The criterion for significance was P < 0.05.

Supplemental Information

Detailed materials and methods (manufacturer, experimental condition, statistical analysis, etc.) are in the online Appendix.

Results

Molecular Expression of Piezo2 in DPA Neurons

We first confirmed the expression of Piezo2 mRNA in rat TGs by reverse transcriptase PCR (RT-PCR) analysis. Lung and white blood cells were used as positive and negative controls, respectively, according to the mRNA expression database for Piezo2 (Coste et al. 2010; www.genecards.org; Fig. 1A). Piezo2 mRNA in mouse DPA neurons was then visualized by fluorescent in situ hybridization analysis in the maxillary division of retrograde-labeled ipsilateral TGs, which appeared as red puncta in the cytosolic compartment of the neurons (Fig. 1B). Piezo2 mRNA fluorescence was observed in 56% of FG-labeled DPA neurons (n = 99/176), with Piezo2 mRNA-positive DPA neurons significantly larger on average than Piezo2 mRNA-negative neurons (Fig. 1C, 1D; 37.0 ± 1.0 µm and 29.2 ± 1.1 µm in diameter, respectively; Student’s t test, P < 0.0001). Further studies were performed with rat DPA neurons.

Molecular Characterization of Piezo2 mRNA-Positive DPA Neurons

Next, we screened single DPA neurons for Piezo2 mRNA expression by single-cell reverse transcriptase polymerase chain reaction (scRT-PCR) (Fig. 2A). Piezo2 mRNA was detected in 71% (n = 120/170) of DPA neurons, with these neurons mostly being medium to large sized (Fig. 2B). Piezo2 mRNA-positive DPA neurons were significantly larger on average than Piezo2 mRNA-negative neurons (Fig. 2C; 31.6 ± 0.7 µm and 24.7 ± 0.9 µm in diameter, respectively; Student’s t test, P < 0.0001).

The neurochemical properties of Piezo2 mRNA-positive DPA neurons were investigated based on nociceptive markers, such as transient receptor potential vanilloid 1 (TRPV1), voltage-gated sodium channel 1.8 (Nav1.8), calcitonin gene-related peptide (CGRP), and IB₄, and myelination markers such as peripherin and neurofilament 200 (NF200) (Fig. 2A). The following mRNA transcripts were coexpressed with Piezo2 mRNA as follows: TRPV1 (n = 45/94, 48%), Nav1.8 (n = 42/47, 89%), CGRP (n = 19/21, 90%), peripherin (n = 12/41, 29%), and NF200 (n = 47/47, 100%). Fourteen percent (n = 3/21) of Piezo2 mRNA-positive DPA neurons were IB₄-positive (Fig. 2D). Among total DPA neurons, mRNA transcripts were detected to express TRPV1 (n = 62/141, 44%), Nav1.8 (n = 49/71, 69%), CGRP (n = 31/27, 87%), peripherin (n = 15/61, 25%), and NF200 (n = 61/71, 86%). Eighteen percent of DPA neurons were IB₄-positive (n = 17/92) (Fig. 2D).

Mechanosensitive Piezo2-Like Currents Are Observed in DPA Neurons

We next measured mechanosensitive currents in DPA neurons by whole-cell patch-clamp recordings (Fig. 3A). Mechanical stimulation evoked mechanosensitive currents in 58% of DPA neurons (n = 131/225, Fig. 3C). When mechanosensitive DPA neurons were classified based on the decaying kinetics of their mechanosensitive currents, Piezo2-like, RI currents with a decay time constant of <10 ms (τ < 10 ms) were observed in 40% (n = 90/225) of DPA neurons tested, occupying 69% (n = 90/131) of the mechanosensitive population (Fig. 3B, C; further information of recorded DPA neurons in Appendix Table 2). The average decay time constant of RI currents was 4.6 ± 0.3 ms, consistent with previous studies (Fig. 3D) (Coste et al. 2010; Ranade et al. 2014). The maximal amplitude of RI current obtained from individual DPA neurons increased according to cell size, whereas maximal RI currents smaller than <400 pA were also observed regardless of cell size (Fig. 3E). scRT-PCR analysis revealed that 100% (n = 10/10) of DPA neurons with mechanosensitive RI currents had Piezo2 mRNA. Among nonmechanically responsive DPA neurons, 38% (n = 3/8) had Piezo2 mRNA (Fig. 3F).

Mechanosensitive RI Current in DPA Neurons Is Mediated by Piezo2 Ion Channels

To confirm whether mechanosensitive RI currents in DPA neurons are generated by Piezo2 activation, we employed 2 approaches: pharmacological blocking of Piezo2 currents with 30 µM ruthenium red and siRNA-mediated Piezo2 knockdown. Consistent with previous studies (Drew et al. 2002; Ikeda et al. 2014), we found that RI currents were reversibly blocked by 30 µM ruthenium red (Fig. 4A, B). We also recorded RI currents in DPA neurons after transfection with either Piezo2 or negative siRNA. Piezo2 mRNA levels were significantly reduced after Piezo2 siRNA transfection (Student’s t test, P = 0.0021, compared with negative siRNA transfection, n = 5, Fig. 4C). After transfection, RI currents were observed in 42% (n = 10/24) of the negative siRNA-transfected DPA neurons but only in 9% (n = 2/22) of the Piezo2 siRNA-transfected DPA neurons (Fig. 4D). When compared with negative siRNA-transfected DPA neurons, the amplitude of the RI currents was reduced after Piezo2 siRNA transfection, although statistically insignificant (Student’s t test, P = 0.3013; Fig. 4E, F).

Mechanosensitive RI Currents Are Robust in Capsaicin-Insensitive and IB₄-Negative DPA Neurons

DPA neurons showing mechanosensitive RI currents were tested for capsaicin sensitivity and IB₄ positivity. Among the RI population, 45% (n = 13/29) were capsaicin-sensitive. However, RI currents were significantly larger in capsaicin-insensitive DPA neurons than capsaicin-sensitive DPA neurons (Student’s t test, P = 0.0094; Fig. 5A, B). The decay constant of the RI currents in capsaicin-insensitive DPA neurons was slightly smaller (3.3 ± 0.7 ms) than the sensitive neurons (4.6 ± 0.9 ms) but not significantly different (Student’s t test, P = 0.2240, figure not shown). All DPA neurons displaying mechanosensitive RI currents were IB₄-negative (100%, n = 22/22), as IB₄-positive DPA neurons failed to show any mechanosensitive RI currents (Fig. 5C).

Discussion

This is the first report identifying the mechanosensitive DPA neurons, the ever searched functional phenotype since the proposal of hydrodynamic theory (Brännström and Åström 1964). By using whole-cell patch-clamp recording and siRNA-mediated Piezo2 knockdown, we have identified that the majority of mechanosensitive DPA neurons produce RI currents in response to mechanical stimulation, which mainly seem attributable to Piezo2 expression. Our results suggest that Piezo2 mediates mechanical transduction in DPA neurons, especially in medium- to large-sized neurons that contain nociceptive neuropeptides such as CGRP. This is consistent with our functional data showing Piezo2-like RI currents exclusively in IB₄-negative DPA neurons. Indeed, larger RI currents were more consistently evoked in capsaicin-insensitive DPA neurons than capsaicin-sensitive neurons.

To determine the neurochemical properties of Piezo2-positive DPA neurons, we characterized Piezo2 mRNA-positive DPA neurons with a range of neuronal markers: TRPV1, Nav1.8, CGRP, IB₄, peripherin, and NF200. TRPV1 is the receptor ion channel for noxious heat and pungent chemical capsaicin and is expressed exclusively in nociceptors with lightly myelinated A_δ-fibers or unmyelinated C-fibers (Tal 1984; Caterina and Julius 2001). Nav1.8 participates in TTX-resistant action potential transduction in nociceptors (Abrahamsen et al. 2008). The expression of CGRP and IB₄ indicates peptidergic and nonpeptidergic nociceptors, respectively (Stucky and Lewin 1999). CGRP has been suggested as a potential mediator for dentin hypersensitivity, as CGRP-positive DPA neurons are mostly medium- to large-sized and NF200-positive, in contrast to CGRP-positive DRG neurons, which are mostly small-sized and NF200-negative (McCarthy and Lawson 1990; Pan et al. 2003; Fried et al. 2011; Chung et al. 2012). Peripherin is mostly expressed in small-sized, unmyelinated C-fiber nociceptors, whereas NF200 is generally expressed in large-sized nonnociceptors with myelinated, fast conducting A-fibers (Goldstein et al. 1991; Lawson et al. 1993).

Our results on the mRNA expression of TRPV1 (44%) and Nav1.8 (69%), as well as IB₄ (18%) positivity in DPA neurons, are similar to previous findings (Pan et al. 2003; Paik et al. 2010; Kim et al. 2011). As our results show that Piezo2 mRNA-positive DPA neurons are mostly medium- to large-sized, NF200-positive, and IB₄-negative, it seems clear that they are rather functionally distinct from the small-sized, unmyelinated DPA neurons that have been conventionally termed nociceptors (Stucky and Lewin 1999). On the other hand, the high coexpression of Piezo2 and CGRP mRNA raises the possibility that these neurons could represent algoneurons, the putative mechanosensitive fibers that transduce weak mechanical stimuli into pain signals (Fried et al. 2011). The actual CGRP contents in Piezo2-positive DPA neurons, and whether this neuropeptide is released by mechanical stimulation, should be investigated further.

In the present study, most Piezo2 mRNA-positive DPA neurons were positive for NF200, which may seem controversial as 89% of Piezo2 mRNA-positive neurons also coexpress Nav1.8 (Abrahamsen et al. 2008). However, a recent study has reported that Nav1.8-positive and NF200-positive/large-sized sensory neurons are not discrete but in fact overlapping populations (Ho and O’Leary 2011). Another recent study has also found that 40% of NF200-positive DRG neurons coexpress Nav1.8, with these neurons innervating Meissner corpuscles and hair follicles, thus indicating Nav1.8 expression among A-beta fibers (Shields et al. 2012). Our results are consistent with these studies, suggesting this might be also the case in DPA neurons.

As above, our scRT-PCR experiments enabled us to characterize Piezo2 mRNA-positive DPA neurons by their size or molecular markers. In situ hybridization results obtained from mice also showed a similar pattern of Piezo2 mRNA in DPA neurons according to cell size. We must caution, however, that the markers for nociceptive neurons used here may have different expression profiles between rat and mouse.

The decay kinetics of mechanosensitive currents revealed Piezo2 expression in nearly 40% of DPA neurons. Previous studies also observed mechanosensitive RI currents in 21% to 44% of DRG neurons, which was mainly attributable to Piezo2 (Coste et al. 2010; Ranade et al. 2014). These studies have also observed RI currents mostly in medium- to large-sized DRG neurons. Interestingly, the percentage of mechanosensitive DPA neurons with RI currents was higher than the population showing mechanosensitive currents with a slower decay kinetics (Coste et al. 2010; Ranade et al. 2014), implying the physiological importance of Piezo2 expression in pulpal mechanosensory function. We were able to compare the expression level of Piezo2 mRNA and Piezo2-like currents in DPA neurons, which accounted for 71% and 40% of inspected DPA neurons, respectively. The presence of Piezo2 mRNA in 37.5% of mechanically insensitive DPA neurons reveals functionally silent Piezo2 mRNA, as confirmed by combining whole-cell patch-clamp and scRT-PCR analysis. Whether quiescent Piezo2 mRNA gains function during pathological changes of the pulp is yet to be found.

When RI currents were quantified according to cell diameter, the recorded currents were quite divergent in size, ranging from a few pA to >3 nA. RI currents of nanoampere sizes were seen mostly in medium- to large-sized DPA neurons. These large RI currents were not observed after Piezo2 siRNA delivery, indicating these currents are derived from Piezo2 activation. RI currents relatively small in amplitude (typically <–100 pA when maximally induced) were also observed, regardless of cell size. The smaller and less mechanosensitive RI currents persisted even after siRNA-mediated Piezo2 knockdown, consistent with previous studies that described small RI currents in Piezo2 knockout mice (Ranade et al. 2014; Florez-Paz et al. 2016). The identity of the mechanoreceptor(s) underlying the residual RI currents evoked after Piezo2 knockdown in DPA neurons remains to be identified.

Functional characterization of DPA neurons by their IB₄ positivity and capsaicin sensitivity revealed 2 features. First, RI currents were observed only in IB₄-negative DPA neurons, which is consistent with the high expression of CGRP mRNA in the Piezo2 mRNA-positive population. Second, the RI currents were significantly larger in capsaicin-insensitive DPA neurons. These 2 findings are also described in a previous report on DRG neurons in which RI currents were observed mostly in IB₄-negative, capsaicin-insensitive neurons (Drew et al. 2002). Moreover, as it has been reported that only few TRPV1-positive DPA neurons coexpress IB₄ (Chung et al. 2012), Piezo2-positive DPA neurons might be mostly IB₄-negative, regardless of their capsaicin sensitivity. Based on these observations, Piezo2-positive DPA neurons are likely to be functionally distinct from thermosensitive, nonpeptidergic DPA neurons, which are generally small-sized, unmyelinated, and IB₄-positive (Park et al. 2006).

In conclusion, we have confirmed that a subpopulation of DPA neurons is indeed mechanically sensitive and provide evidence suggesting that Piezo2 contributes in part to the mechanical responsiveness of DPA neurons. Piezo2-positive DPA neurons are likely to be medium- to large-sized with myelinated A-fibers, possibly mediating nociceptive peptidergic transmission when activated. Whether these properties reflect the function of algoneurons, the putative low-threshold mechanoreceptors among DPA neurons that are paradoxically engaged in nociception (Fried et al. 2011; Ranade et al. 2014), remains to be investigated. Further confirmation on whether Piezo2 activation can trigger action potential firings in DPA neurons, and if these Piezo2-positive populations do release nociceptive neurotransmitters, will be helpful in advancing our understanding regarding dental nociception.

Author Contributions

J. Won, contributed to design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; H. Vang, P.R. Lee, contributed to data acquisition and analysis, critically revised the manuscript; Y.H. Kim, H.W. Kim, contributed to data acquisition, critically revised the manuscript; Y. Kang, contributed to data interpretation, critically revised the manuscript; S.B. Oh, contributed to conception, design, and data interpretation, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Footnotes

Acknowledgements

We thank Prof. Sun Wook Hwang at Korea University for introducing the piezoelectric device, Byeong-Min Lee for technical consultation, and Ji-A Park for statistical consultation. We also thank Dr. Alexander J. Davies for the English correction and editing.

This research was supported by the National Research Foundation of Korea grants (NRF-2016M3A9B6021209) funded by the Korea government (Ministry of Science, ICT and Future Planning). Y. Kang was supported by the Brain Pool Program through the Korean Federation of Science and Technology Societies (KOFST) funded by the Ministry of Science, ICT and Future Planning.

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

A supplemental appendix to this article is available online.

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Supplementary Material

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Piezo2 Expression in Mechanosensitive Dental Primary Afferent Neurons

Abstract

Keywords

Introduction

Materials and Methods

Animals

Primary Culture of Rat Trigeminal Ganglion Neurons after Retrograde Labeling of DPA Neurons

In Situ Hybridization after Retrograde Labeling of Mouse DPA Neurons

Single-Cell Reverse Transcriptase Polymerase Chain Reaction

Whole-Cell Patch-Clamp Recordings

Mechanical Stimulation and Analysis

Piezo2 Knockdown and Quantitative PCR

Statistical Analysis

Supplemental Information

Results

Molecular Expression of Piezo2 in DPA Neurons

Molecular Characterization of Piezo2 mRNA-Positive DPA Neurons

Mechanosensitive Piezo2-Like Currents Are Observed in DPA Neurons

Mechanosensitive RI Current in DPA Neurons Is Mediated by Piezo2 Ion Channels

Mechanosensitive RI Currents Are Robust in Capsaicin-Insensitive and IB4-Negative DPA Neurons

Discussion

Author Contributions

Footnotes

Acknowledgements

References

Supplementary Material

Mechanosensitive RI Currents Are Robust in Capsaicin-Insensitive and IB₄-Negative DPA Neurons