ASICs mediate fast excitatory synaptic transmission for tactile discrimination

Akihiro Yamada; Jennifer Ling; Ayaka I Yamada; Hidemasa Furue; Jianguo G Gu

doi:10.1016/j.neuron.2024.01.018

. Author manuscript; available in PMC: 2025 Apr 17.

Published in final edited form as: Neuron. 2024 Feb 14;112(8):1286–1301.e8. doi: 10.1016/j.neuron.2024.01.018

ASICs mediate fast excitatory synaptic transmission for tactile discrimination

Akihiro Yamada ¹, Jennifer Ling ¹, Ayaka I Yamada ¹, Hidemasa Furue ³, Jianguo G Gu ^1,^2,^4,^*

PMCID: PMC11031316 NIHMSID: NIHMS1962828 PMID: 38359825

SUMMARY

Tactile discrimination, the ability to differentiate objects’ physical properties such as texture, shape, and edges, is essential for environmental exploration, social interaction, and early childhood development. This ability heavily relies on Merkel cell-neurite complexes (MNCs), the tactile end-organs enriched in the fingertips of humans and the whisker hair follicles of non-primate mammals. Although recent studies have advanced our knowledge on mechanical transduction in MNCs, it remains unknown how tactile signals are encoded at MNCs. Here, using rodent whisker hair follicles, we show that tactile signals are encoded at MNCs as fast excitatory synaptic transmission. This synaptic transmission is mediated by acid-sensing ion channels (ASICs) located on the neurites of MNCs, with protons as the principal transmitters. Pharmacological inhibition or genetic deletion of ASICs diminish the tactile encoding at MNCs and impair tactile discrimination in animals. Together, ASICs are required for tactile encoding at MNCs to enable tactile discrimination in mammals.

Keywords: Sense of touch, tactile discrimination, Merkel cell-neurite complex, acid-sensing ion channel, proton, fast excitatory synaptic transmission, excitatory postsynaptic current, Piezo2 channel, node of Ranvier, whisker hair follicle

Graphical Abstract

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In brief

Yamada et al. show that acid sensing ion channels (ASICs) mediate fast excitatory synaptic transmission at the tactile end organs, Merkel cell-neurite complexes (MNCs), with protons serving as the principal transmitters. This suggests a unique role of ASICs in the sense of touch and tactile discrimination in mammals.

INTRODUCTION

Merkel cell-neurite complexes (MNC), also known as Merkel discs^1–3, along with Meissner’s corpuscles and other tactile end organs, are crucial for sensing touch and performing sophisticated sensory tasks, such as tactile discrimination of an object’s texture, shape, and edges⁴. MNCs are clustered at touch sensitive areas of the skin and hair follicles in mammals^2,5–7. They are most abundant in whisker hair follicles of non-primates and fingertips of primates^2,5–7, two functionally equivalent tactile organs displaying high tactile sensitivity and acuity^8–10. Structurally, an MNC consists of a Merkel cell and its associated Aβ-afferent neurite^2,3,6. MNCs are highly sensitive to skin indentation, pressure, and hair movement. Tactile stimuli to MNCs of fingertips and whisker hair follicles result in firing slowly adapting type 1 (SA1) impulses of action potentials (APs) on Aβ-afferent fibers, which are characteristic tactile responses^2,4,11,12 essential for tactile discrimination^4,8,12. Tactile functions of MNCs can be impaired under clinical conditions such as diabetes and chemotherapy^13,14, which may contribute to the loss of touch sensation or numbness in extremities in patients with these clinical conditions.

Although MNCs were discovered about one and a half centuries ago¹, the molecular mechanisms underlying tactile encoding by MNCs remain incompletely understood. Recent studies have identified Piezo2 channels¹⁵ as mechanical transducers at MNCs, demonstrating that Piezo2 is a molecular sensor of touch in mammals^16–18. However, currently, the molecular mechanism underlying the conveyance of tactile signals from Merkel cells to Aβ-afferent fibers, generating SA1 impulses for tactile encoding and enabling tactile discrimination, remains unknown. One hypothesis is that tactile signals may be conveyed from Merkel cells to Aβ-afferent fibers via excitatory synaptic transmission with classical neurotransmitters such as glutamate, 5-HT, and norepinephrine at MNCs^2,19,20. However, so far, there has been no direct evidence indicating that excitatory synaptic transmission indeed occurs at MNCs between Merkel cells and the neurites of Aβ-afferent fibers.

ASICs are a proton-gated subclass of the degenerin/epithelial Na⁺ channel (DEG/ENaC) family expressed in mammals^21,22. Four ASIC subunits have been identified in mammalian afferent neurons²³, and functional ASICs are formed by ASIC1, ASIC2, and ASIC3 as homomers or heteromers in trimeric proteins in afferent neurons²⁴. ASICs are activated by extracellular protons in an acidic pH range of pH 7.0 to 4.5²⁵, resulting in the entry of Na⁺ ions into cells through ASIC channels. Initially, ASICs were thought to be mechanical transducers of mammals, similar to their homolog degenerin Mec-4/Mec-10 in C. elegans^21,26–28. However, numerous studies over the past several decades could not establish that ASICs are mechanical transducers in mammals^27,29–32. In the present study we have discovered that, instead of serving as mechanical transducers, ASICs mediate fast excitatory synaptic transmission at MNCs to encode tactile signals and enable tactile discrimination.

RESULTS

Mechanical stimulation elicits fast excitatory synaptic transmission at MNCs in whisker hair follicles

MNCs are located in the front part of whisker hair follicles, before the ringwulst (Rw), and innervated by Aβ-afferent terminals (Figure 1A). Each Aβ-afferent terminal subdivided into a cluster of enlarged neurites aligning with Merkel cells to form MNCs (Figure 1A). The axon segment prior to the neurites of MNCs is the heminode of the Aβ-afferent terminal, and the first node of Ranvier (1st node) is in a short distance from the heminode (Figure 1A&B). To detect fast excitatory synaptic transmission at MNCs, patch-clamp recordings are required to apply to the axon sites near MNCs. However, patch-clamp recordings have never been previously performed on Aβ-afferent axons near MNCs due to technical difficulties because most parts of Aβ-afferent axons are heavily myelinated and inaccessible to patch-clamp recording electrodes. Since the 1^st nodes or heminodes are axon segments without myelination and adjacent to MNCs (Figure 1B–C), we applied pressure-patch-clamp recordings to the heminodes or 1^st nodes of MNCs (Figure 1A–C) in attempting to detect synaptic transmission at MNCs.

In our ex vivo whisker hair follicle preparation, the 1^st nodes and heminodes could be visualized under a microscope (Figure 1B). The neurites of the MNCs could not be normally visualized, but could be seen under a fluorescent microscope after whole-cell patch-clamp recordings were applied to the 1^st nodes or heminodes of MNCs with electrodes containing the fluorescent dye Alexa Fluor 555 (Figure 1C). This allowed us to observe morphological details of MNCs in fresh whisker hair follicles. Each Aβ-afferent terminal gave rise to approximately 28 enlarged neurites (27.8 ± 1.5, n = 6 recordings) in rat whisker hair follicles (Figure 1C), and these enlarged neurites were inter-connected by finer linker axons (video S1). While heminodes were the starting points of the neurites of MNCs, the 1^st nodes were 72 ± 7 μm (n = 12 recordings) away from the heminodes (Figure 1C). Therefore, both heminodes and 1^st nodes were close to the neurites of MNCs, and patch-clamp recordings applied at these sites would provide a technical solution to detect synaptic transmission if it does occur at MNCs.

The first finding from the pressure-patch-clamp recordings at either heminodes or 1^st nodes of MNCs in rat whisker hair follicles was the transient currents that occurred spontaneously (Figure 1D&E). The transient currents were in the inward direction (i.e., excitatory currents), had variable amplitudes, showed fast kinetics, and occurred randomly (Figure 1D). These properties highly resemble those of the fast excitatory postsynaptic currents (EPSCs) occurring spontaneously at various types of excitatory synapses^33,34, due to spontaneous vesicular release of transmitters and subsequent activation of postsynaptic receptors^33,34. Therefore, hereafter, we will use the term ‘EPSCs’ for the transient currents recorded at MNCs. The averaged frequency of the spontaneous EPSCs was low at approximately 1 to 2 Hz, and the averaged amplitude was approximately 50 pA (Figure 1E). Both the frequency and amplitude of spontaneous EPSCs were not significantly different between recordings made from heminodes and the 1^st nodes of MNCs (Figure 1E). Most recordings in this study were performed at the 1^st nodes because it was relatively easier. To address the key question of whether tactile stimuli to MNCs elicit fast excitatory synaptic transmission at MNCs, we next investigated whether mechanical stimulation could elicit EPSCs. We recorded EPSCs at MNCs in the whisker hair follicles of both rats and mice (Figure 1F–M), while mechanical steps were applied to MNCs. As exemplified in Figure 1F&G, a mechanical step evoked a mechanically activated current (MA) at the beginning or the dynamic phase of a mechanical step, which was immediately followed by many EPSCs through the entire static phase of the mechanical step (Figure 1F). The MA currents recorded from MNCs decayed rapidly with the decay τ of approximately 13 ms in rats and 9 ms in mice (Figure 1G&H), consistent with Piezo2-mediated currents^17,18. The MA currents at MNCs of both rats and mice had large peak amplitudes, which rapidly decayed to very small steady-state currents (Figure S1). For the EPSCs evoked mechanically, they decayed very fast with the decay τ of approximately 3 ms in rat MNCs and 2 ms in mouse MNCs, much faster than the decay of MA currents (Figure 1G&H). In addition to the distinct kinetics described above, EPSCs and MA currents exhibited significant differences in their reversal potentials, pharmacological properties, and responsiveness following genetic deletion of ASIC3, as presented later in the result section. The amplitudes of individual EPSCs varied in a broad range from a few pA to over 150 pA when EPSCs were evoked by mechanical step at 10 μm (Figure 1I). The frequency of EPSCs evoked by mechanical steps increased in a mechanical step-dependent manner in experiments with rat whisker hair follicles (Figure 1J). Large increases in the EPSC frequency were also observed with the 10-μm mechanical step in experiments using mouse whisker hair follicles (Figure 1L). On the other hand, the averaged amplitudes of EPSCs evoked by mechanical steps were not significantly changed (Figure 1K&M). While the above recordings were performed at 24°C, we also recorded EPSCs in experiments with rat whisker hair follicle preparations at 34°C, a more physiological temperature. The EPSCs evoked by 10 μm mechanical steps had a frequency of 23.2 ± 4.5 Hz (n = 5) and an amplitude of 119.5 ± 22.5 pA (n = 5) at 34°C, significantly higher than the EPSC frequency (12.4 ± 2.3 Hz, n = 9, p < 0.001) and EPSC amplitude (41.7 ± 5.3 pA, n = 9, p < 0.001) evoked by 10 μm mechanical steps with recordings conducted at 24°C (not shown in a figure). The frequency but not the amplitude of EPSCs was also significantly increased following the application of caffeine (Figure 1N–Q), an activator of intracellular Ca²⁺ stores that was previously found to act on Merkel cells to cause an increase of SA1 impulses³⁵. Taken together, mechanical stimulation and intracellular Ca²⁺ store activation trigger fast excitatory synaptic transmission at MNCs.

Fast excitatory synaptic transmission at MNCs is mediated by ASICs located on the neurites of MNCs

To investigate the nature of the receptors that mediated fast synaptic transmission at MNCs, we characterized the reversal potentials of EPSCs at MNCs of both rats and mice. EPSCs were evoked by 10-μm mechanical steps and recorded at MNCs with axon membranes voltage-clamped at −70 mV to 50 mV (Figure 2A–C). At negative voltages, mechanical steps evoked MA currents followed by multiple EPSCs, and both MA currents and EPSCs were in the inward direction (Figure 2A–C). The MA currents were reversed at the holding voltage above 0 mV (Figure 2A–C), consistent with the property of Piezo2 channels^15,16. In contrast, mechanically evoked EPSCs remained to be inward currents at the positive holding voltages of up to 50 mV (Figure 2A–C). The very positive reversal potentials suggested that the receptors mediating the EPSCs were ligand-gated Na⁺ channels. Consistently, the reversal potentials of EPSCs shifted from ~50 mV to ~0 mV when Na⁺ concentration gradients between extracellular and intracellular sites were changed from a normal concentration gradient of 5:1 to an equal concentration gradient of 1:1, respectively (Figure 2D&E).

Figure 2. — A) Traces exemplify MA currents and EPSCs of rat MNCs recorded at −70, −40, −10, 20, and 50 mV. Mechanical step of 10 μm was applied for 5 s to MNCs. Blue arrows indicate MA currents and red arrows indicate EPSCs. B) I-V curve (n = 6) of both MA currents (blue circles) and EPSCs (red squares) at different voltages. C) Similar to B except that the EPSCs were recorded from mouse MNCs (n = 4). D) Traces exemplify EPSCs of a rat MNC recorded at −70 and 20 mV in [Na⁺]_o/[Na⁺]_in = 5 (top) and [Na⁺]_o/[Na⁺]_in = 1 (bottom). E) I-V curve (n = 5) shows a shift of the reversal potential with the change of [Na⁺]_o/[Na⁺]_in. The recordings were performed in the presence of 10 mM caffeine. F) Traces exemplify mechanically evoked EPSCs of rat MNCs in the absence (control, top) and presence of the ASIC blocker amiloride (200 μM, bottom). G) Pooled data of the amplitudes (left) and frequency (right) of mechanically evoked EPSCs of rat MNCs in the absence (control) and presence of 200 μM amiloride. H) Similar to G, except EPSCs of mouse MNCs were tested. I) Summary data of the amplitude (left) and frequency (right) of mechanically evoked EPSCs of MNCs in the absence (control) and presence of 200 μM amiloride, 100 μM diminazene, and 100 μM nafamostat. J) Summary data of the amplitude (left) and frequency (right) of mechanically evoked EPSCs in the absence (control) and presence of the adrenergic β2 receptor antagonist ICI118551 (50 μM), and 5-HT3 receptor blocker VUF10166 (20 μM). The mechanical step was applied to MNCs at 10 μm for 5 s. The 1^st nodes of MNCs were voltage-clamped at −72 mV (F-J) or −69 mV (H). Data represent independent observations and mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significantly different, paired Student’s t-test or one-way ANOVA with Bonferroni post hoc test. See also Figure S2–4.

Acid-sensing ion channels (ASICs) are Na⁺ channels gated by protons^25,28. To investigate whether fast excitatory synaptic transmission at MNCs was mediated by ASICs, we examined whether mechanically evoked EPSCs at MNCs could be inhibited by ASIC blockers. Amiloride, a widely used ASIC blocker^28,36, significantly inhibited amplitudes and frequency of EPSCs at MNCs of both rats (Figure 2F&G) and mice (Figure 2H). Similarly, diminazene and nafamostat, two other ASIC blockers³⁶, also significantly inhibited amplitude and frequency of EPSCs at rat MNCs (Figure 2I). In addition to mechanically evoked EPSCs, the amplitudes and frequency of EPSCs induced by caffeine were also significantly inhibited by these three ASIC blockers (Figure S2). While the ASIC blockers largely abolished EPSCs, Piezo2-mediated MA currents on the neurites of MNCs were not significantly affected by these blockers (Figure S3). Furthermore, neither amiloride nor diminazene had any effect on action potential (AP) firing and voltage-activated currents in Merkel cells (Figure S3). Thus, the inhibitory effects of ASIC blockers on EPSCs were not a non-specific action.

Previous studies proposed that β2 adrenergic receptors and 5-HT₃ receptors may mediate synaptic transmission at MNCs^19,20, but these studies did not record synaptic transmission at MNCs. We recorded EPSCs at MNCs and found that neither ICI118551, a potent β2 adrenergic receptor inhibitor, nor VUF10166, a potent 5-HT₃ inhibitor, had any effect on the amplitude and frequency of EPSCs evoked by mechanical steps (Figure 2J). Similarly, the amplitude and frequency of EPSCs induced by caffeine were not affected by ICI118551 and VUF10166 (Figure S2). The amplitude and frequency of spontaneous EPSCs were also not affected by CNQX and APV, the blockers of ionotropic glutamate receptors (Figure S2). These results indicated that fast excitatory synaptic transmission at MNCs was not mediated by β2 adrenergic receptors, 5-HT₃ receptors, or ionotropic glutamate receptors. Although blockage of 5-HT₃ receptors did not affect EPSCs at MNCs, 5-HT (100 μM) significantly increased the frequency but not the amplitude of EPSCs (Figure S4), suggesting that 5-HT may modulate ASIC-mediated fast excitatory synaptic transmission at MNCs.

To further support the idea that the fast excitatory synaptic transmission at MNCs is mediated by ASICs on the neurites of MNCs, we performed immunostaining to investigate ASIC expression at Aβ-afferent terminals in whisker hair follicles. Immunoreactivity of ASIC1 (ASIC1-ir) was observed in Aβ-afferent terminals in the region of MNCs in rat whisker hair follicles, and some of ASIC1-ir was shown to be on the neurites of MNCs (Figure 3A). The ASIC1-ir on axons in the regions of MNCs was abolished by a blocking peptide to the ASIC1 antibody (Figure S4). Similar to ASIC1, immunoreactivity of ASIC2a (ASIC2a-ir, Figure 3B) and ASIC3 (ASIC3-ir, Figure 3C) were observed on Aβ-afferent axons in the region of MNCs in whisker hair follicles. This region contains the axons that innervate both MNCs and lanceolate endings. Previous immunostaining studies have shown the presence of ASIC2 and ASIC3 at both the axons of MNCs and lanceolate endings^29,37. Our results in Figure 3B&C are consistent with these earlier findings.

We focally puff-applied acidified Krebs solution to MNCs to see if protons could directly evoke excitatory currents, mimicking EPSCs (Figure 3D–F). Puff-application of acidified Krebs solution (pH 5) to the MNCs evoked strong inward currents (Figure 3D), which had reversal potentials near 50 mV (Figure 3D). The proton-evoked currents were pH-dependent, and the amplitudes increased progressively in the pH range from 6.5 to 5 (Figure 3E). In contrast to protons, puff applications of 5-HT (2 mM), NE (2 mM), glutamate (2 mM), and ACh (2 mM) did not evoke any current (Figure 3E). While protons evoked large inward currents from the neurites of MNCs, these currents were largely inhibited by the ASIC blockers amiloride and diminazene (Figure 3F–H). These results indicated that ASICs are located at the neurites of MNCs.

ASIC-mediated fast excitatory synaptic transmission at MNCs drives SA1 impulses at Aβ-afferent terminals

We next addressed another key question as whether ASIC-mediated fast excitatory synaptic transmission drives SA1 impulses at MNCs. Excitatory postsynaptic potentials (EPSPs) and AP firing were recorded at MNCs under the current-clamp mode. Low-frequency spontaneous impulses (0.26 ± 0.05 Hz, n = 25) were observed in the majority of Aβ SA1-LTMRs in the absence of mechanical stimulation. A mechanical step of 10 μm evoked EPSPs and many of them reached a threshold, leading to AP firing (Figure 4A). The AP firing at MNCs was slowly adapting and highly irregular (Figure 4A&B) with a coefficient of variance of inter-impulse interval near 1 (Figure 4C), featuring SA1 impulse properties³⁸. The number of APs (Figure 4D) and EPSPs (Figure 4E) showed a mechanical step-dependent manner from the step of 2 to 10 μm. While the above recordings were conducted at 24°C, we also recorded APs and EPSPs at 34°C. The numbers of APs (Figure 4D) and EPSCs (Figure 4E) evoked by 10 μm mechanical steps were not significantly different between experiments performed at 24°C and 34°C. Mechanically evoked APs during static phase of mechanical steps were almost completely abolished by amiloride (Figure 4F–H) and diminazene (Figure 4I–K). The amplitude of mechanically evoked EPSPs during mechanical steps was also largely reduced by amiloride (Figure 4F&H) and diminazene (Figure 4I&K). In the dynamic phase of mechanical step, some APs remained in the presence of the ASIC blockers (Figure 4F&I), indicating that Piezo2 activation on the neurites of MNCs partially contributes to impulse generation at the dynamic phase. Collectively, these results indicate that tactile stimulation to MNCs evoked ASIC-mediated EPSPs, which drives SA1 impulses on MNCs during the entire static phase of mechanical steps.

Figure 4. — A) Trace exemplifies APs and EPSPs evoked by a mechanical step at 10 μm applied to rat MNCs. Red and orange arrows indicate an AP and an EPSP, respectively. B) Plot of instantaneous frequency of APs shown in A during the 5 s of 10-μm mechanical step. C) Coefficient of variance of the inter-event intervals of APs evoked by the 10-μm mechanical step in 5 s. **D&E**) Numbers of APs (D) and EPSPs (E) evoked by mechanical steps at 0, 1, 2, 5, and 10 μm each for 5 s (circles). The squares in D and E are responses to 10-μm mechanical steps in recordings performed at 34°C. F) Traces exemplify APs evoked by a 10-μm mechanical step in the absence (top) and presence (bottom) of 200 μM amiloride. G) Pooled data of the number of APs (left) and EPSP amplitude (right) in 5 s following the 10-μm mechanical step in the absence (control) and presence of 200 μM amiloride. H) Pooled data of the number of APs (left) and EPSP amplitude (right) in 5 s following the 10-μm mechanical step in the absence (control) and presence of 100 μM diminazene. In A-H, Current-clamp recordings were applied to the 1^st nodes. **I-K**) Single fiber recordings from afferent fibers in whisker hair follicles (I) show that 200 μM amiloride (J) and 100 μM diminazene (K) abolished AP impulses evoked by whisker hair deflection at 200 μm. Data represent independent observations and mean ± SEM, *p < 0.05, ***p < 0.05, the paired Student’s t-test.

Mechanically evoked fast excitatory synaptic transmission at MNCs is associated with action potential firing on Merkel cells and mediated by protons

Merkel cells are presynaptic sites of MNCs, and we have previously shown that Merkel cells respond to tactile stimulation with Piezo2 activation and Ca²⁺-action potential (Ca²⁺-AP) firing¹⁶. To determine how Merkel cells contribute to the fast excitatory synaptic transmission at MNCs, we conducted patch-clamp recordings from Merkel cells in whisker hair follicles and investigated their electrophysiological properties and mechanical responsiveness. Depolarizing current steps induced membrane depolarization and sustained AP firing in Merkel cells (Figure 5A–C). Merkel cells fired APs in response to depolarizing currents as small as 5 pA, and all Merkel cells fired APs in response to 10 pA or larger depolarizing currents (Figure 5C). This result indicated that Merkel cells had a very low current threshold for AP firing. We next characterized mechanical responsiveness of Merkel cells (Figure 5D–G). Mechanical steps evoked sustained AP firing on Merkel cells (Figure 5D&E). Under voltage-clamp recording mode, mechanical steps applied to Merkel cells evoked MA currents that decayed rapidly, but a small steady-state current component lasted until the end of mechanical steps (Figure 5D&F). The MA currents recorded from Merkel cells were previously shown to be mediated by Piezo2 channels^16–18. The peak amplitudes of the MA currents were nearly 200 pA, and the steady-state currents were approximately 20 pA when 5-μm mechanical steps were applied to Merkel cells (Figure 5D&F). The steady-state components of MA currents, although small, were sufficient to drive sustained AP firing in Merkel cells during the static phase of the mechanical step (Figure 5B&C). The averaged mechanical threshold for triggering APs was ~2.6 μm, and the number of AP increased in a mechanical step-dependent manner (Figure 5G). Collectively, the electrophysiological properties and mechanical responsiveness of Merkel cells are well suited for driving sustained EPSCs at postsynaptic sites of MNCs.

Figure 5. — A) Illustration of recordings from Merkel cells for experiments in A-G, or from 1^st nodes of MNCs for experiments in H-N. B) Trace exemplifies a Merkel cell firing sustained APs following a 15-pA current step (5 s). C) Pooled results of AP numbers recorded from Merkel cells following the current steps at 5, 10, and 15 pA. D) Sample traces of APs (top, current-clamp) and MA currents (bottom, voltage-clamp at −75 mV) evoked by mechanical steps at 5 μm. E) Pooled results of AP numbers evoked by mechanical step of 3 and 4 μm. F) Pooled results of MA current amplitudes at the peak and the steady-state following a mechanical step of 5 μm. G) AP impulses recorded from Merkel cells following mechanical steps at threshold (2.6 ± 0.4 μm) and increased distances (n = 8). Whole-cell configuration in A-F and cell-attached configuration in G. H) Traces exemplify EPSCs recorded from 1^st nodes of MNCs following mechanical step (10 μm) in normal Ca²⁺ Krebs solution (control, [Ca²⁺]_out = 2 mM, top) and a low Ca²⁺ Krebs solution ([Ca²⁺]_out = 20 μM, bottom). **I-K**) Summary data of the frequency (I) and amplitude (J) of EPSCs as well as MA currents (K) recorded at rat MNCs in normal Krebs solution (control), low Ca²⁺ Krebs solution, in the presence of Cd²⁺ (300 μM), NH₄Cl (10 mM), or quinine (450 μM). In each test, EPSCs were evoked by the 10-μm mechanical step for 5 s. L) Trace exemplify caffeine-induced EPSCs of rat MNCs in the absence (top) and presence (bottom) of 10 mM NH₄Cl. **M&N**) Pooled data of the frequency (M) and amplitude (N) of caffeine-induced EPSCs in the absence (control), presence of 10 mM NH₄Cl, 400 μM bafilomycin A1, 450 μM quinine, 500 μM mefloquine, and 500 μM chloroquine. In L-N, caffeine (10 mM) was present in the bath solution. In H-N, 1^st nodes were voltage-clamped at −72 mV. Data represent independent observations and mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significantly different, one-way ANOVA with the Dunnett’s post hoc test, the paired Student’s t-test, or Kruskal-Wallis test with Dunn’s post hoc test.

Ca²⁺-APs of Merkel cells would increase Ca²⁺ levels in Merkel cells¹⁶, which may trigger the release of protons from Merkel cells to activate ASICs and generate EPSCs at postsynaptic sites of MNCs. To test this idea, we recorded EPSCs at MNCs (Figure 5A) and investigated whether mechanically evoked EPSCs were Ca²⁺-dependent. While mechanical step evoked robust EPSCs in Krebs solution with physiological concentrations of 2 mM Ca²⁺, the mechanically evoked EPSCs at MNCs were almost completely abolished in the Krebs solution containing a low level of 20 μM Ca²⁺ (Figure 5H–J). Since Ca²⁺-APs on Merkel cells depended on voltage-gated Ca²⁺ channels¹⁶, we further determined whether blocking voltage-gated Ca²⁺ channels would also abolish mechanically evoked EPSCs. Indeed, mechanically evoked EPSCs were almost completely abolished by the voltage-gated Ca²⁺ channel blocker Cd²⁺ (Figure 5I&J). While the low Ca²⁺ concentrations and Cd²⁺ largely abolished EPSCs, they had no effect on MA currents (Figure 5K). The Ca²⁺-dependence of mechanically evoked EPSCs supports the notion that vesicular release of protons and/or other ASIC ligands, such as the neuropeptide nocistatin³⁹ and FMRFamide-related peptides⁴⁰, may occur at MNCs in response to mechanical stimulation.

Merkel cells contain numerous synaptic vesicles, and synaptic vesicles typically contain high concentrations of protons⁴¹ which may be released from Merkel cells to activate ASICs to generate EPSCs. To further support this idea, we investigated whether alkalizing agents, which can reduce proton concentrations, can suppress, or abolish EPSCs. We treated the whisker hair follicle preparations with NH₄Cl and quinine, two pharmacological agents widely used to alkalize intracellular and vesicular pH^42–45, and assessed their effects on mechanically evoked EPSCs. NH₄Cl and quinine almost completely abolished mechanically evoked EPSCs (Figure 5I&J). On the other hand, the two alkalizing agents had no effect on MA currents (Figure 5K). In addition to mechanically evoked EPSCs, caffeine-evoked EPSCs were also largely abolished by alkalizing agents, including NH₄Cl, quinine, and quinine derivatives mefloquine, and chloroquine (Figure 5L&M). Furthermore, bafilomycin A1, a specific vacuolar H⁺ ATPase (V-ATPase) inhibitor⁴², significantly inhibited caffeine-evoked EPSCs of MNCs (Figure 5M&N). These results suggest that protons are the transmitters mediating fast excitatory synaptic transmission at MNCs.

Genetic deletion of ASIC3 impairs fast excitatory synaptic transmission at MNCs

To further support that ASICs mediate fast excitatory synaptic transmission at MNCs, we used mice in which ASIC3 channels were genetically deleted (ASIC3^−/−). The rationale for choosing ASIC3^−/− mice is that functional ASIC channels at the neurites of MNCs may be ASIC₁₊₂₊₃ heteromeric channels in wide type mice, and the deletion of ASIC3 subunits would result in ASIC₁₊₂ heteromeric channels, which would have lower proton sensitivity²⁴. Therefore, ASIC3^−/− mice provide a useful genetic tool to demonstrate the reduction of proton-evoked currents at the neurites of MNCs and the EPSC amplitudes in MNCs of ASIC3^−/− mice. This would allow us to establish that EPSCs at MNCs are mediated by ASIC3-containing channels. We first examined whether proton-evoked currents at the neurites of MNCs were affected following the genetic deletion of ASIC3. At the neurites of MNCs of wild type (WT) mice (ASIC3^+/+), focal puff-application of acidified Krebs solution (pH 5.5) to the MNCs evoked large inward currents (Figure 6A&B), which decayed rapidly (Figure 6A&C). In contrast, at the neurites of MNCs of ASIC3^−/− mice, the inward currents evoked by pH 5.5 Krebs solution were significantly smaller and the current decay was slower than those of ASIC3^+/+ mice (Figure 6A–C). Furthermore, the recovery from desensitization of proton-evoked currents took a significantly longer time in ASIC3^−/− mice than in ASIC3^+/+ mice (Figure S5). These results suggested that functional ASICs consisting of ASIC3 were localized at the neurites of MNCs in WT mice.

Figure 6. — A) Traces exemplify currents evoked by protons (pH 5.5) at the neurites of MNCs of an ASIC3^+/+ mouse (left) and an ASIC3^−/− mouse (right). **B&C**) Pooled data of the amplitudes (B) and decay τ (C) of proton-evoked currents in ASIC3^+/+ mice and ASIC3^−/− mice. D) Traces exemplify spontaneous EPSCs recorded at MNCs in an ASIC3^+/+ mouse (top) and an ASIC3^−/− mouse (bottom). Right panel, two scaled EPSCs, one from an ASIC3^+/+ mouse (blue) and another from an ASIC3^−/− mouse (red). **E-G**) Pooled data of the amplitude (E), frequency (F), and decay τ (G) of the spontaneous EPSCs recorded at MNCs of ASIC3^+/+ mice and ASIC3^−/− mice. H) Traces exemplify EPSCs and MA currents recorded at MNCs following a 10-μm mechanical step applied to MNCs of an ASIC3^+/+ mouse (left) and an ASIC3^−/− mouse (right). **I&J**) Pooled data of the amplitude (I) and frequency (J) of mechanically evoked EPSCs at MNCs of ASIC3^+/+ mice and ASIC3^−/− mice. Mechanical step, 10 μm. The 1^st nodes were voltage-clamped at −69 mV. K) Proportion of afferent fibers displaying RA, SA1, and SA2 impulses following whisker hair deflection (400 μm) in ASIC3^+/+ (left, n = 35) and ASIC3^−/− mice (right, n = 74). L) Traces exemplify SA1 impulses recorded from a whisker afferent fiber of an ASIC3^+/+ mice (top) and an ASIC3^−/− mouse (bottom) following whisker hair deflection for 400 μm. M) Left: Summary data of the number of SA1 impulses recorded from whisker afferent nerves of ASIC3^+/+ mice (blue, n = 11) and ASIC3^−/− mice (red, n = 11) in the static phase of mechanical step. Whisker hairs were deflected at 25, 50, 100, 200, and 400 μm. Right: Similar to the left, except that impulse numbers in the dynamic phase are shown. Data represent independent observations and mean ± SEM, *p < 0.05, **p < 0.01, *** p < 0.001, unpaired Student’s t-test, Chi-squared test, or two-way ANOVA; ^#p < 0.05, ^##p < 0.01, Bonferroni post hoc test. **See also** Figure S5–7.

We investigated whether deletion of ASIC3 impaired fast excitatory synaptic transmission at MNCs. The amplitudes of spontaneous EPSCs recorded from the MNCs of ASIC3^−/− mice were significantly smaller than those recorded from the MNCs of ASIC3^+/+ mice (Figure 6D&E). EPSC kinetics were also significantly slower (longer τ) in the MNCs of ASIC3^−/− mice than in the MNCs of ASIC3^+/+ mice (Figure 6D&G). Moreover, the EPSCs evoked by mechanical stimulation had significantly smaller amplitudes in the MNCs of ASIC3^−/− mice compared to those in the MNCs of ASIC3^+/+ mice (Figure 6H&I). The frequency of the evoked EPSCs was also significantly lower in the MNCs of ASIC3^−/− mice compared to those of ASIC3^+/+ mice (Figure 6H&J). While the genetic deletion of ASIC3 significantly diminished mechanically evoked EPSCs (Figure 6H–J), the MA currents, known to be mediated by Piezo2^17,18, were not significantly different in amplitudes (Figure 6H) between ASIC3^−/− mice (624 ± 130 pA, n = 7) and ASIC3^+/+ mice (730 ± 98 pA, n = 8).

We investigated whether the genetic deletion of ASIC3 impaired SA1 impulses in Aβ-afferent fibers. In this set of experiments, afferent impulses elicited by mechanical deflection of whisker hairs were recorded using pressure-clamped single-fiber recordings⁴⁶. In both ASIC3^+/+ mice and ASIC3^−/− mice, whisker deflections elicited three types of tactile responses: rapidly adapting (RA), slowly adapting type 1 (SA1), and slowly adapting type 2 (SA2) impulses (Figure 6K, L and Figure S6). In ASIC3^+/+ mice, the proportions of RA, SA1, and SA2 were 52%, 24%, and 24%, respectively. However, in ASIC3^−/− mice, the proportion of SA1 was significantly lower compared to ASIC3^+/+ mice, which was accompanied by a significant increase of RA proportion in ASIC3^−/− mice, whereas the proportion of SA2 remained the same as that in ASIC3^+/+ mice (Figure 6K). While SA1 impulses in ASIC3^+/+ mice displayed slowly adapting responses, the SA1 impulses in ASIC3^−/− mice showed relatively faster adapting (Figure 6L). Quantifying SA1 impulses showed that the impulse numbers during the static phase of mechanical displacements were significantly fewer in ASIC3^−/− mice compared to ASIC3^+/+ mice (Figure 6M). The impulse numbers in the dynamic phase were also fewer in ASIC3^−/− mice compared to ASIC3^+/+ mice (Figure 6M), suggesting that fast excitatory synaptic transmission at MNCs also drives some impulses in the dynamic phase. The genetic deletion of ASIC3 resulted in an increase in the proportion (Figure 6K) and responsiveness (Figure S6) of RA, most likely due to the diminishment of SA1 impulses in the static phase (Figure 6H–M), leading to many MNCs displaying RA-like impulses.

The genetic deletion of ASIC3 channels did not completely abolish proton-evoked currents (Figure 6A–C) and EPSCs (Figure 6D–J) at the MNCs, suggesting that functional ASICs may consist of ASIC1 and ASIC2 to form ASIC₁₊₂₊₃ heteromeric channels. For ASIC₁₊₂₊₃ channels²⁴, the deletion of ASIC3 subunits resulted in ASIC₁₊₂ channels, which were previously shown to have smaller proton-evoked currents, slower current kinetics, and slower recovery from desensitization compared to ASIC₁₊₂₊₃ channels²⁴. These differences in proton-evoked currents were observed at MNCs of ASIC3^+/+ and ASIC3^−/− mice in the present study (Figure 6A&B). We next Used ASIC2^−/− mice and found that the proton-evoked currents and EPSCs at the MNCs were comparable between ASIC2^−/− and ASIC2^+/+ mice (Figure S7), which is also consistent with functional ASICs being ASIC₁₊₂₊₃ in WT mice²⁴. Proton-evoked currents and EPSCs at MNCs of ASIC2^−/− mice, but not those of ASIC2^+/+ mice, were significantly inhibited by both psalmotoxin 1 and APETx2 (Figure S7), the two venom toxins that could block heteromeric ASIC₁₊₃ channels but not heteromeric ASIC₁₊₂₊₃ channels^36,47–49. These results support that the functional ASICs at MNCs were mainly heteromeric ASIC₁₊₂₊₃ channels in WT mice.

ASIC channels are required for whisker tactile discrimination

SA1 impulses generated at MNCs of Aβ-afferent terminals play a crucial role in tactile discrimination^4,8,12. Since ASICs mediate EPSCs at MNCs to drive SA1 impulses, we investigated whether ASICs are required for whisker tactile discrimination in rats using the tactile recognition behavioral test^50–52. In this set of experiments, two identical rectangular objects with smooth surfaces were placed at diagonal corners in an open field box, and animals were introduced into the box for tactile familiarization (Figure 7A, left). As innate tactile behaviors, control animals used their whiskers to palpate and recognize the two objects, and the time spent palpating the two smooth-surface-objects did not significantly differ (Figure 7B). Similarly, animals administered with amiloride and diminazene also spent equal amounts of time palpating the two smooth-surface-objects (Figure 7B). Subsequently, one of the smooth-surface-objects was replaced with a novel object featuring an identical shape but with a rough surface, allowing assessment of the animals’ ability to discriminate the roughness of the novel project (Figure 7A, right). In the control group, animals spent approximately twice as much time palpating the rough-surface-object compared to the smooth-surface-object (Figure 7C), with a preference index of approximately 70% (Figure 7D). This result indicates that the control animals discriminated the rough-surface-object as a novel tactile stimulus that differed from the smooth-surface-object previously explored. In contrast, animals administered with amiloride or diminazene spent approximately equal amounts of time palpating the smooth-surface-object and the rough-surface-object (Figure 7C), with the preference index near 50% (i.e., no preference to the rough-surface-object, Figure 7D). Amiloride and diminazene at the doses tested had no effect on animal’s motor functions, as indicated by the comparable travel distances in the open field box between the control and drug treatment groups (Figure S8). These results suggest that the animals treated with ASIC blockers were unable to discriminate the rough surface object as a novel tactile stimulus.

Figure 7. — A) Schematic illustration of the whisker tactile discrimination test, which consists of a tactile familiarization phase (left) with two identical smooth-surface-objects (S1 and S2) and a tactile discrimination phase with S1 and a rough-surface-object (R) (right). B) Time of palpation of S1 and S2 objects during the tactile familiarization phase in the control group, amiloride-treated group, and diminazene-treated group. C) Time of palpation of S1 and R objects during the tactile discrimination phase in the control group, amiloride-treated group, and diminazene-treated group. D) Preference index of control, amiloride-treated, and diminazene-treated groups. The dose of amiloride was 5 mg/kg and diminazene was 2.5 mg/kg. E) Time of palpation of S1 and S2 objects during the tactile familiarization phase in ASIC3^+/+ mice and ASIC3^−/− mice. F) Time of palpation of S1 and R objects during the tactile discrimination phase in ASIC3^+/+ mice and ASIC3^−/− mice. G) Preference index of ASIC3^+/+ mice and ASIC3^−/− mice. Data represent independent observations and mean ± SEM, *p < 0.05, ns, not significantly different, one-way ANOVE with Bonferroni or Dunnett’s post hoc tests. **See also** Figure S8.

To further support the idea that ASIC channels are involved in whisker tactile discrimination, we used ASIC3^−/− mice in the tactile discrimination experiments (Figure 7E–G) since ASIC3 channels are primarily expressed in the sensory neurons of the peripheral nervous system of mice^53–55. We tested whether animals’ tactile discrimination was impaired by the genetic deletion of ASIC3 in mice. Both ASIC3^+/+ and ASIC3^−/− mice were tested for whisker tactile discrimination in a manner similar to that described above for rats (Figure 7A). Both ASIC3^+/+ and ASIC3^−/− mice used their whiskers to palpate the two objects in the open field box (Figure 7A), and the time spent palpating the two smooth-surface-objects was not significantly different during the tactile familiarization phase (Figure 7E). In the subsequent tactile discrimination phase, after one of the smooth-surface-objects was replaced with a rough-surface-object (Figure 7A, right), ASIC3^+/+ mice spent approximately twice as much time palpating the rough-surface-object compared to the smooth-surface-object (Figure 7F), with a preference index of approximately 65% (Figure 7G). This result indicates that ASIC3^+/+ mice discriminated the rough-surface-object as a novel tactile stimulus. In contrast, ASIC3^−/− mice spent equal amounts of time palpating the smooth-surface-object and the rough-surface-object (Figure 7F), with a preference index of approximately 50% (i.e., no preference, Figure 7G). The total travel distances in the open field box for ASIC3^+/+ and ASIC3^−/− mice were not significantly different, indicating that the motor functions of ASIC3^−/− mice were not affected by the genetic deletion of ASIC3 channels (Figure S8). These results suggest that ASIC3^−/− mice could not discriminate the rough-surface-object as a novel tactile stimulus.

DISCUSSION

We demonstrate that following the initial mechanical transduction in Merkel cells^16–18, tactile signals are conveyed from Merkel cells to Aβ-afferent fibers through fast excitatory synaptic transmission at MNCs. The fast excitatory synaptic transmission at MNCs is mediated by protons/ASICs. Furthermore, ASICs are required for tactile-elicited SA1 impulses and whisker tactile discrimination, indicating the essential role of ASICs in sophisticated tactile tasks. Our findings have rekindled the curiosity in the role of ASICs in the sense of touch. Although ASICs were initially thought to be mechanical transducers for the sense of touch³⁰, extensive investigations for decades have failed to establish this role in mammals^{29–32,56,57}. Here we show that ASICs mediate fast excitatory synaptic transmission at MNCs to drive SA1 impulses in Aβ-afferent terminals. Thus, ASICs are key molecules for the sense of touch, even though they are not mechanoreceptors. Several lines of evidence support the role of ASICs in mediating fast excitatory synaptic transmission at MNCs. First, EPSCs at MNCs are mediated by Na⁺-selective channels based on their positive reversal potentials. Second, EPSCs at MNCs are abolished by ASIC blockers and reduced in ASIC3^−/− mice. Third, ASICs are expressed at the Aβ-afferent terminals innervating MNCs. Fourth, focal application of protons to the neurites of MNCs directly evokes excitatory currents. Fifth, neutralizing endogenous protons and blocking vesicular releases abolish EPSCs at MNCs. ASIC-mediating fast excitatory synaptic transmission indicates that protons serve as the principal transmitters at MNCs. Merkel cells contain releasable synaptic vesicles and other presynaptic release machinery^3,58, and synaptic vesicles usually contain high concentrations of protons built by v-ATPases^42,59. Protons have never been previously reported as principal transmitters in any synapse, and their role as the principal transmitters for tactile transmission at MNCs is highly unique.

ASIC-mediated fast synaptic transmission is responsible for generating SA1 impulses through the entire static phase of tactile stimulation since APs occurring during the static phase are completely abolished by ASIC blockers. ASIC-mediated fast synaptic transmission at MNCs is attributed to the Piezo2-mediated mechanical transduction in Merkel cells as demonstrated in the present study. Additionally, we have directly recorded Piezo2-mediated MA currents in the neurites of MNCs, providing support to the two-receptor site model of mechanical transduction at MNCs proposed in earlier studies^17,18. ASICs that mediate fast excitatory synaptic transmission at MNCs may be primarily heteromeric channels consisting of ASIC1, ASIC2, and ASIC3 subunits. This is supported by the immunochemical results in the current study and previous studies^29,37, previous electrophysiological studies on medium- to large-sized afferent neurons in mice²⁴, as well as our current study with ASIC3^−/− mice and ASIC2^−/− mice in combination with the venom toxin psalmotoxin-1 and APETx2. Our results align with the pharmacological profile of heteromeric ASIC channels shown in previous studies^36,47.

We show that whisker tactile discrimination is impaired by ASIC blockers or genetic deletion of ASIC3. This outcome is consistent with ASICs-mediating fast excitatory synaptic transmission at MNCs to drive SA1 impulses. SA1 impulses generated at MNCs are critical to tactile discrimination function^4,8,12. Interestingly, genetic deletion of Merkel cells impaired cutaneous tactile discrimination but not whisker tactile discrimination⁶⁰. This discrepancy is thought to be due to a functional compensation by other types of tactile end organs in whisker hair follicles⁶⁰. Previous studies with ASIC knockout (KO) mice have produced a range of tactile responses, including no significant effects, decreases or increases of tactile responses^30,31,56,57. The inconsistent tactile responses observed in previous studies with ASIC KO mice is thought to be at least partially due to functional compensation³¹. Additionally, the previous behavioral tests in ASIC KO mice used the von Frey test^29,31,56, which may not be an appropriate approach to determine behavioral outcomes with respect to functional changes in MNCs. Through whisker tactile discrimination tests and pharmacological inhibition of ASICs, along with the genetic deletion of ASIC3, our results suggest a role of ASICs in tactile discrimination. One limitation for the systemic administration of ASIC blockers in the present study is their potential CNS actions, which may affect the analysis of the tactile discrimination test. However, in our study using ASIC3^−/− mice, a CNS effect is unlikely to account for the tactile behavioral outcomes since ASIC3 channels are primarily expressed in primary afferent nerves and not in the CNS of wild type mice^53–55. Mice whose ASIC3 channels are conditionally knocked out from Aβ-afferent nerves that innervate MNCs of whisker hair follicles, if available in the future, will provide more conclusive evidence to confirm the essential role of ASIC channels in tactile discrimination.

Clinical conditions such as diabetes and chemotherapy^13,14 often lead to numbness and poor tactile discrimination in patients’ fingertips, presenting a significant clinical problem that affects patients’ quality of life. ASIC-mediated fast excitatory synaptic transmission and tactile encoding at MNCs for tactile discrimination may offer insight into pathological mechanisms underlying sensory dysfunctions in these clinical conditions, with potential clinical implications.

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jianguo Gu (jianguogu@uabmc.edu)

Materials availability

No new unique reagents were generated in this study.

Data and code availability

This paper does not report original code.
Data reported in this paper will be shared by the lead contact upon request.
All data and any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Sprague Dawley rats (Envigo, Prattville, AL, USA) aged 24–60 days (both male and female) were used in this study. ASIC2 gene knockout mice (ASIC2^−/−, both male and female) and their littermate wild type mice (ASIC2^+/+) were generated from heterozygous ASIC2 mouse breeders (ASIC2^+/−, Jackson Labs). ASIC3 gene knockout mice (ASIC3^−/−, both male and female) and their littermate wild type mice (ASIC3^+/+) were generated from heterozygous ASIC3 mouse breeders (ASIC3^+/−, Jackson Labs). C57BL/6J mice were also used in some experiments (wild type, Jackson Labs). The mice were used at the aged of 6–13 weeks in this study. All animals were housed in a temperature-controlled room at 23°C and maintained on a 12-hour light/dark cycle. Animal care and use adhered to the guidelines for the care and use of experimental animals set forth by the National Institutes of Health (NIH). The experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Alabama at Birmingham.

METHOD DETAILS

Ex vivo Whisker hair follicle preparations

Ex vivo whisker hair follicle preparations were made from both rats and mice in a similar manner as described in our previous studies^16,20,61. In brief, animals were anesthetized with 5% isoflurane and then sacrificed by decapitation. Whisker pads were dissected out, and whisker hair follicles were carefully pulled out from the whisker pads and placed in a Petri dish filled with a Krebs bath solution that contained (in mM): 117 NaCl, 3.5 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 1.2 NaH₂PO₄, 25 NaHCO₃ and 11 glucose. The pH of the solution was adjusted to 7.3, and the osmolarity was set at 325 mOsm. The Krebs bath solution was saturated with 95% O₂ and 5% CO₂ and maintained at a temperature of 32°C. The capsules of the whisker hair follicles were cut open and removed with a pair of fine scissors, and the whisker hair follicles (without the capsules) were perfused with the Krebs bath solution at 32°C for 15 min. The temperature of the Krebs bath solution was then gradually reduced to room temperature (24°C). Whisker hair follicles were then anchored on the bottom of a recording chamber. To facilitate the penetration of recording electrode through tissues surrounding axons of Aβ-afferent fibers, whisker hair follicles were incubated with 0.05% collagen plus 0.01% to 0.06% dispaseⅡ in the Krebs bath solution for 8 – 20 min. The enzymes were then washed off with the Krebs bath solution, and the connective tissues covering axon terminals were gently removed as much as possible by a glass pipette controlled by a micromanipulator. Whisker hair follicle preparations were continuously perfused with the Krebs bath solution saturated with 95% O₂ and 5% CO₂ at room temperature (24°C) during experiments.

Pressure-patch-clamp recordings at the 1^st nodes and heminodes on Aβ-afferent terminals that innervate MNCs of whisker hair follicles

The 1^st nodes or heminodes of Aβ-afferent terminals that innervate MNCs of whisker hair follicles were identified in the ex vivo whisker hair follicle preparations based on morphological features observed under the bright field microscope with a 40x (NA 0.80) water immersion objective and an infrared CCD camera (IR-2000, Dage-MTI, USA). The 1^st nodes and the heminodes of the Aβ-afferent terminals were in front of the ringwulst of whisker hair follicles and could be directly observed under the 40x objective.

Patch-clamp recordings were applied at the 1^st nodes or the heminodes using the pressure-patch-clamp recording technique described in our previous study with modification^62,63. In brief, recording electrodes were pulled with a Flaming/Brown Micropipette Puller (P-97, Shutter Instruments, USA). The electrode resistance after filling recording electrode internal solutions ranged from 4.5 to 7 MΩ for patch-clamp recordings. For most voltage-clamp experiments, a Cs⁺-based internal solution was used and the solution contained (in mM): 140 CsSO₄, 20 NaCl, 0.5 CaCl₂, 2 MgCl₂, 5 EGTA, 5 HEPES, 5 Na₂ATP and 0.5 GTP-TRIS salt; the pH of the solution was adjusted to 7.35 with CsOH. For some experiments, recording electrodes were filled with a K⁺-based internal solution containing (in mM): 105 K-gluconate, 30 KCl, 0.5 CaCl₂, 2.4 MgCl₂, 5 EGTA, 10 HEPES, 5 Na₂ATP and 0.33 GTP-TRIS salt; the pH of the solution was adjusted to 7.35 with KOH. The use of a high Cl⁻ concentration in recording electrode internal solution was because trigeminal afferent nerves normally have high intracellular Cl⁻ concentrations. The junction potentials were 9.1 mV and 12 mV for Cs⁺- and K⁺-based recording electrode internal solutions, respectively. To access axon membranes of the 1^st nodes or heminodes by the recording electrode and achieve high quality membrane seals, a high-speed pressure-clamp device (HSPC-1, ALA Scientific instruments, USA) was connected to the patch-clamp recording electrode to finely control the internal pressures of the patch-clamp recording electrode. Initially, a high positive pressure of 200 mmHg was applied to the recording electrode to pressure-clean the surface areas around the 1^st node or heminodes. Intra-electrode positive pressures were maintained at 200 mmHg while the electrode penetrated the tissues covering the axons of 1^st nodes of heminodes. Once the recording electrode penetrated through the tissue layer, intra-electrode pressures were reduced to 20–30 mmHg, and the recording electrode further penetrated a thinner membrane layer to approach the axons of the 1^st nodes or heminodes. Optimal access to axon membranes was determined by the reduction of seal-test currents, the appearance of a small current oscillation, and a two- to three-fold increase in recording electrode resistance. Once the electrode tip optimally accessed nodal membranes, intra-electrode positive pressure was gradually reduced, and a negative pressure of −2 to −10 mmHg was applied to the recording electrodes until gigaohm seals (usually > 5 GΩ) were formed between the recording electrodes and nodal axon membranes. The formation of gigaohm seals usually took less than 3 min. To achieve the whole-cell configuration, axon membranes were ruptured by a few short electrical pulses at an intensity of 280 – 325 mV and a duration of 100 ms for each pulse. The electrical pulses were delivered through the patch-clamp recording electrodes, and the intra-electrode pressures were held at a constant negative pressure of −20 to −35 mmHg during the application of electric pulses. After establishing the whole-cell configuration, the negative pressure was reduced to −3 to −8 mmHg and maintained during recordings.

Spontaneous EPSCs, EPSCs evoked by mechanical stimulation, and EPSCs induced by caffeine were recorded under the voltage-clamp configuration with axon membranes of the 1^st nodes or the heminodes held at −72 mV (command voltage: −60 mV) with the K⁺-based recording electrode internal solution or at −69 mV (command voltage: −60 mV) with the Cs⁺-based recording electrode internal solution, unless otherwise indicated. Proton-evoked whole-cell currents on the neurites of MNCs were also recorded under the same voltage-clamp configuration. To investigate the nature of ion channels that mediated EPSCs at MNCs, reversal potentials of EPSCs were determined with axons of the 1^st nodes or the heminodes held at −70, −40, −10, 20, 50 mV. Furthermore, to determine whether the EPSCs were mediated by a Na⁺ selective ion channels, reversal potentials of EPSCs were determined with Na⁺ concentration gradients ([Na⁺]_out/[Na⁺]_in) being approximately 5:1 ([Na⁺]_out=143 mM, [Na⁺]_in= 30 mM) and approximately 1:1 ([Na⁺]_out=26.2 mM, [Na⁺]_in= 30 mM). The reversal potentials for a Na⁺ selective ion channels would be > 50 mV with [Na⁺]_out/[Na⁺]_in being 5:1 and near 0 mV with [Na⁺]_out/[Na⁺]_in being 1:1. In all whole-cell patch-clamp recording experiments, unless otherwise indicated, membrane voltages mentioned in the texts have been corrected for calculated junction potentials. Signals of both voltage-clamp and current-clamp experiments were recorded and amplified using a MultiClamp 700B amplifier with a 3 kHz Bessel filter, digitized by the Digidata 1550B (Molecular Devices), and sampled at 25 kHz using the pCLAMP 11 software (Molecular Devices, Sunnyvale, USA). Unless otherwise specified, all experiments were carried out at 24°C.

Morphology of neurites of MNCs

To determine the morphological features of the neurites of MNCs, the axons of Aβ-afferent fibers that innervated MNCs were intra-axon-labeled with the fluorescent dye Alexa Fluor 555 (ThermoFisher Scientific, Waltham, USA). Alexa Fluor 555 was prepared at a concentration of 85 μM in the electrode internal solution, and filled to the recording electrode, which was first back-filled with a small amount of normal electrode internal solution at the tip of the electrode. The dye-containing electrode was then applied to the 1^st nodes or heminodes to make whole-cell patch-clamp recordings. Following the establishment of the whole-cell configuration for over 50 min, the neurites of MNCs were directly visualized under a fluorescent microscope. The fluorescence excitation was provided by a mercury lamp. A filter set with an excitation wavelength of 500 to 550 nm and an emission wavelength of 565 to 625 nm was used for imaging Alexa Fluor 555 fluorescence. The images of dye-labeled axons were captured with a peltier-cooled charge-coupled device (CCD) camera (Photometrics Cool SNAP^™ HQ²). Stacked fluorescent images of MNCs with a series of sections along the z-axis were captured by the MetaFluor Imaging System software (Molecular Devices, USA). The images were then analyzed using the ImageJ software (National Institutes of Health, USA) to measure the lengths and thickness of the neurites of MNCs, as well as the distance between the 1^st nodes and heminodes. In some cases, after finishing the recordings, the dye-containing electrode was slowly removed and the whisker hair follicle was fixed with 4% paraformaldehyde for 30 min and washed 3 times with PBS solution. Antifading solution (ProLong^™ Diamond Antifade Mountant, invitrogen, USA) was then applied to the whisker hair follicle preparation, and the sample was covered with a cover-glass. The labeled MNCs were examined, and images captured under a confocal microscope (Nikon A1 Plus, Nikon, Japan).

Patch-clamp recordings from Merkel cells in situ in whisker hair follicles

Patch-clamp recordings were made from Merkel cells in situ in whisker hair follicles using a method described in a previous study¹⁶. In brief, preparations of rat whisker hair follicles were made in the same manner as described above. After the exposure of whisker hair follicles to dispase II (0.05%) and collagenase (0.01%-0.06) in Krebs solution for 8~15 min, the enzymes were washed off with the Krebs bath solution. The ring sinus and the glassy membranes were removed using a glass electrode controlled by a manipulator, and the procedure was performed under a 40X objective lens. Patch-clamp recordings were made at room temperature of 24°C from visually identified Merkel cells. Recording electrodes were filled with an internal solution containing (in mM): 135 K-gluconate, 5 KCl, 0.5 CaCl₂, 2 MgCl₂, 5 EGTA, 5 HEPES, 5 Na₂ATP and 0.5 GTP-TRIS salt. The pH of the solution was adjusted to 7.3 with KOH. Signals were amplified and filtered at 3 kHz using the Multiclamp 700B amplifier and sampled at 25 kHz using pCLAMP 11 software (Molecular Devices, USA). Membrane and action potential (AP) properties of Merkel cells were determined under the whole-cell current-clamp mode following step current pulses injected into Merkel cells through patch-clamp electrodes. The step currents ranged from 5 to 15 pA in increments of 5 pA, with a pulse duration of 5 s for each pulse. APs evoked by mechanical stimulation were recorded under the whole-cell current-clamp configuration following membrane displacements applied to Merkel cells with the ramp-and-hold mechanical steps that ranged from 3 to 5 μm in increments of 1 μm. In a subset of experiments, AP spikes evoked by the mechanical steps were recorded under the cell-attached recording configuration with membranes held at 0 mV. In a different set of experiments, voltage- and mechanical stimulation-evoked currents (MA currents) were recorded with Merkel cells voltage-clamped at −75 mV. For voltage-evoked currents, voltage steps were applied from −80 mV to 50 mV in increments of 10 mV and a duration of 500 ms. For mechanically evoked currents, membrane displacements were applied to Merkel cells using the ramp-and-hold steps that ranged from 0.5 to 5 μm in increments of 1 μm. In all Merkel cell recordings, unless otherwise indicated, membrane voltages mentioned in the texts have been corrected for calculated junction potentials of 15 mV.

Mechanical Stimulation in patch-clamp recording experiments

Mechanical stimulation was applied by poking hair follicle tissues using a mechanical probe fabricated from a glass pipette (1.1 mm inner diameter and 1.5 mm outer diameter, thin-wall). The glass pipette was first pulled by a Brown-Flaming P-97 pipette puller and then fire polished to create a blunt and smooth tip. The tip sizes (diameters) were approximately 20 μm for stimulating MNCs to evoke EPSCs and approximately 2 μm for stimulating Merkel cells to evoke mechanically activated responses in Merkel cells. The mechanical probe was mounted onto a pipette holder and stably attached to a piezo actuator (P841.20; Physik Instrumente, USA) which was connected to a computer-programmable piezo device (E-625.SR; Physik Instrumente, USA). The piezo device was connected to the Digidata 1440A, allowing the movement of the mechanical probe to be controlled by the pClamp11 software. The mechanical probe was positioned at an angle of 30 degrees to the surface of the hair follicle preparation. The distance from the probe tip to the surface of the hair follicle tissue was set in such a way that the tip would contact the surface when the probe had a forward movement. In recordings of mechanical responses in Merkel cells, Merkel cells were indirectly poked by the mechanical probe, achieved by poking adjacent non-recorded cells¹⁶. For evoking EPSCs with mechanical probes, mechanical stimuli were applied by poking the region (receptive field) of MNCs, which is within the radius of approximately 30 μm around the heminode. Note that the number of Merkel cells activated by mechanical stimulation to evoke EPSCs depends on several factors, including the maximum number of Merkel cells in each receptive field, the size of the probes, and the distance of mechanical displacement. Unless otherwise stated, mechanical stimulation was applied in a ramp-and-hold step, with step sizes ranging from 1 to 10 μm for evoking EPSCs and 0.5 to 5 μm for evoking mechanical responses in Merkel cells. For each displacement step, the forward movement was exponentially increased to reach the targeted displacement distance with an equation f(t) = D(1-e^−t/τ), where D was the targeted displacement distance, τ was the time constant of distance increase and was at the same value of 2 ms for different displacement distances. The duration of the step of static phase of the mechanical stimulation was 5 sec.

Pressure-clamped single-fiber recordings of whisker afferent impulses evoked by whisker hair deflections

Ex vivo whisker hair follicle preparations were made from ASIC3^+/+ and ASIC3^−/− mice, following a similar procedure as described above, except that the capsule of each whisker hair follicle with attached afferent bundle (approximately 2 mm) was kept intact. The whisker hair follicle was placed in a 30-mm recording chamber with a Sylgard Silicone-coated bottom and affixed to the bottom of the chamber with tissue pins, while the nerve bundle was affixed with a tissue anchor within the same recording chamber. The cutting end of the nerve bundle was briefly exposed to a mixture of 0.05% dispase II plus 0.05% collagenase for 30 s, after which the enzyme was washed off using normal Krebs solution. This gentle enzyme treatment aimed to facilitate the separation of individual afferent fibers at the cutting end of the nerve bundle, allowing for the aspiration of a single fiber into the recording electrode for pressure-clamped single-fiber recordings (see below). Subsequently, the recording chamber was mounted on the stage of an Olympus BX51WI upright microscope. The whisker hair follicle preparation was continuously superfused with normal Krebs bath solution saturated with 95% O₂ and 5% CO₂. Unless otherwise indicated, the Krebs bath solution in the recording chamber was maintained at 24°C throughout the experiments.

The pressure-clamped single-fiber recording was conducted in a manner similar to our previous study^46,64 for detecting action potential (AP) impulses elicited by mechanical displacement of whisker hairs. Briefly, the recording electrodes for the pressure-clamped single-fiber recordings were made with thin-walled borosilicate glass tubing without filament (inner diameter 1.12 mm, outer diameter 1.5 mm, World Precision Instruments, Sarasota, FL). These electrodes were fabricated using a P-97 Flaming/Brown Micropipette Puller (Sutter Instrument Co., Novato, CA), and the tip of each electrode was fire-polished to a final size of 4 to 10 μm in diameter using a microforge (MF-900, Narishige). The recording electrode, filled with Krebs bath solution, was mounted onto an electrode holder connected to a high-speed pressure-clamp (HSPC) device (ALA Scientific Instruments, Farmingdale, NY) to allow fine controls of intra-electrode pressures. Under a 40x objective, the end of the individual afferent nerve was visualized and isolated by applying a low positive pressure (approximately 10 mmHg or 0.19 Psi) to the recording electrode. Subsequently, a single nerve fiber’s end was aspirated into the recording electrode under a negative pressure of approximately 10 mmHg. Once the nerve fiber’s end entered the recording electrode in a length of approximately 10 μm, the electrode pressure was readjusted to −3 ± 2 mmHg and maintained at this pressure throughout the experiment. Nerve impulses in the single afferent fiber were recorded under the I₀ configuration and amplified using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA) in the AC recording mode (100 × AC membrane potential, 5 × gain, 0.1 Hz AC filter, 3 kHz Bessel filter). The analog signals were digitized by the Digidata 1550B (Molecular Devices) and sampled at a rate of 25 kHz with the Axon Clampex 11 software (Molecular Devices). Unless otherwise specified, all experiments were carried out at 24°C.

Mechanical stimulation was applied by deflecting whisker hairs in a manner described in our previous study⁶⁴. Briefly, whisker hair was trimmed to have the length of approximately 3 mm. A mechanical probe, made with an L-shaped glass pipette, was mounted on a pipette holder and controlled by a programmable manipulator (MPC-385, Sutter). The trimmed hair shaft was fit into the end of the mechanical probe, and the position of the whisker hair shaft was adjusted to a neutral position at which the hair shaft was not bended toward any direction. The whisker hair was then deflected in 4 directions at 90° increments (360° total) using the mechanical probe, and in each direction, the mechanical displacement was applied in a ramp-and-hold manner. The interval between any two angular deflections was 60 seconds. The ramp-and-hold deflection consisted of a 200 ms ramp-up to 400 μm (dynamic phase), 6.2 s hold at 400 μm (static phase), and a 200 ms ramp-down to the original position. All 4 angular deflections were made by a programmable manipulator that was controlled by a software (Multi-Link^™, Sutter). Nerve impulses evoked by the mechanical stimulation were recorded using the pClamp 11 software. Following the angular stimulation described above, the angle that evoked the highest impulse numbers was chosen as the angle for classifying mechanical response types, including rapidly adapting (RA), slowly adapting type 1 (SA), and slowly adapting type 2 (SA2) responses, as described in our previous study⁶⁴. The angle with the largest responses was also chosen for further investigating mechanical responses evoked by difference distances of whisker hair displacements. The displacement distances tested were 25, 50, 100, 200, and 400 μm, with dynamic phases (ramp-up) of 50, 60, 80, 120, and 200 ms, respectively, and a 6.2-s static phase for holding the displacements, followed by a ramp-down to the original position.

Pharmacology

Pharmacological agents were applied to whisker hair follicle preparations either by focal puff-applications or bath applications. For focal puff-application, glass pipettes with a 1.1 mm inner diameter and 1.5 mm outer diameter were pulled by a Flaming/Brown Micropipette Puller (P-97, Shutter Instruments, CA, USA) to generate narrow tips with diameters of approximately 2 μm for the fabrication of puff pipettes. Testing compounds were loaded into the puff pipettes. The puff pipette was mounted on a micropipette holder, and connected to a valve pressure control system (Picospritzer II, Parker Hannifin Corporation, USA). The tip of the puff pipette was positioned at an angle of 30 degrees to the surface of the hair follicle preparation, aimed at the area of MNCs, and placed approximately 20 μm away from the heminode. Each testing compound was puff-applied with a pressure of 20 psi. The duration of focal puff-application was 1 s, unless otherwise indicated.

For bath application of testing compounds, each compound was perfused to ex vivo whicker hair follicle preparations for 10 min unless otherwise indicated. The testing compounds were delivered through a tube with an internal diameter of 1 mm, and the outlet of the tube was positioned 0.5 cm away from the recording site. The tube was connected to a peristaltic pump (Miniplus 3 Peristaltic Pumps, Gilson, USA), and testing compounds were bath applied by the drug delivery pump at a rate of 2 ml/min. Testing compounds were made in Krebs bath solution for both focal puff-application and bath applications.

To test the effects of Ca²⁺ released from caffeine-sensitive Ca²⁺ stores on EPSCs, caffeine (10 mM) was bath-applied and EPSCs were recorded for 10 – 30 min. To examine the effects of ASIC channel blockers on mechanically evoked or caffeine-induced EPSCs, amiloride (200 μM), diminazene (100 μM), or nafamostat (100 μM) were bath-applied. EPSCs were determined before (control) and 10 min following the applications of these ASIC blockers. EPSCs recorded from whisker hair follicles of mice were also tested with 1 μM psalmotoxin 1 and 1 μM APETx2, two subtype specific ASIC channel blockers. To assess the role of Ca²⁺ entry on EPSCs, EPSCs were recorded before and 10 min following the bath application of 300 μM Cd²⁺ (a voltage-gated Ca²⁺ channel blocker) or a low Ca²⁺ Krebs bath solution to see whether EPSCs could be abolished by Cd²⁺ or low Ca²⁺. The low Ca²⁺ Krebs bath solution was similar to the normal Krebs bath solution, except the Ca²⁺ concentration was reduced to 20 μM. To explore whether protons may mediate EPSCs, the effects of alkalization on EPSCs were examined. In this set of experiments, EPSCs were recorded before and 10 min following the bath application of agents that could alkalize intracellular pH. The alkalizing agents being tested included NH₄Cl (10 mM), quinine (450 μM), chloroquine (500 μM), or mefloquine (200 μM), and bafilomycin A1 (400 μM). To investigate whether EPSCs may be mediated by 5-HT₃ receptors, adrenergic β₂-receptors, or ionotropic glutamate receptors, EPSCs were tested in the absence (control) and presence of the 5-HT₃ receptor blocker VUF10166 (20 μM), the adrenergic β₂-receptor blocker ICI118551 (50 μM), or the ionotropic glutamate receptor blockers CNQX (10 μM) + APV (50 μM). To test whether protons could activate ASIC channels to directly evoke inward currents at the neurites of MNCs, normal Krebs bath solution (pH of 7.4, control), and acidified Krebs bath solutions with pH values of 6.5, 6, 5.5, 5, and 4.5 were puff-applied to MNC regions while recordings were performed with axons held at −69 mV. Acidified Krebs solutions were made with the addition of citric acid into the normal Krebs bath solution. In a different set of experiments, the currents evoked by puff-applications of pH 5 Krebs solution were recorded at the holding voltage of −70, −40, −10, 20 and 50 mV. The inhibitory effects of ASIC channel blockers, including amiloride (200 μM) and diminazene (100 μM), were tested on the inward currents evoked by the pH 5 Krebs solution. In this set of experiments, inward currents evoked by the puff-application of pH 5 Krebs solution were recorded before (control) and 10 min following the bath application of amiloride (200 μM) or diminazene (100 μM).

To determine whether 5-HT, NA, glutamate, and Ach can directly excite neurites of MNCs, 2 mM of each of these compounds were puff-applied to MNC areas of whisker hair follicle preparations while recordings were conducted at the 1^st nodes under the voltage-clamp configuration to detect whether these testing compounds could evoke depolarizing (inward) currents. All recordings were performed with nodal axons held at −72 mV, unless otherwise indicated.

To test whether ASIC channel blockers may have non-specific effects on the electrophysiological properties of Merkel cells, whole-cell patch-clamp recordings were performed from Merkel cells. Under the current-clamp configurations, membrane depolarization and APs were evoked by the injection of depolarizing currents into Merkel cells via recording electrodes. Under the voltage-clamp configuration, voltage-activated currents were recorded following voltage steps. The above experiments were performed before (control) and following the bath application of 200 μM amiloride or 100 μM deminazene to see whether there were changes in electrophysiological properties and membrane currents following the applications of these two ASIC channel blockers.

Immunohistochemistry

Animals were anesthetized with isoflurane and decapitated. Whisker pads were cut off and placed in a petri dish that contained 2 ml ice-cold L-15 medium. Whisker hair follicles were removed from whisker pads with a pair of forceps, embedded in OCT compound medium (O.C.T. Compound, Sakura-finetek, Japan), and fresh frozen with powered dry ice. The whisker hair follicles were then sectioned into 18, 30 or 60 μm sections at −20°C using a Leica CM1860 cryomicrotome (Leica Biosystems, Nusslock, Germany). Whisker hair follicle sections were fixed by 4% paraformaldehyde (PFA) at 4°C for 1 hour, then washed 3 times with 1x phosphate buffer solution (PBS), 3 times (20 min each) with the PBS that contained 1% triton-100 (PBST), and kept in the PBST solution at 4°C overnight. The next day, the sections were incubated with primary antibodies in the PBST solution that contained 5% heat-inactivated normal goat serum, 0.125% sodium azide, and 20% DMSO (antibody buffer solution, ABS) for 72 hours at 4°C. After washing the sections in the PBST 3 times at the room temperature, the sections were further washed with PBST 3 times each for 20 minutes at 4°C. The sections were then incubated with a secondary antibody for 4 hours at 4°C in the ABS solution. Following 3 washes in PBST at the room temperature, the sections were further washed with PBST 3 times each for 20 minutes at 4°C. Then, anti-fade mountant medium (ProLong Diamond Antifade Mountant, Invitrogen, Carlsbad, CA, USA) was added, and sections were covered by cover-glasses. The following primary antibodies were used for the immunostaining experiments: guineapig anti-ASIC1 (1:400, AGP-053, Alomone Labs, Israel), rabbit anti-ASIC2a (1:500, PA5–77729, Invitrogen, USA), and rabbit ASIC3 (1:250, PA5–77734, Invitrogen, USA). For ASIC1 immunostaining experiment (ASIC1-ir), a different set of experiment was performed in the presence of a blocking peptide to the ASIC1 antibody (4 μg/ml, BLP-SC014, Alomone Labs, Israel). The following secondary antibodies were used for visualizing the immunostaining results: Alexa Fluor 594-conjugated goat anti-rabbit (1:500, A-11076, Invitrogen, Carlsbad, CA, USA), Alexa Fluor 488-conjugated goat anti-rabbit (1:500, A-11008, Invitrogen, Carlsbad, CA, USA), Alexa Fluor 488-conjugated goat anti-guineapig (1:500; ab150169, Abcam, Cambridge, MA, USA). Images of immunostaining were captured under a confocal microscope (LSM800, Zeiss, Germany).

Whisker texture discrimination test

Whisker texture discrimination tests were performed with a method modified from the novel object recognition test^51,65. In brief, rats were first randomly assigned into three groups, control group, amiloride-treated group, and diminazene-treated group. These three groups of rats would later be i.p. administered with saline (control), amiloride (5 mg/kg), and diminazene (2.5 mg/kg). Prior to the whisker texture discrimination test, each rat first underwent two sessions of habituation, each for 10 min a day for two consecutive days in an open field test box without any object. The open field box was made with a black acrylic material, with a dimension of 60 cm (length) × 60 cm (width) × 40 cm (height) (Maze engineers, USA). On the day of performing the whisker texture discrimination test, two identical smooth-surface-objects were affixed in the two diagonal corners of the open field box. Each object was 20 cm away from the corner. The smooth-surface-objects were made of transparent acrylic material in a bookstand shape, and the standing part of each object had a dimension of 18 cm (height) × 12 cm (length) × 0.3 cm (width). The surface on the lower half of the standing part of the objects was rubbed thoroughly⁶⁵ with a fine sandpaper of high grit of P3000 to generate a smooth texture on the surface. In addition to the two smooth-surface-objects, a rough-surface-object was made in the same materials and identical shape, except the object was rubbed by a coarse sandpaper of low grit of P400 to generate a rough surface on the lower half of the stand. The lower half of the stands were treated with sandpaper because those are the areas where animals palpate the objects with their whisker hairs. The rough-surface-object would be used later to replace one of the smooth-surface-object during the texture discrimination test performed below. In experiments, after the two smooth-surface-objects were first affixed in the open field box, rats were i.p. administered with saline (control group), amiloride at the dose of 5 mg/kg (amiloride group), or diminazene at the dose of 2.5 mg/kg. Fifteen (15) min after the injections, a rat was placed in the open field box for 5 min, and the animal palpated the two smooth-surface-objects with their whiskers and traveled within the open field box, which were video-recorded by a camera (Logitech BRIO). The rat was then removed from the open field box and returned to its home cage, and one of the two smooth-surface-objects was replaced with the rough-surface object. Five min later, the animal was placed back into the open field box, and its palpation behaviors with its whiskers and travel within the open field box were video-recorded for 5 min. Animals’ behaviors, including their palpation of the two objects and travel within the open field box, were analyzed offline. Whisker palpation time for each animal was measured manually using a timer^51,65 in a blinded manner in which the examiner did not know the experimental groups. The total distance traveled within the open field box was measured using an artificial intelligence-based tracking system (Deeplabcut) (Mathis et al., 2018) on a Cheaha supercomputer with GPU (Pascalnode: UAB facility). Whisker texture discrimination tests were also performed with ASIC3^+/+ and ASIC3^−/− mice using the similar method described above for rats, except that the open field box for mice had the dimension of 40 cm (length) × 40 cm (width) × 30 cm (height) (Maze engineers, USA). All behavioral experiments were performed in a room illuminated with a red light at an illuminance of 10 lux, and the temperature of the room was 24°C. The objects and open field box were thoroughly cleaned with 70% ethanol prior to each test.

QUANTIFICATION AND STATISTICAL ANALYSIS

Electrophysiological data were collected from hair follicles of 91 male and 67 female rats, and 48 male and 30 female mice. The data of tactile behavioral experiments were collected from of 14 male and 13 female rats, and 13 male and 11 female mice. We observed no signs of differences in electrophysiological and tactile behavioral results and between the male and female animals. Thus, their data were combined for analysis. Electrophysiological data were analyzed using the CLAMPFIT 11 software. Synaptic transmission data were analyzed using the Minianalysis software. Statistical analyses were performed using Graph Pad Prism (version 9). In the tactile discrimination behavioral test, the preference index was calculated as the percentage of palpation time for the rough-surface-object in the total palpation time for both rough-surface-object and smooth-surface-object during the tactile discrimination phase. Unless otherwise indicated, data were reported as individual values as well as mean ± SEM of n independent observations. Data normality was analyzed by the Shapiro Wilk test. Statistical significance was evaluated using unpaired and paired Student’s t-tests, one-way ANOVA with Bonferroni or Dunnett’s post hoc tests, Kruskal-Wallis test with Dunn’s post hoc test, two-way ANOVA with Bonferroni post hoc test. Differences were significant with *p < 0.05, **p < 0.01, ***p < 0.001, and not significant (ns) with p ≥ 0.05.

Supplementary Material

NIHMS1962828-supplement-1.pdf^{(1.6MB, pdf)}

Video S1. Detailed morphological features of MNCs in a fresh whisker hair follicle of a rat following Alexa fluor 555 intra-axon-labeling under the whole-cell patch-clamp recording configuration, related to Figure 1.

Download video file^{(25.6MB, mp4)}

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
ASIC1	Invitrogen	Cat#PIPA526278
ASIC2a	Invitrogen	Cat#PIPA577729
ASIC3
Alexa Fluor 594-conjugated goat anti-rabbit	Invitrogen	A-11076
Alexa Fluor 488-conjygated goat anti-rabbit	Invitrogen	A-11008
Alexa Fluor 594-conjugated goat anti-mouse	Invitrogen	A-11032
Chemicals, Peptides, and Recombinant Proteins
Acetylcholine chloride	Sigma-Aldrich	Cat# A6625
Alexa Fluor 555	ThermoFisher Scientific	A20501MP
Amiloride hydrochloride	Tocris	Cat#0890
Amiloride hydrochloride hydrate	Sigma-Aldrich	Cat#A7410
Ammonium chloride	Sigma-Aldrich	Cat#213330
Antifade Mountant	ThermoFisher Scientific	P36965
APETx2	Almone labs	Cat#STA-160
Bafilomycin A1	Tocris	Cat#1334
Cadmium Chloride hydrate	Sigma	Cat#C3141
Caffeine	Sigma-Aldrich	Cat#C0750
Cesium chloride	MilliporeSigma	C3139
Chloroquine	Sigma-Aldrich	Cat#C6628
Citric Acid	Sigma	Cat#C0759
Collagenase	MilliporeSigma	C0130
Diminazene aceturate	Tocris	Cat#6705
Dispase II	Roche	Cat#4942078001
DL-Norepinephrine hydrochloride	Sigma-Aldrich	Cat# A7256
Goat serum	Jackson Immuno Research	005-000-121
ICI118551 hydrochloride	Sigma-Aldrich	Cat#I127
Isoflurane	Vet One	V1–502017
L-Glutamic acid	Sigma	Cat# G8415
Mefloquine hydrochloride	Sigma-Aldrich	Cat#M2319
Nafamostat mesylate	Sigma-Aldrich	Cat#N0289
Paraformaldehyde (PFA)	Sigma	P6148
PBS	ThermoFisher Scientific	28374
Psalmotoxin 1	Tocris	Cat#5042
Quinine	Sigma	Cat#145904
Serotonin hydrochloride	Sigma-Aldrich	Cat#H9523
Serotonin creatinine sulfate monohydrate	Sigma-Aldrich	Cat# H7752
Tetrodotoxin	Tocris	Cat# 1078
VUF10166	Tocris	Cat#4532
Experimental Models: Animals
Sprague-Dawley rats	Envigo	Cat#Hsd:
C57BL/6J	Jackson Laboratory (USA)
ASIC2^+/− mice	Jackson Laboratory (USA)	JAX: # 013126
ASIC3^+/− mice	Jackson Laboratory (USA)	JAX: # 013127
ASIC2^+/+ mice	In-house breed from ASIC2^+/− mice	N/A
ASIC2^−/− mice	In-house breed from ASIC2^+/− mice	N/A
ASIC3^+/+ mice	In-house breed from ASIC3^+/− mice	N/A
ASIC3^−/− mice	In-house breed from ASIC3^+/− mice	N/A
Oligonucleotides
Primers for ASIC2^+/+ and ASIC2^−/− mice genotyping:
AGT CCT GCA CGG TGG GAG CTT CTA	Common
GAA GAG GAA GGG AGC CAT GAT GAG	Wild type Forward
TGG ATG TGG AAT GTG TGC GA	Mutant Forward
Primers for ASIC3^+/+ and ASIC3^−/− mice genotyping:
GAA CCT GGA AAA CAG AGG CAG GAA GGA T	Wild type Forward
CAG GGA GTA AGA TCT TAT GTA GCC TGG C	Wild type Reverse
CCC TGG GCA CCA GAG TTG AAG GTG TAG C	Mutant Reverse
TGG ATG TGG AAT GTG TGC GA	Mutant Forward
Software and Algorithms
Clampfit 11	Molecular Devices	www.moleculardevices.com
Deeplabcut	Mathis Laboratory	www.mackenziemathislab.org
Fiji	NIH	N/A
Matlab	Mathworks	www.mathworks.com
Metaflour	Molecular Devices	www.moleculardevices.com
Minianalysis	Blue Cell	http://bluecell.co.kr
pCLAMP11	Molecular Devices	www.moleculardevices.com
Prism 9	GraphPad	www.graphpad.com
Other
Cheha supercomputer	UAB	N/A
LSM800	Zeiss	https://www.zeiss.com
Nikon A1 Plus	Nikon	https://www.microscope.com
Digidata 1440A	Molecular Devices	www.moleculardevices.com
Flaming/Brown micropipette puller	Shutter Instruments	Cat# P-97
Glass capillary (thin wall, OD 1.5 mm, filament)	World Precision Instruments	Cat#TW150F-4
High-speed pressure-clamp device	ALA Scientific instruments	https://alascience.com/
IR-2000	Dagi-MTI	https://dagemti.com
Leica CM1860 cryomicrotome	Leica Biosystems	www.leicabiosystems.com
Master-8 Stimulator	A.M.P.I	www.ampi.co.il
MPC-385	Sutter instrument	https://www.sutter.com
Minipuls 3 Peristaltic Pumps	Gilson	Cat# GFAM00051
Multiclamp 700B	Molecular Devices	www.moleculardevices.com
Olympus BX51	Olympus	www.olympus-lifescience.com
Open field box	Maze engineering	https://conductscience.com
Recording chamber	Custom made	N/A
Syringe filters (0.22 mm)	Research Products International	Cat#256130
Piezoelectric actuator and controller	Physik Instrumente	CAT# E-625.SR, P841.20
Picospritzer II	Parker Hannifin	https://ph.parker.com
Tissue anchor	Warner Instruments	Cat#WI 64–1413
Vibration isolation table (63–500 series)	TMC Vibration Control	www.techmfg.com
40x water immersion objective	Olympus	Cat# LUMPLFLN40XW

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Highlights.

Merkel cell-neurite complexes (MNCs) encode tactile signals using synaptic transmission
This synaptic transmission is mediated by acid-sensing ion channels (ASICs) on MNCs
Proton serve as the principal transmitter for synaptic transmission at MNCs
ASICs play a role in the sense of touch and tactile discrimination in mammals

ACKNOWLEDGMENTS

We thank Drs. Wenqin Luo and Jacques Wadiche for providing scientific advice on this work, Drs. Ryan Vaden and Lucas Miller-Pozzo for their technical assistance in confocal microscopy. This study was supported by the National Institute of Health grants NS109059, DE018661 and DE023090 to J.G.G.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Declaration of interests

The authors declare no competing interests.

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Supplementary Materials

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Data Availability Statement

This paper does not report original code.
Data reported in this paper will be shared by the lead contact upon request.
All data and any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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PERMALINK

ASICs mediate fast excitatory synaptic transmission for tactile discrimination

Akihiro Yamada

Jennifer Ling

Ayaka I Yamada

Hidemasa Furue

Jianguo G Gu

SUMMARY

Graphical Abstract

In brief

INTRODUCTION

RESULTS

Mechanical stimulation elicits fast excitatory synaptic transmission at MNCs in whisker hair follicles

Figure 1. Mechanical stimulation triggers fast excitatory synaptic transmission at MNCs in whisker hair follicles of rats and mice.

Fast excitatory synaptic transmission at MNCs is mediated by ASICs located on the neurites of MNCs

Figure 2. Fast excitatory synaptic transmission at MNCs is mediated by ASICs.

Figure 3. ASIC channels are localized at the neurites of MNCs and can be directly activated by protons to elicit excitatory inward currents.

ASIC-mediated fast excitatory synaptic transmission at MNCs drives SA1 impulses at Aβ-afferent terminals

Figure 4. Mechanically evoked and ASIC-mediated fast excitatory synaptic transmission at MNCs drives SA1 impulses at Aβ-afferent terminals.

Mechanically evoked fast excitatory synaptic transmission at MNCs is associated with action potential firing on Merkel cells and mediated by protons

Figure 5. Mechanically evoked fast excitatory synaptic transmission at MNCs is associated with action potential firing on Merkel cells and mediated by protons.

Genetic deletion of ASIC3 impairs fast excitatory synaptic transmission at MNCs

Figure 6. Genetic deletion of ASIC3 channels impairs fast excitatory synaptic transmission at MNCs and diminishes SA1 impulses.

ASIC channels are required for whisker tactile discrimination

Figure 7. ASIC channels are required for whisker tactile discrimination.

DISCUSSION

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Materials availability

Data and code availability

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

METHOD DETAILS

Ex vivo Whisker hair follicle preparations

Pressure-patch-clamp recordings at the 1st nodes and heminodes on Aβ-afferent terminals that innervate MNCs of whisker hair follicles

Morphology of neurites of MNCs

Patch-clamp recordings from Merkel cells in situ in whisker hair follicles

Mechanical Stimulation in patch-clamp recording experiments

Pressure-clamped single-fiber recordings of whisker afferent impulses evoked by whisker hair deflections

Pharmacology

Immunohistochemistry

Whisker texture discrimination test

QUANTIFICATION AND STATISTICAL ANALYSIS

Supplementary Material

Highlights.

ACKNOWLEDGMENTS

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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Links to NCBI Databases

Pressure-patch-clamp recordings at the 1^st nodes and heminodes on Aβ-afferent terminals that innervate MNCs of whisker hair follicles