Merkel cells transduce and encode tactile stimuli to drive Aβ-afferent impulses

Abstract

Sensory systems for detecting tactile stimuli have evolved from touch-sensing nerves in invertebrates to complicated tactile end organs in mammals. Merkel discs are tactile end organs consisting of Merkel cells and Aβ-afferent nerve endings and are localized in fingertips, whisker hair follicles, and other touch-sensitive spots. Merkel discs transduce touch into slowly adapting impulses to enable tactile discrimination, but their transduction and encoding mechanisms remain unknown. Using rat whisker hair follicles, we show that Merkel cells rather than Aβ-afferent nerve endings are primary sites of tactile transduction and identify the Piezo2 ion channel as the Merkel cell mechanical transducer. Piezo2 transduces tactile stimuli into Ca(2+)-action potentials in Merkel cells, which drive Aβ-afferent nerve endings to fire slowly adapting impulses. We further demonstrate that Piezo2 and Ca(2+)-action potentials in Merkel cells are required for behavioral tactile responses. Our findings provide insights into how tactile end-organs function and have clinical implications for tactile dysfunctions.

Figure 1. Merkel cells in situ fire action potentials

(A) Whisker hair follicle structure. (B) Image shows a fresh whisker hair follicle anchored in a recording chamber for patch-clamp recordings; the capsule of the hair follicle was removed. (C) Top, Merkel cell layer after peeling off the glassy membrane. Bottom, Quinacrine vital-staining for pre-identifying Merkel cells for patch-clamp recordings. (D) A quinacrine-stained cell in situ was filled with both Alexa 555 and Fluo-3 through a recording electrode (indicated by *). The arrow in the first image indicates a cell process viewed with Alexa 555. The Ca²⁺ imaging shows Fluo-3 fluorescence before (2^nd image), during (3^rd image), and after (4^th image) action potential (AP) firing (illustrated in E). (E) Injection of depolarizing currents elicited AP firing (superimposed colored traces) in the Merkel cell. The red trace is the response to a 40-pA current step. (F) Time course (left) of Fluo-3 intensity of the cell in E and summary data (right, n = 7, Ctr, control before APs). Colored line in left panel indicates the period of 10 supra-threshold depolarizing steps. (G&H) Sample traces of membrane responses and AP firing in response to depolarizing current steps in a Merkel cell (G) and summary data of V-I relationship of 48 Merkel cells (H, n = 48). (I&J) Sample traces of membrane responses to depolarizing current steps in a non-Merkel cell (I) and summary data of V-I relationship of 19 non-Merkel cells (J, n = 19). Data represent the mean ± SEM. *** P < 0.001, paired Student t-test. See also Table S1.

Figure 2. Merkel cells fire slowly adapting Ca²⁺-action potentials

(A) Merkel cell APs in the absence (left) and presence (middle) of 0.5 μM TTX. Right panel, summary data (n = 6). (B) Merkel cell APs depend on extracellular Ca²⁺. Left panel, APs in normal Krebs solution ([Ca²⁺]_o = 2.5 mM). Middle panel, failure to fire APs in a bath solution with low extracellular Ca²⁺ (20 μM). Right Panel, summary data (n = 6). (C) Merkel cell APs are abolished by Ca²⁺ channel blocker Cd²⁺. Left, in the absence of Cd²⁺; Middle panel, in the presence of 300 μM Cd²⁺, Right panel, summary data (n = 9). (D) Merkel cell APs are abolished by L-type VGCC blocker felodipine (Felo). Left panel, in the absence of felodipine; Middle panel, in the presence of 0.1 μM felodipine, Right panel, summary data (n = 9). (E) Merkel cell APs in response to a 1-min depolarizing current step at 40 pA. The recording was performed in normal Krebs solution. (F) APs at an expanded scale in the a, b, and c locations indicated in E. (G) Representative plots of instantaneous AP frequency over the 1-min recording shown in E. (H) Summary data for the experiments represented in E. The frequency at each point is calculated with a time bin of 3 sec. Results are pooled from 11 Merkel cells (n = 11). Data represent the mean ± SEM. NS, no significant difference; *** P < 0.001, paired Student t-test. See also Figure S1.

Figure 3. Touching follicle tissues evokes MA currents in Merkel cells

(A–C) The configuration of indirect displacement stimulation during patch-clamp recordings from Merkel cells in situ. The arrow indicates a quinacrine-stained Merkel cell in fluorescent image (A) and bright field (B). The Merkel cell and two adjacent cells are outlined in C. The mechanical impact is transmitted to the recorded Merkel cell via adjacent cells when the stimulation probe moves forward. (D&E) Whole-cell mechanically activated currents (MA) recorded from two Merkel cells stimulated by either indirect displacement (D) or direct displacement (E). Displacement step, 1 μm. Vh = −75 mV. (F) Summary data of MA amplitude at different distances of indirect displacement (n = 28) or direct displacement (n = 9). Displacement step, 0.5 μm. (G) Sample traces of dual recording of piezo probe movement (top) and MA current (bottom) at an expanded time scale. (H–J) Summary data of latency, rising slope, and decay time constant (τ) at different displacement distances. Closed circle, indirect displacement (n = 28); open circle, direct displacement (n = 9). (K–N) I-V relationships of MA currents under normal recording condition (K, E_rev = 1.1 ± 1.2 mV, n = 21), under [Ca²⁺]_out/[Cs⁺]_in (L, E_rev = 7.0 ± 1.3 mV, n = 7), [Na⁺]_out/[Cs⁺]_in (M, E_rev = 1.3 ± 1.9 mV, n = 7) and [Na⁺]_out/[K⁺]_in (N, E_rev = 5.1 ± 4 mV, n = 7) recording conditions. Insets in K and L are sample traces of MA currents. (N) Ion permeability: PCa²⁺/PCs⁺ = 1.1 ± 0.1 (n = 7), PNa⁺/PCs⁺ = 1.1 ± 0.1 (n = 7), and PNa⁺/PK⁺ = 1.3 ± 0.2 (n = 7). Data represent the mean ± SEM. See also Figure S2.

Figure 4. Expression of Piezo2 ion channels in Merkel cells and pharmacological properties of MA currents in Merkel cells

(A) RT-PCR shows Piezo2 mRNA in Merkel cells. (B&C) Inhibition of MA currents in Merkel cells by 30 μM Gd³⁺ (B, n = 11) and 30 μM RR (C, n = 9). Sample traces (inset) represent MA currents before (gray line), 10 min following the bath application of Gd³⁺ or RR (black line), and after wash off (dashed line). The graphs are MA currents before (○) and following (●) the bath applications of the blockers. Indirect displacements were applied. (D) Sample traces of MA currents in the absence (control), presence of a Piezo2 antibody (Piezo2Ab), and the presence of the Piezo2Ab plus its blocking peptide (BP). MA currents were recorded 10 min after establishing the whole-cell mode and indirect displacement was applied at 3.5 μm. (E) Comparison of MA current amplitudes recorded 10 min after establishing the whole-cell mode. Control, n = 9; Piezo2Ab, n = 17; piezo2Ab+BP, n = 12. In D and E Piezo2Ab or Piezo2Ab+BP was applied through the patch-clamp internal recording solution. Data represent the mean ± SEM. * P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni post-hoc tests. See also Figure S3.

Figure 5. MA currents in Merkel cells are specifically reduced by knockdown of Piezo2 ion channels

(A) Left, schematic illustration of intra-follicle injection. Right, a whisker hair follicle after the injection of a blue dye solution (~3 μl), it shows that the solution is injected into the whisker hair follicle and remains inside. (B) Top, lentiviral particle-mediated GFP expression in a whisker hair follicle 10 days after intra-follicle injection of GFP lentiviral particles. Bottom, the same field following quinacrine staining. Note that quinacrine fluorescent intensity is stronger than GFP so that GFP and quinacrine staining could be imaged sequentially. (C) Top, percentage of GFP-positive and -negative cells among 218 quinacrine-stained cells (8 follicles). Bottom, percentage of quinacrine-stained or non-stained cells among 202 GFP-positive cells (8 follicles). (D) Quantitative PCR measurement of the changes of Piezo2 mRNA in the enlargement segments of whisker hair follicles. Open bar: control group following intra-follicle injection of scrambled shRNA lentiviral particles (n = 4, triplicate for each sample). Solid bar: whisker hair follicles that received intra-follicle injection of Piezo2 shRNA lentiviral particles (n = 4, triplicate for each sample). (E) Traces represent averaged MA currents in Merkel cells following intra-follicle application of lentiviral particles with either scrambled shRNA (left, n = 17) or Piezo2 shRNA (right, n = 20). (F) Summary data for scrambled or Piezo2 shRNA groups. (G) Percentage of Merkel cells with different thresholds following scrambled or Piezo2 shRNA. (H) Summary of mechanotransduction thresholds for scrambled or Piezo2 shRNA groups. From F to H, cell numbers are 28 for scrambled shRNA group and 43 for Piezo2 shRNA group. (I) MA amplitudes of high threshold Merkel cells (≥ 2.0 μm, 21 cells) or low threshold Merkel cells (≤ 1.5 μm, 22 cells) in Piezo2 shRNAs group. Data represent the mean ± SEM. * P < 0.05, ** P < 0.01, ***P < 0.001, Student’s t-test or two-way ANOVA with Bonferroni post-hoc tests. See also Figure S4 and Figure S5.

Figure 6. Gently touching whisker hairs or follicle tissues induces action potential firing in Merkel cells

(A–F) Gently touching whisker hairs induces MA currents and AP firing in Merkel cells in situ. (A) Recording setup. (B) MA currents in a Merkel cell elicited by hair displacement (1.0 μm increments). Inset, at an expanded time scale. (C) Summary data (n = 15). (D) Left, a single AP spike in a Merkel cell evoked by a single 500-ms hair movement. Right, 5 spikes elicited by 5 25-ms stimuli. Recordings were under cell-attached mode with hair displacement of 4 μm. (E) Multiple AP spikes in a Merkel cell induced by a 500-ms hair displacement at 3 μm. (F) Summary data. The single closed circle shows threshold (2.4 ± 0.4 μm, n = 5) for the single AP spike cells. The open circles show the relationship between hair displacement distance and AP spike number for the cells with multiple AP spikes. The mean threshold is 2.2 ± 0.3 μm (n = 5). (G–J) Indirectly displacing Merkel cells induces AP firing in Merkel cells in situ. (G) AP spike currents recorded from a Merkel cell in response to indirect stimulation. The AP spikes are recorded under the cell-attached (c/a) mode. Traces from the top to the bottom are baseline, and responses following displacement steps of 2 and 3 μm. The bottom panel shows the displacement steps. (H) Similar to G except that this cell has graded responses with multiple AP spikes. Displacement steps are 1 and 2 μm. (I) Same cell as H after breaking into the whole-cell (w/c) mode, a 2-μm displacement step elicits APs (top trace) in the current-clamp mode and an inward current (bottom trace) in voltage-clamp mode (Vh = −75 mV). Similar results were obtained in 9 other Merkel cells. (J) Summary of AP spikes recorded under the cell-attached mode. The single closed circle shows the threshold (1.9 ± 0.2 μm, n = 20) for the Merkel cells that only fired a single spike. Single AP spike cells are arbitrarily defined as the Merkel cells that fired only a single spike following an additional 3 forward displacement steps (0.5 μm increment each) above the threshold. The open circles show the relationship between displacement distances and spike numbers in the cells that had graded responses (n = 8); the threshold is 1.4 ± 0.2 μm (n = 8). Displacement steps were applied for 250 ms in each test. Data represent the mean ± SEM.

Figure 7. Inhibition of Merkel cell Ca²⁺-action potentials suppresses SAI responses, and Ca²⁺-action potentials and Piezo2 are required for behavioral tactile sensitivity

(A) Setup for whisker afferent recordings. (B) Left panel, sample traces of SAI responses before (Ctr, top) and following bath application of 0.5 μM TTX (bottom). Right panel, Summary data (n = 5). (C) Sample traces of SAI responses before (Ctr, top) and following bath application of 300 μM Cd²⁺ (bottom). (D) Summary data (n = 7) of the experiments represented in C. Open and closed bars are SAI frequency before and following Cd²⁺ application, respectively. Left panel, dynamic phase; Right panel, static phase. (E) Sample traces of SAI responses before (Ctr, left) and following bath application of 0.1 μM felodipine (right). (F) Summary data (n = 6) of the experiments represented in E. (G) Sample traces of SAI responses before (Ctr, left) and following bath application of 1 μM ω-conotoxin (right). (H) Summary data (n = 6) of the experiments represented in G. (I) Sample traces of SAI responses in scrambled shRNA group (left) and Piezo2 shRNA group (right). (J) Summary data of the experiments represented in I, n = 12 for scrambled shRNA group, n = 12 for Piezo2 shRNA group. Hair displacement was 38-μm From B–J. (K) Schematic illustration of the whisker tactile test. (L) Behavioral tactile responses to whisker tactile stimulation under the following conditions: no injection (n = 8), intra-follicle injections of saline (3 μl, n = 8), TTX (0.048 μg, n = 8), Cd²⁺ (33 μg, n = 8), felodipine (0.058 μg, n = 6), or ω-conotoxin MVIIC (2.8 μg, n = 5). (M) Behavioral tactile responses to whisker tactile stimulation in rats following intra-follicle injection of Piezo2 shRNA lentiviral particles (n = 6) or scrambled shRNA lentiviral particles (n = 6). In Both L and M, prior to each behavioral experiment, capsaicin was injected subcutaneously into facial areas of the testing rats to facilitate quantitatively measuring tactile responses. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.001, paired or unpaired Student’s t-test or two-way ANOVA with Bonferroni post-hoc tests. See also Figure S6 and Figure S7.