Molecular profiling reveals synaptic release machinery in Merkel cells

Abstract

Merkel cell-neurite complexes are somatosensory receptors that initiate the perception of gentle touch. The role of epidermal Merkel cells within these complexes is disputed. To ask whether Merkel cells are genetically programmed to be excitable cells that may participate in touch reception, we purified Merkel cells from touch domes and used DNA microarrays to compare gene expression in Merkel cells and other epidermal cells. We identified 362 Merkel-cell-enriched transcripts, including neuronal transcription factors, presynaptic molecules, and ion-channel subunits. Antibody staining of skin sections showed that Merkel cells are immunoreactive for presynaptic proteins, including piccolo, Rab3C, vesicular glutamate transporter 2, and cholecystokinin 26-33. These data indicate that Merkel cells are poised to release glutamate and neuropeptides. Finally, by using Ca(2+) imaging, we discovered that Merkel cells have L- and P/Q-type voltage-gated Ca(2+) channels, which have been shown to trigger vesicle release at synapses. These results demonstrate that Merkel cells are excitable cells and suggest that they release neurotransmitters to shape touch sensitivity.

Fig. 1.

Purifying Merkel cells. (A) A plot of red versus green fluorescence of 5 × 10⁵ epidermal cells from Math1/nGFP mice was used to set regions around GFP⁺ cells (R1) and GFP^- cells (R2). (B and C) Confocal micrographs show epidermal cells collected from R1 (B) or R2 (C). Nuclei were stained with 4′,6-diamidino-2-phenylindole (shown in red). GFP⁺ cells appear yellow. (Scale bar, 50 μm.) (D) PCR products were amplified from sorted cells. Templates were dH₂O, GFP⁺ cell cDNA, GFP^- cell cDNA, and whole-skin cDNA (control cDNA). Cell-type-specific markers were amplified with the primers indicated. GAPDH product confirmed that comparable amounts of template were used from sorted cells.

Fig. 2.

Histograms of the log₂ ratio of signals from GFP⁺ cells to GFP^- cells (GFP⁺/GFP^-). For each array type, data from each trial are plotted in a different color. Gray bars indicate the enrichment values exceeded by clones that were analyzed further. (A) Results from five trials with glass-slide cDNA microarrays. Microarray elements shown had sums of median Cy3 and Cy5 signals ≥500 fluorescence units. (B) Results from two trials with Affymetrix oligonucleotide microarrays. Elements shown were scored as “present” by the absolute-call algorithm.

Fig. 3.

Merkel cells express presynaptic proteins in vivo. Confocal micrographs show immunohistochemical staining of touch domes in Math1/nGFP skin cryosections. Each row includes antibody staining (red, Left), GFP fluorescence (green, Center) and a merged image (Right). (A) An anti-NF200 antibody labeled sensory afferents that contacted Merkel cells. Dermal fluorescence reflects autofluorescence that is independent of GFP expression. (Scale bar, 10 μm.) (B) Low-magnification micrographs demonstrate that, in the skin, Rab3C staining was detectable only in Merkel cells (arrowhead). (Scale bar, 25 μm.) (C-E) High-magnification images show immunoreactivity of Rab3C (C), CCK8 (D), and Piccolo (Pclo, E) in Merkel cells. (Scale bar in C, 5 μm, applies to C-E.)

Fig. 4.

Merkel cells express VGLUT2 protein. An antibody against VGLUT2 (red in A, D, and E) labeled KRT1-18-positive (blue in B, D, and E), GFP-expressing Merkel cells (green in C-E) in a touch dome. Dorsal root ganglion fibers that contacted Merkel cells and those that formed palisade endings around hair shafts displayed weak VGLUT2 staining (arrowheads in E). The image in E is a projection of a confocal z series collected with 2-μm axial steps. (Scale bars, 5 μm in D; 20 μm in E.)

Fig. 5.

Merkel cells have functional P/Q- and L-type Ca²⁺ channels. (A) PCR products were amplified from sorted Merkel cells with primers specific for the indicated voltage-gated Ca²⁺-channel α₁-subunits. (B) An epifluorescence micrograph shows sorted GFP⁺ cells after 2 d in culture. (Scale bar, 100 μm.) (C and D) Pseudocolor images of fura-2 fluorescence ratios (F₃₄₀/F₃₈₀) of the cells in B just before (C) and 6 s after (D) perfusion with high-K⁺ Ringer's solution. Pseudocolor scale denotes F₃₄₀/F₃₈₀ from 0.1 (black) to 3 (white). (E-G) Plots of mean fura-2 ratios versus time in the absence (dashed line) or presence (solid line) of Ca²⁺-channel antagonists. Cells were exposed to drugs for 15-20 min before depolarization. Application of high-K⁺ solution began at t = 0 and lasted throughout the recording. Each trace represents the average fura-2 ratio of 57-119 cells. Error bars indicate SEM. Antagonists used were 10 μM nimodipine (Nim) (E), 1 μM ω-agatoxin IVA (Aga) (F), and 10 μM nimodipine plus 1 μM ω-agatoxin IVA (G). (H) Quantification of the effects of Ca²⁺-channel antagonists (n = 4-5 experiments per group). Responses of Merkel cells exposed to antagonists were normalized to those measured from control cells. The effect of ω-agatoxin IVA or nimodipine was significantly different from that of ω-conotoxin GVIA (Ctx) (P ≤ 0.002). Inhibition by ω-agatoxin IVA plus nimodipine was significantly greater than that achieved with either alone (P ≤ 8 × 10^-4).