Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors (original) (raw)

References

  1. Johnson, K. O. The roles and functions of cutaneous mechanoreceptors. Curr. Opin. Neurobiol. 11, 455–461 (2001)
    Article CAS Google Scholar
  2. Lumpkin, E. A. & Caterina, M. J. Mechanisms of sensory transduction in the skin. Nature 445, 858–865 (2007)
    Article ADS CAS Google Scholar
  3. Kwan, K. Y., Glazer, J. M., Corey, D. P., Rice, F. L. & Stucky, C. L. TRPA1 modulates mechanotransduction in cutaneous sensory neurons. J. Neurosci. 29, 4808–4819 (2009)
    Article CAS Google Scholar
  4. Haeberle, H. et al. Molecular profiling reveals synaptic release machinery in Merkel cells. Proc. Natl Acad. Sci. USA 101, 14503–14508 (2004)
    Article ADS CAS Google Scholar
  5. Maksimovic, S., Baba, Y. & Lumpkin, E. A. Neurotransmitters and synaptic components in the Merkel cell-neurite complex, a gentle-touch receptor. Ann. NY Acad. Sci. 1279, 13–21 (2013)
    Article ADS CAS Google Scholar
  6. Hartschuh, W. & Weihe, E. Fine structural analysis of the synaptic junction of Merkel cell-axon-complexes. J. Invest. Dermatol. 75, 159–165 (1980)
    Article CAS Google Scholar
  7. Iggo, A. & Muir, A. R. The structure and function of a slowly adapting touch corpuscle in hairy skin. J. Physiol. (Lond.) 200, 763–796 (1969)
    Article CAS Google Scholar
  8. Woodbury, C. J. & Koerber, H. R. Central and peripheral anatomy of slowly adapting type I low-threshold mechanoreceptors innervating trunk skin of neonatal mice. J. Comp. Neurol. 505, 547–561 (2007)
    Article Google Scholar
  9. Wellnitz, S. A., Lesniak, D. R., Gerling, G. J. & Lumpkin, E. A. The regularity of sustained firing reveals two populations of slowly adapting touch receptors in mouse hairy skin. J. Neurophysiol. 103, 3378–3388 (2010)
    Article Google Scholar
  10. Piskorowski, R., Haeberle, H., Panditrao, M. V. & Lumpkin, E. A. Voltage-activated ion channels and Ca2+-induced Ca2+ release shape Ca2+ signaling in Merkel cells. Pflugers Arch. 457, 197–209 (2008)
    Article CAS Google Scholar
  11. Chalfie, M. Neurosensory mechanotransduction. Nature Rev. Mol. Cell Biol. 10, 44–52 (2009)
    Article CAS Google Scholar
  12. Hu, J. & Lewin, G. R. Mechanosensitive currents in the neurites of cultured mouse sensory neurones. J. Physiol. (Lond.) 577, 815–828 (2006)
    Article CAS Google Scholar
  13. Coste, B. et al. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330, 55–60 (2010)
    Article ADS CAS Google Scholar
  14. Walker, R. G., Willingham, A. T. & Zuker, C. S. A Drosophila mechanosensory transduction channel. Science 287, 2229–2234 (2000)
    Article ADS CAS Google Scholar
  15. Woo, S. H. et al. Piezo2 is required for Merkel cell mechanotransduction. Nature http://dx.doi.org/10.1038/nature13251(this issue)
  16. Lesniak, D. R. et al. Computation identifies structural features that govern neuronal firing properties in slowly adapting touch receptors. eLife 3, e01488 (2014)
    Article Google Scholar
  17. Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nature Neurosci. 8, 1263–1268 (2005)
    Article CAS Google Scholar
  18. Taniguchi, H. et al. A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron 71, 995–1013 (2011)
    Article CAS Google Scholar
  19. Madisen, L. et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nature Neurosci. 15, 793–802 (2012)
    Article CAS Google Scholar
  20. Quiroga, R. Q., Nadasdy, Z. & Ben-Shaul, Y. Unsupervised spike detection and sorting with wavelets and superparamagnetic clustering. Neural Comput. 16, 1661–1687 (2004)
    Article Google Scholar
  21. Dassule, H. R., Lewis, P., Bei, M., Maas, R. & McMahon, A. P. Sonic hedgehog regulates growth and morphogenesis of the tooth. Development 127, 4775–4785 (2000)
    Article CAS Google Scholar
  22. Han, X. et al. A high-light sensitivity optical neural silencer: development and application to optogenetic control of non-human primate cortex. Front. Syst. Neurosci. 5, 18 (2011)
    Article Google Scholar
  23. Morrison, K. M., Miesegaes, G. R., Lumpkin, E. A. & Maricich, S. M. Mammalian Merkel cells are descended from the epidermal lineage. Dev. Biol. 336, 76–83 (2009)
    Article CAS Google Scholar
  24. Maricich, S. M. et al. Merkel cells are essential for light-touch responses. Science 324, 1580–1582 (2009)
    Article ADS CAS Google Scholar
  25. Meyers, J. R. et al. Lighting up the senses: FM1–43 loading of sensory cells through nonselective ion channels. J. Neurosci. 23, 4054–4065 (2003)
    Article CAS Google Scholar
  26. Koltzenburg, M., Stucky, C. L. & Lewin, G. R. Receptive properties of mouse sensory neurons innervating hairy skin. J. Neurophysiol. 78, 1841–1850 (1997)
    Article CAS Google Scholar
  27. Johnson, K. O. & Lamb, G. D. Neural mechanisms of spatial tactile discrimination: neural patterns evoked by braille-like dot patterns in the monkey. J. Physiol. (Lond.) 310, 117–144 (1981)
    Article CAS Google Scholar
  28. Phillips, J. R. & Johnson, K. O. Tactile spatial resolution. II. Neural representation of Bars, edges, and gratings in monkey primary afferents. J. Neurophysiol. 46, 1192–1203 (1981)
    Article CAS Google Scholar
  29. Maricich, S. M., Morrison, K. M., Mathes, E. L. & Brewer, B. M. Rodents rely on Merkel cells for texture discrimination tasks. J. Neurosci. 32, 3296–3300 (2012)
    Article CAS Google Scholar
  30. Yamashita, Y. & Ogawa, H. Slowly adapting cutaneous mechanoreceptor afferent units associated with Merkel cells in frogs and effects of direct currents. Somatosens. Mot. Res. 8, 87–95 (1991)
    Article CAS Google Scholar
  31. Lumpkin, E. A. et al. Math1-driven GFP expression in the developing nervous system of transgenic mice. Gene Expr. Patterns 3, 389–395 (2003)
    Article CAS Google Scholar
  32. Yang, Y., Lacas-Gervais, S., Morest, D. K., Solimena, M. & Rasband, M. N. βIV spectrins are essential for membrane stability and the molecular organization of nodes of Ranvier. J. Neurosci. 24, 7230–7240 (2004)
    Article CAS Google Scholar
  33. Ertürk, A. et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nature Protocols 7, 1983–1995 (2012)
    Article Google Scholar
  34. Drew, L. J., Wood, J. N. & Cesare, P. Distinct mechanosensitive properties of capsaicin-sensitive and -insensitive sensory neurons. J. Neurosci. 22, RC228 (2002)
    Article Google Scholar
  35. Holt, J. R., Corey, D. P. & Eatock, R. A. Mechanoelectrical transduction and adaptation in hair cells of the mouse utricle, a low-frequency vestibular organ. J. Neurosci. 17, 8739–8748 (1997)
    Article CAS Google Scholar
  36. Lin, J. Y. A user’s guide to channelrhodopsin variants: features, limitations and future developments. Exp. Physiol. 96, 19–25 (2011)
    Article Google Scholar
  37. Haeberle, H., Bryan, L. A., Vadakkan, T. J., Dickinson, M. E. & Lumpkin, E. A. Swelling-activated Ca2+ channels trigger Ca2+ signals in Merkel cells. PLoS ONE 3, e1750 (2008)
    Article ADS Google Scholar
  38. Woo, S. H., Baba, Y., Franco, A. M., Lumpkin, E. A. & Owens, D. M. Excitatory glutamate is essential for development and maintenance of the piloneural mechanoreceptor. Development 139, 740–748 (2012)
    Article CAS Google Scholar

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Acknowledgements

Thanks to R. Axel, A. MacDermott and the Lumpkin laboratory for helpful discussions and to D. Florez and R. Piskorowski for advice on whole-cell recordings. Funding was provided by NIH/NIAMS grants R01AR051219 and R21AR062307 (to E.A.L.), R01DE022358 (to A.P.) and fellowships to S.M. (5T32HL087745-05 and NIH/NINDS F32NS080544), M.N. (JSPS Research Fellowships for Young Scientists 24-7585), and A.M.N. (McNair Foundation). Microscopy and flow cytometry was performed with core support from the Columbia SDRC (P30AR044535) and Cancer Center (P30CA013696). Initial studies were performed at Baylor College of Medicine with assistance from Flow Cytometry and Genetically Engineered Mouse Shared Resources (P30CA125123).

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Author notes

  1. Srdjan Maksimovic, Masashi Nakatani and Yoshichika Baba: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Dermatology, Columbia University, New York, 10032, New York, USA
    Srdjan Maksimovic, Masashi Nakatani, Yoshichika Baba, Aislyn M. Nelson, Kara L. Marshall & Ellen A. Lumpkin
  2. Graduate School of System Design and Management, Keio University, Yokohama 223-8526, Japan,
    Masashi Nakatani
  3. Department of Neuroscience, Baylor College of Medicine, Houston, 77006, Texas, USA
    Aislyn M. Nelson, Scott A. Wellnitz & Pervez Firozi
  4. Howard Hughes Medical Institute, Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, 92037, California, USA
    Seung-Hyun Woo, Sanjeev Ranade & Ardem Patapoutian
  5. Department of Physiology & Cellular Biophysics, Columbia University, New York, 10032, New York, USA
    Ellen A. Lumpkin
  6. Program in Neurobiology & Behavior, Columbia University, New York, 10032, New York, USA
    Ellen A. Lumpkin

Authors

  1. Srdjan Maksimovic
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  2. Masashi Nakatani
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  3. Yoshichika Baba
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  4. Aislyn M. Nelson
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  5. Kara L. Marshall
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  6. Scott A. Wellnitz
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  7. Pervez Firozi
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  8. Seung-Hyun Woo
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  9. Sanjeev Ranade
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  10. Ardem Patapoutian
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  11. Ellen A. Lumpkin
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Contributions

S.M. screened transgenic mouse lines, and performed and analysed all ex vivo optogenetic experiments (Fig. 2 and Extended Data Figs 3–6). M.N. performed and analysed all whole-cell recordings (Fig. 1, Extended Data Fig. 1a, b and Extended Data Table 1). Y.B. performed and analysed recordings from Atoh1 and Piezo2 strains (Fig. 3, Extended Data Fig. 8 and Extended Data Table 2). A.M.N. performed qRT-PCR (Fig. 1i) and calcium imaging (Extended Data Fig. 1c–i). K.L.M. performed immunohistochemistry in Atoh1 strains (Extended Data Fig. 7) and assisted in preparation of all figures. S.A.W. and E.A.L. conceived optogenetic strategies. P.F. generated initial ChR2 transgenic mouse lines. E.A.L. conceived and supervised the project. During this manuscript’s peer-review process, we entered into a collaboration with S.H.W., S.R. and A.P., to analyse unpublished Piezo2 CKO mice. S.R. generated Piezo2 flox/flox mice and S.-H.W. generated and validated Krt14 Cre ;Piezo2 flox/flox mice in the laboratory of A.P. The manuscript was written by S.M., M.N., Y.B. and E.A.L. and edited by A.M.N., K.L.M., S.A.W., P.F., S.-H.W. and A.P.

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Correspondence toEllen A. Lumpkin.

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Extended data figures and tables

Extended Data Figure 1 Mechanically activated responses in Merkel cells.

a, Representative trace of mechanically evoked current induced by 1-µm mechanical displacement. Application of Ruthenium red (RR, 100 µM) attenuated mechanically activated current. b, Peak currents (_I_peak) were estimated from 250 µs around peak and steady-state currents (_I_ss) were estimated from the last 5 ms (black bar in 1a) of mechanical displacements. Data were normalized by _I_peak for each cell. With Ruthenium red, _I_peak was reduced to 38 ± 7% of control condition. Steady state currents were also reduced by Ruthenium red (n = 4; control: 9 ± 1% of _I_peak; RR: 2 ± 1% of _I_peak). **P < 0.01; paired Student’s _t_-test (two-tailed). c–i, Merkel cells display reversible Ca2+ responses to focal displacements applied to somata. c, Representative pseudocolour images of fura-2 ratios (340:380) of a Merkel cell at rest. d, A Merkel cell activated by depolarizing (high-K+) solution. e, A brightfield image showing the position of the stimulus probe. f–h, Peak responses corresponding to each displacement. ‘Fold Δ’ is the fold change in fluorescence ratio from baseline. Scale bar, 10 µm. i, Representative time course of mean fura-2 ratios during the touch stimuli shown above. Stimulus onset in f–h is indicated by arrows. Calcium responses were stimulus-dependent. Similar responses were observed from 11 Merkel cells.

Extended Data Figure 2 ChR2+ Merkel cells display light-activated inward currents.

a, Light-activated currents were recorded with whole-cell, tight-seal voltage clamp methods. b, Fluorescent image of a ChR2-tdTomato expressing Merkel cell. Scale bar, 10 µm. c, Representative trace for light-activated inward currents at a holding potential of −70 mV. Inactivation kinetics were measured by fitting a single exponential curve (red).

Extended Data Figure 3 Immunostaining of ChR2-expressing touch domes.

a–e, Whole-mount staining and confocal axial projection of the touch dome shown in Fig. 2d. a, Merged image. b–d, Expression of ChR2-tdTomato was present in Merkel cells (Krt8), but absent from sensory terminals (neurofilament heavy, NFH). e, Some terminal Schwann cells (Nestin)38 also expressed ChR2 (arrowheads in b and e). f–i, Immunostaining of skin cryosections. f, Merged image. g–i, ChR2-tdTomato was present in some S100+ Schwann cells that also expressed Nestin, a marker for type II terminal Schwann cells38 (arrowheads in f–i). Scale bars, 20 µm.

Extended Data Figure 4 Light-evoked activity is specific to touch-dome illumination.

a, f, Responses to light stimuli centred on a touch dome. b–e, When the light stimulus was positioned around the touch dome, no light-evoked activity was observed. Illuminating a cluster of ChR2+ dermal cells did not evoke any responses (c). f, To confirm that the absence of light-evoked activity was not due to the loss of Merkel cells and/or neuronal fibres, the experiment ended by re-positioning the light stimulus over the touch dome to re-elicit light-evoked activity. Images have been thresholded for clarity. Scale bars, 200 µm.

Extended Data Figure 5 K14 Cre ;ChR2 loxP/+ mice exhibit light-evoked SAI activity.

a, Confocal image of a touch dome illustrating ChR2-tdTomato expression driven by K14 Cre. ChR2-tdTomato expressed much stronger in Merkel cells than in neighbouring keratinocytes. b, Light-evoked responses from the touch dome shown in a to seven light intensities as indicated. Spike sorting and clustering analysis were used to identify the unit that fired in phase with light (lower trace with spike positions and their amplitudes). c, Mean IFFs for light with varying illumination intensities on a log-intensity scale (n = 3 recordings). Scale bar, 20 µm.

Extended Data Figure 7 Structure of touch-dome afferents in Atoh1 CKO mice.

Immunostaining of skin cryosections from Atoh1 CKO and control genotypes are shown. Antibodies labelling myelinated afferents (NFH; cyan), Merkel cells (Krt8; yellow), nodes of Ranvier (βIV spectrin; magenta) show that the general structure of touch-dome afferents, including myelinated branches and Nodes of Ranvier (arrowheads), appears normal even in the absence of Merkel cells. Scale bar, 20 µm.

Extended Data Figure 8 Comparison of ISI distributions in Atoh1 CKO, Piezo2 CKO and control genotypes.

a, Histogram of ISI distribution during saturating responses in Atoh1 CKO (mean ± s.d., 43.4 ± 59.2 ms, median: 29.8 ms; n = 466 intervals from n = 6 units) and control genotypes (mean ± s.d., 16.5 ± 12.9 ms, median: 13.8 ms; n = 1,412 intervals from n = 5 units). Inset on the left illustrates all ISIs, including those > 150 ms, which were excluded from the main histograms (14/466 intervals in Atoh1 CKO and 1/1,412 in control genotypes). At right, bar graphs show the minimum ISIs during dynamic and static phases. Minimum ISIs were longer in Atoh1 CKO than control mice for both phases, indicating a loss of high-frequency firing during dynamic stimuli and static displacement (**P < 0.02, ***_P_ < 0.01; Student’s _t_-test). Mann–Whitney tests indicated that median values were also significantly different (_P_ < 0.001). **b**, Histogram of ISI distribution for _Piezo2_ _CKO_ (Mean ± s.d., 41.9 ± 32.3 ms, median: 23.4 ms; _n_ = 792 intervals from _N_ = 6 units) and control genotypes (mean ± s.d., 13.9 ± 1.4 ms, median: 11.8 ms; _n_ = 1,845 intervals from _n_ = 5 units). Main histograms excluded long intervals (>150 ms; 4/792 intervals in Piezo2 CKO and 2/1,845 in control mice.) Minimum ISIs were not significantly different in the dynamic phase (P ≥ 0.76; Student’s _t_-test and Mann–Whitney test); indicating that high-frequency firing is preserved in touch-dome afferents in these mice. For static phase firing, the means were not significantly different (P = 0.095; Student’s _t_-test); however, non-parametric analysis indicated that medians differed between genotypes (P = 0.0043; Mann–Whitney test).

Extended Data Table 1 Properties of mechanically and light-evoked currents in Merkel cells

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Extended Data Table 2 Summary of touch-dome responses from Atoh1 CKO and control mice

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Maksimovic, S., Nakatani, M., Baba, Y. et al. Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors.Nature 509, 617–621 (2014). https://doi.org/10.1038/nature13250

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