Hair-bearing human skin generated entirely from pluripotent stem cells.

The skin is a multilayered organ, equipped with appendages (that is, follicles and glands), that is critical for regulating body temperature and the retention of bodily fluids, guarding against external stresses and mediating the sensation of touch and pain1,2. Reconstructing appendage-bearing skin in cultures and in bioengineered grafts is a biomedical challenge that has yet to be met3-9. Here we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use stepwise modulation of the transforming growth factor ß (TGFß) and fibroblast growth factor (FGF) signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During an incubation period of 4-5 months, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis and pigmented hair follicles that are equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking the neural circuitry associated with human touch. Single-cell RNA sequencing and direct comparison to fetal specimens suggest that the skin organoids are equivalent to the facial skin of human fetuses in the second trimester of development. Moreover, we show that skin organoids form planar hair-bearing skin when grafted onto nude mice. Together, our results demonstrate that nearly complete skin can self-assemble in vitro and be used to reconstitute skin in vivo. We anticipate that our skin organoids will provide a foundation for future studies of human skin development, disease modelling and reconstructive surgery.

Three-dimensional correlative light and focused ion beam scanning electron microscopy reveals the distribution and ultrastructure of lanceolate nerve endings surrounding terminal hair follicles in human scalp skin.

Lanceolate nerve endings (LNEs) surrounding hair follicles (HFs) play an important role in detecting hair deflection. Complexes of the LNEs form a palisade-like structure along the longitudinal axis of hair roots in which axons are sandwiched between two processes of terminal Schwann cells (tSCs) at the isthmus of HFs. The structure and molecular mechanism of LNEs in animal sinus hair, pelage, and human vellus hairs have been investigated. Despite the high density of HFs in human scalp skin, the LNEs in human terminal HFs have not been investigated. In this study, we aimed to reveal the distribution and ultrastructure of LNEs in terminal HFs of human scalp skin. Using light-sheet microscopy and immunostaining, the LNEs were observed at one terminal HF but not at the other terminal HFs in the same follicular unit. The ultrastructure of the LNEs of terminal HFs in human scalp skin was characterized using correlated light and electron microscopy (CLEM). Confocal laser microscopy and transmission electron microscopy of serial transverse sections of HFs revealed that LNEs were aligned adjacent to the basal lamina outside the outer root sheath (ORS), at the isthmus of terminal HFs, and adjacent to CD200-positive ORS cells in the upper bulge region. Moreover, axons with abundant mitochondria were sandwiched between tSCs. Three-dimensional CLEM, specifically confocal laser microscopy and focused ion beam scanning electron microscopy, of stained serial transverse sections revealed that LNEs were wrapped with type I and type II tSCs, with the processes protruding from the space between the Schwann cells. Moreover, the ultrastructures of LNEs at miniaturized HFs were similar to those of LNEs at terminal HFs. Preembedding immunoelectron microscopy revealed that Piezo-type mechanosensitive ion channel component 2 (Piezo2), a gated ion channel, was in axons and tSCs and adjacent to the cell membrane of axons and tSCs, suggesting that LNEs function as mechanosensors. The number of LNEs increased as the diameter of the ORS decreased, suggesting that LNEs dynamically adapt to the HF environment as terminal HFs miniaturize into vellus-like hair. These findings will provide insights for investigations of mechanosensory organs, aging, and re-innervation during wound healing.

EGFL6 expression in hair follicle central isthmus is dependent on the presence of terminal Schwann cells.

Hair follicle central isthmus is surrounded by dense nerve endings and terminal Schwann cells (TSCs), forming a specialized sensory structure called lanceolate complexes. Extracellular matrix protein EGFL6 expressed from epidermis has been found closely associated with lanceolate complexes and important for proper alignment of nerve fibres and TSCs processes, and for proper response to light touch. However, how EGFL6 itself is specifically induced/deposited/maintained at the central isthmus remains to be elucidated. Previous reports and our results showed that nerve endings and TSCs docking at the central isthmus during hair follicle development occur before the specific depositing of EGFL6 protein. Furthermore, we found nude mice rarely maintain the lanceolate complex, and EGFL6 is lost in their aberrant hair follicle. Instead, reconstituted hair follicle in nude mice by stem cells chamber grafting assay expresses EGFL6 at the central isthmus area after hair follicle innervation. At last, long-term but not short-term cutaneous denervation leads to degeneration of TSCs and loss of EGFL6 expression. Together, our results demonstrate that EGFL6 expression in the central isthmus is dependent on the presence of TSCs, proposing that the interplay of epidermis and neuronal components is important for maintaining functional structure of lanceolate complexes.

KCNQ Potassium Channels Modulate Sensitivity of Skin Down-hair (D-hair) Mechanoreceptors.

M-current-mediating KCNQ (Kv7) channels play an important role in regulating the excitability of neuronal cells, as highlighted by mutations in Kcnq2 and Kcnq3 that underlie certain forms of epilepsy. In addition to their expression in brain, KCNQ2 and -3 are also found in the somatosensory system. We have now detected both KCNQ2 and KCNQ3 in a subset of dorsal root ganglia neurons that correspond to D-hair Aδ-fibers and demonstrate KCNQ3 expression in peripheral nerve endings of cutaneous D-hair follicles. Electrophysiological recordings from single D-hair afferents from Kcnq3(-/-) mice showed increased firing frequencies in response to mechanical ramp-and-hold stimuli. This effect was particularly pronounced at slow indentation velocities. Additional reduction of KCNQ2 expression further increased D-hair sensitivity. Together with previous work on the specific role of KCNQ4 in rapidly adapting skin mechanoreceptors, our results show that different KCNQ isoforms are specifically expressed in particular subsets of mechanosensory neurons and modulate their sensitivity directly in sensory nerve endings.

Deflection of a vibrissa leads to a gradient of strain across mechanoreceptors in a mystacial follicle.

Rodents use their vibrissae to detect and discriminate tactile features during active exploration. The site of mechanical transduction in the vibrissa sensorimotor system is the follicle sinus complex and its associated vibrissa. We study the mechanics within the ring sinus (RS) of the follicle in an ex vivo preparation of the mouse mystacial pad. The sinus region has a relatively dense representation of Merkel mechanoreceptors and longitudinal lanceolate endings. Two-photon laser-scanning microscopy was used to visualize labeled cell nuclei in an ∼ 100-nl vol before and after passive deflection of a vibrissa, which results in localized displacements of the mechanoreceptor cells, primarily in the radial and polar directions about the vibrissa. These displacements are used to compute the strain field across the follicle in response to the deflection. We observe compression in the lower region of the RS, whereas dilation, with lower magnitude, occurs in the upper region, with volumetric strain ΔV/V ∼ 0.01 for a 10° deflection. The extrapolated strain for a 0.1° deflection, the minimum angle that is reported to initiate a spike by primary neurons, corresponds to the minimum strain that activates Piezo2 mechanoreceptor channels.

Excitatory glutamate is essential for development and maintenance of the piloneural mechanoreceptor.

The piloneural collar in mammalian hairy skin comprises an intricate pattern of circumferential and longitudinal sensory afferents that innervate primary and secondary pelage hairs. The longitudinal afferents tightly associate with terminal Schwann cell processes to form encapsulated lanceolate nerve endings of rapidly adapting mechanoreceptors. The molecular basis for piloneural development, maintenance and function is poorly understood. Here, we show that Nefh-expressing glutamatergic neurons represent a major population of longitudinal and circumferential sensory afferents innervating the piloneural collar. Our findings using a VGLUT2 conditional-null mouse model indicate that glutamate is essential for innervation, patterning and differentiation of NMDAR(+) terminal Schwann cells during piloneural collar development. Similarly, treatment of adult mice with a selective NMDAR antagonist severely perturbed piloneural collar structure and reduced excitability of these mechanosensory neurons. Collectively, these results show that DRG-derived glutamate is essential for the proper development, maintenance and sensory function of the piloneural mechanoreceptor.

Hairy sensation.

The hairs of the skin not only function to prevent heat loss but also have important sensory functions. Recent work has now established that each hair of the skin is innervated by one or more of three types of mechanoreceptor ending. Each of these three mechanoreceptor types possesses distinct molecular features and detects distinctive information about skin touch, which is relayed to specific brain locations in a somatotopic fashion.

Overview of alopecia areata.

Alopecia areata is a complex genetic, immune-mediated disease that targets anagen hair follicles. The disease affects children and adults and is characterized by round or oval patches of hair loss, loss of all scalp hair (alopecia totalis), body hair (alopecia universalis), or ophiasis pattern hair loss. Patients may also present with patchy loss in multiple hair-bearing areas. Commonly associated diseases include asthma, allergic rhinitis, atopic dermatitis, thyroid disease, and automimmune diseases, such as thyroiditis and vitiligo. Nail abnormalities may precede, follow, or occur concurrently with hair loss activity. Alopecia areata has no known age, race, or ethnic preponderance and in contrast to other autoimmune diseases such as thyroiditis or lupus, the hair follicle does not usually sustain permanent injury and maintains its potential to regrow hair. It is estimated that alopecia areata affects between six and seven million individuals in the United States. Genes, the immune and nervous systems have all been implicated in the pathogenesis of alopecia areata. Although many treatments are available, there is still no cure. Bolstered by new scientific and translational opportunities from recently published genome-wide association studies, an ambitious treatment development program has recently been initiated by the National Alopecia Areata Foundation.

Follicle-innervating Aδ-low threshold mechanoreceptive neurons form receptive fields through homotypic competition.

The mammalian somatosensory system is comprised of multiple neuronal populations that form specialized, highly organized sensory endings in the skin. The organization of somatosensory endings is essential to their functions, yet the mechanisms which regulate this organization remain unclear. Using a combination of genetic and molecular labeling approaches, we examined the development of mouse hair follicle-innervating low-threshold mechanoreceptors (LTMRs) and explored competition for innervation targets as a mechanism involved in the patterning of their receptive fields. We show that follicle innervating neurons are present in the skin at birth and that LTMR receptive fields gradually add follicle-innervating endings during the first two postnatal weeks. Using a constitutive Bax knockout to increase the number of neurons in adult animals, we show that two LTMR subtypes have differential responses to an increase in neuronal population size: Aδ-LTMR neurons shrink their receptive fields to accommodate the increased number of neurons innervating the skin, while C-LTMR neurons do not. Our findings suggest that competition for hair follicles to innervate plays a role in the patterning and organization of follicle-innervating LTMR neurons.

Touching and feeling: differences in pleasant touch processing between glabrous and hairy skin in humans.

Previous functional magnetic resonance imaging studies in two rare patients, together with microneurography and psychophysical observations in healthy subjects, have demonstrated a system of mechanosensitive C-fiber tactile (CT) afferents sensitive to slowly moving stimuli. They project to the posterior insular cortex and signal pleasant aspects of touch. Importantly, CTs have not been found in the glabrous skin of the hand, yet it is commonly observed that glabrous skin touch is also perceived as pleasant. Here we asked if the brain processing of pleasant touch differs between hairy and glabrous skin by stroking the forearm and glabrous skin of the hand during positron emission tomography. The data showed that, when contrasting slow brush stroking on the forearm with slow brush stroking on the palm, there were significant activations of the posterior insular cortex and mid-anterior orbitofrontal cortex. The opposite contrast showed a significant activation of the somatosensory cortices. Although concurrent psychophysical ratings showed no differences in intensity or pleasantness ratings, a subsequent touch questionnaire in which subjects used a newly developed 'touch perception task' showed significant difference for the two body sites. Emotional descriptors received higher ratings on the forearm and sensory descriptors were rated more highly on the palm. The present findings are consistent with the hypothesis that pleasant touch from hairy skin, mediated by CT afferents, is processed in the limbic-related cortex and represents an innate non-learned process. In contrast, pleasant touch from glabrous skin, mediated by A-beta afferents, is processed in the somatosensory cortex and represents an analytical process dependent on previous tactile experiences.

A mechanism of rat vibrissal movement based on actual morphology of the intrinsic muscle using three-dimensional reconstruction.

The vibrissal capsular muscle (VCM) of the rat is known to differ from the arrector pili muscle. The purpose of the present study was to characterize the rat VCM morphologically using three-dimensional reconstruction. The rat snout skin was fixed, processed with routine histological methods, sectioned serially at a thickness of 10 µm, and then stained with Masson's trichrome. The sectioned images were reconstructed three-dimensionally using 'Reconstruct' software. The findings confirmed that the VCM is a skeletal muscle attached to the vibrissal follicle such that the latter is rooted within the former. The VCM encircles the follicle almost entirely, from base to apex, and hooks around the follicle caudally. Each one of these capsular muscles is connected to two adjacent follicles in the same row. They overlap each other in the lower part, as the rostral follicular muscle that surrounds the caudal follicle. The present findings suggest that the vibrissae are able to move more freely (under voluntary control) than other general arrector pili muscles, in line with their sensory function.

Fluorescence-labeled reporter gene in transgenic mice provides a useful tool for investigating cutaneous innervation.

B6.Cg-Tg(Thy1-YFP)16Jrs/J transgenic mice were created to express the yellow fluorescent protein gene driven by a mouse Thy1 promoter that labeled motor and sensory neurons such that individual nerves could be followed. These mice were used to identify nerves in the skin that innervate the erector pili and panniculus carnosus muscle. Whole mounts demonstrated yellow fluorescent protein expression in nerves of the skin, which was confirmed by labeling the neuromuscular junction with fluorescinated α-bungarotoxin. Frozen and paraffin-embedded skin sections revealed innervation of the panniculus carnosus muscle. Paraffin sections labeled with an anti-green fluorescent protein antibody revealed innervation of the panniculus carnosus as well as the erector pili muscle and around the hair follicle bulge.

How many hair follicles are innervated by one afferent axon? A confocal microscopic analysis of palisade endings in the auricular skin of thy1-YFP transgenic mouse.

Hairs are known as a sensory apparatus for touch. Their follicles are innervated predominantly by palisade endings composed of longitudinal and circumferential lanceolate endings. However, little is known as to how their original primary neurons make up a part of the ending. In this study, innervation of the palisade endings was investigated in the auricular skin of thy1-YFP transgenic mouse. Major observations were 1) Only a small portion of PGP9.5-immunopositive axons showed YFP-positivity, 2) All of thy1-YFP-positive sensory axons were thick and myelinated, 3) Individual thy1-YFP-positive trunk axons innervated 4-54 hair follicles, 4) Most palisade endings had a gap of lanceolate ending arrangement, 5) PGP9.5-immunopositive 10-32 longitudinal lanceolate endings were closely arranged. Only a part of them were thy1-YFP-positive axons that originated from 1-3 afferents, and 6) Single nerve bundles of the dermal nerve network included both bidirectional afferents. Palisade endings innervated by multiple sensory neurons might be highly sensitive to hair movement.

Dynamic plasticity of axons within a cutaneous milieu.

The skin is a repository of sensory axons immersed within the turnover of epidermal, follicular, and dermal cellular constituents. We show that epidermal and perifollicular axons within intact hairy skin of mice possess a remarkable dynamic plasticity linked to their microenvironment. For example, the majority of epidermal axons express the growth protein GAP43. Unexpectedly, we induced new cutaneous axogenesis by simple and noninvasive hair clipping, a response linked to a series of changes in their cutaneous neighbors. In thy-1 YFP transgenic mice with fluorescent axons, superficial epidermal and perifollicular cells newly acquired YFP, indicating diffuse activation by clipping despite the absence of skin injury. At 48 h after clipping, this activation was accompanied by a rise in the number of epidermal cells, transient rises in mRNA of Sox2, a marker of follicular stem cells, and a rise in mRNA of glial fibrillary acidic protein, a marker of glial cells. Axons responded with rises in their numbers in the epidermis and around dermal hair follicles. Linking these responses were early, large, and selective rises in hepatic growth factor (HGF) mRNA, with its protein identified in epidermal cells, perifollicular cells, and sensory axons. Moreover, these elements also expressed the HGF receptor c-Met, especially in small caliber sensory neurons. Finally, we identified concurrent rises in Rac1 activation, a downstream target of ligated c-Met. Together, these results confirm critical linkages between sensory axons and their cutaneous milieu. We believe that the plasticity is provoked by follicular-originating cutaneous activation with HGF and Rac1 signaling, allowing cross talk and axonal remodeling.

Allodynia mediated by C-tactile afferents in human hairy skin.

We recently showed a contribution of low-threshold cutaneous mechanoreceptors to vibration-evoked changes in the perception of muscle pain. Neutral-touch stimulation (vibration) of the hairy skin during underlying muscle pain evoked an overall increase in pain intensity, i.e. allodynia. This effect appeared to be dependent upon cutaneous afferents, as allodynia was abolished by intradermal anaesthesia. However, it remains unclear whether allodynia results from activation of a single class of cutaneous afferents or the convergence of inputs from multiple classes. Intriguingly, no existing human study has examined the contribution of C-tactile (CT) afferents to allodynia. Detailed psychophysical observations were made in 29 healthy subjects (18 males and 11 females). Sustained muscle pain was induced by infusing hypertonic saline (HS: 5%) into tibialis anterior muscle (TA). Sinusoidal vibration (200 Hz­200 µm) was applied to the hairy skin overlying TA. Pain ratings were recorded using a visual analogue scale (VAS). In order to evaluate the role of myelinated and unmyelinated cutaneous afferents in the expression of vibration-evoked allodynia, compression block of the sciatic nerve, and low-dose intradermal anaesthesia (Xylocaine 0.25%) were used, respectively. In addition, the modulation of muscle pain by gentle brushing (1.0 and 3.0 cm s(−1))--known to excite CT fibres--was examined. Brushing stimuli were applied to the hairy skin with all fibres intact and following the blockade of myelinated afferents. During tonic muscle pain (VAS 4­6), vibration evoked a significant and reproducible increase in muscle pain (allodynia) that persisted following compression of myelinated afferents. During compression block, the sense of vibration was abolished, but the vibration-evoked allodynia persisted. In contrast, selective anaesthesia of unmyelinated cutaneous afferents abolished the allodynia, whereas the percept of vibration remained unaffected. Furthermore, allodynia was preserved in the adjacent non-anaesthetized skin. Conformingly, gentle brushing produced allodynia (at both brushing speeds) that persisted during the blockade of myelinated afferents. Prior to the induction and following cessation of muscle pain, all subjects reported vibration and brushing as non-painful (VAS = 0). These results demonstrate that CT fibres in hairy skin mediate allodynia, and that CT-mediated inputs have a pluripotent central effect.

The morphology of the rat vibrissal follicle-sinus complex revealed by three-dimensional computer-aided reconstruction.

The vibrissal follicle-sinus complex (FSC) is a sensory receptor of the mammalian integumentary system that is located around the mouth. The purpose of the present study was to identify the actual 3-dimensional structure of the rat vibrissal FSC. Rat skin tissue was serially sectioned at a thickness of 10 µm and then stained with Masson's trichrome. The serial sections were reconstructed 3-dimensionally using Reconstruct software. The rat vibrissal follicle is a spindle-shaped structure that is embedded within a blood sinus and enveloped within a thick collagenous capsule. The vibrissal FSC is innervated by the deep vibrissal and superficial vibrissal nerves. The deep vibrissal nerve, travelling in the basal-to-apical direction, penetrates the thick collagenous capsule of the vibrissal FSC. The sinus system can be divided into a superior portion, known as the ring sinus, and an inferior portion, known as the cavernous sinus. The ring sinus contains a C-shaped structure, the ringwulst, which is suspended from the mesenchymal sheath of the follicle. Collagenous trabeculae can be seen in the cavernous sinus but not in the ring sinus. The ring sinus encircles the follicle obliquely and asymmetrically. The ringwulst encircles the follicle incompletely, in a C-shaped fashion. This study has demonstrated the previously underappreciated 3-dimensional structure of the vibrissal FSC, which differs from previously reported descriptions, and provides data that will enhance the understanding of vibrissal function.

Sensations evoked by microstimulation of single mechanoreceptive afferents innervating the human face and mouth.

Intraneural microneurography and microstimulation were performed on single afferent axons in the inferior alveolar and lingual nerves innervating the face, teeth, labial, or oral mucosa. Using natural mechanical stimuli, 35 single mechanoreceptive afferents were characterized with respect to unit type [fast adapting type I (FA I), FA hair, slowly adapting type I and II (SA I and SA II), periodontal, and deep tongue units] as well as size and shape of the receptive field. All afferents were subsequently microstimulated with pulse trains at 30 Hz lasting 1.0 s. Afferents recordings whose were stable thereafter were also tested with single pulses and pulse trains at 5 and 60 Hz. The results revealed that electrical stimulation of single FA I, FA hair, and SA I afferents from the orofacial region can evoke a percept that is spatially matched to the afferent's receptive field and consistent with the afferent's response properties as observed on natural mechanical stimulation. Stimulation of FA afferents typically evoked sensations that were vibratory in nature; whereas those of SA I afferents were felt as constant pressure. These afferents terminate superficially in the orofacial tissues and seem to have a particularly powerful access to perceptual levels. In contrast, microstimulation of single periodontal, SA II, and deep tongue afferents failed to evoke a sensation that matched the receptive field of the afferent. These afferents terminate more deeply in the tissues, are often active in the absence of external stimulation, and probably access perceptual levels only when multiple afferents are stimulated. It is suggested that the spontaneously active afferents that monitor tension in collagen fibers (SA II and periodontal afferents) may have the role to register the mechanical state of the soft tissues, which has been hypothesized to help maintain the body's representation in the central somatosensory system.

Follicle-sinus complexes in muzzle skin of domestic and wild animals as diagnostic material for detection of rabies.

We previously reported a novel diagnostic method using follicle-sinus complexes (FSCs) in the muzzle skin for postmortem diagnosis of rabies in dogs. However, whether this method works in other animal species remains unclear. Here, FSCs were collected from a wolf, a red fox, 2 bats, and a cat, and examined for the presence of viral antigen, viral mRNA, and viral particles. Viral antigen and viral mRNA were confirmed in Merkel cells (MCs) in FSCs of all species. Electron microscopy performed using only samples from wolf and cat confirmed viral particles in MCs of FSCs. These results suggested that this novel diagnostic method using FSCs might be useful for detection of rabies not only in domestic but also wild animals.

Cutaneous overexpression of NT-3 increases sensory and sympathetic neuron number and enhances touch dome and hair follicle innervation.

Target-derived influences of nerve growth factor on neuronal survival and differentiation are well documented, though effects of other neurotrophins are less clear. To examine the influence of NT-3 neurotrophin overexpression in a target tissue of sensory and sympathetic neurons, transgenic mice were isolated that overexpress NT-3 in the epidermis. Overexpression of NT-3 led to a 42% increase in the number of dorsal root ganglia sensory neurons, a 70% increase in the number of trigeminal sensory neurons, and a 32% increase in sympathetic neurons. Elevated NT-3 also caused enlargement of touch dome mechanoreceptor units, sensory end organs innervated by slowly adapting type 1 (SA1) neurons. The enlarged touch dome units of the transgenics had an increased number of associated Merkel cells, cells at which SA1s terminate. An additional alteration of skin innervation in NT-3 transgenics was an increased density of myelinated circular endings associated with the piloneural complex. The enhancement of innervation to the skin was accompanied by a doubling in the number of sensory neurons expressing trkC. In addition, measures of nerve fibers in cross-sectional profiles of cutaneous saphenous nerves of transgenics showed a 60% increase in myelinated fibers. These results indicate that in vivo overexpression of NT-3 by the epidermis enhances the number of sensory and sympathetic neurons and the development of selected sensory endings of the skin.

Functional properties of mechanoreceptors in mouse whisker hair follicles determined by the pressure-clamped single-fiber recording technique.

Several types of mechanoreceptors have been identified anatomically in rodent whisker hair follicles, but their functional properties have not been fully studied. Here we developed a pressure-clamped single-fiber recording technique to record impulses on mouse whisker hair follicle afferent nerves following displacements of whisker hair follicles. On the basis of the patterns of impulses evoked by the mechanical stimulation, three functional types of mechanoreceptors were identified, including rapidly adapting (RA), slowly adapting type 1 (SA1), and slowly adapting type 2 (SA2) mechanoreceptors. Impulses of all these mechanoreceptors were almost completely abolished by 30 nM TTX, and were largely suppressed by cooling temperatures at 15°C. Tested at different displacement distances as different stimulation intensity, RA mechanoreceptors showed a limited capacity for stimulation intensity encoding, but both SA1 and SA2 mechanoreceptors displayed linear increases of impulse numbers with increased stimulation intensity. Tested with different ramp speed of displacements, RA impulses were only evoked by rapid ramp stimulation but SA1 and SA2 impulses could be evoked by both rapid and slow ramp stimulation. Tested with different stimulation frequency, all three types of mechanoreceptors well followed the stimulation at 10-100 Hz. Taken together, this study revealed some important functional properties of RA, SA1 and SA2 mechanoreceptors, which helps better understand the encoding of tactile information by different types of low-threshold mechanoreceptors.