Summary Innocuous touch of the skin is detected by distinct populations of low-threshold mechanoreceptors (LTMRs), which are classified as Aβ-, Aδ- and C-LTMRs. Here, we report genetic labeling of LTMR subtypes and visualization of their relative patterns of axonal endings in hairy skin and the spinal cord. We found that each of the three major hair follicle types of trunk hairy skin; guard, awl/auchene, and zigzag hairs, is innervated by a unique and invariant combination of LTMRs; thus, each hair follicle type is a functionally distinct mechanosensory end organ. Moreover, the central projections of Aβ-, Aδ- and C-LTMRs that innervate the same or adjacent hair follicles form narrow LTMR columns in the dorsal horn. These findings support a model of mechanosensation in which the activities of Aβ-, Aδ- and C-LTMRs are integrated within dorsal horn LTMR columns and processed into outputs that underlie the perception of myriad touch sensations.

Mechanical pain contributes to the morbidity associated with inflammation and trauma, but primary sensory neurons that convey the sensation of acute and persistent mechanical pain have not been identified. Dorsal root ganglion (DRG) neurons transmit sensory information to the spinal cord using the excitatory transmitter glutamate1, a process that depends on glutamate transport into synaptic vesicles for regulated exocytotic release. Here we report that a small subset of cells in the DRG expresses the low abundance vesicular glutamate transporter VGLUT3. In the dorsal horn of the spinal cord, these afferents project to lamina I and the innermost layer of lamina II, which has previously been implicated in persistent pain caused by injury2. Since the different VGLUT isoforms generally exhibit a nonredundant pattern of expression3, we used VGLUT3 knock-out (KO) mice to assess the role of VGLUT3+ primary afferents in the behavioral response to somatosensory input. The loss of VGLUT3 specifically impairs mechanical pain sensation and in particular the mechanical hypersensitivity to normally innocuous stimuli that accompanies inflammation, nerve injury and trauma. Direct recording from VGLUT3+ neurons in the DRG further identifies them as a poorly understood population of unmyelinated, low threshold mechanoreceptors (C-LTMRs)4,5. The analysis of VGLUT3 KO mice now indicates a critical role for C-LTMRs in the mechanical hypersensitivity caused by injury.

Summary Touch perception begins with activation of low-threshold mechanoreceptors (LTMRs) in the periphery. LTMR terminals exhibit tremendous morphological heterogeneity that specifies their mechanical receptivity. In a survey of mammalian skin, we found a preponderance of neurofilament-heavy chain+ circumferential endings associated with hair follicles, prompting us to develop a genetic strategy to interrogate these neurons. Targeted in vivo recordings revealed them to be Aβ Field-LTMRs, identified 50 years ago but largely elusive thereafter. Remarkably, while Aβ Field-LTMRs are highly sensitive to gentle stroking of the skin, they are unresponsive to hair deflection, and they encode skin indentation in the noxious range across large, spotty receptive fields. Individual Aβ Field-LTMRs form up to 180 circumferential endings, making them the most anatomically expansive LTMR identified to date. Thus, Aβ Field-LTMRs are a major mammalian LTMR subtype that forms circumferential endings in hairy skin, and their sensitivity to gentle skin stroking arises through integration across many low-sensitivity circumferential endings.

Vanilloid receptor 1 (TRPV1) has been proposed to be the principal heat-responsive channel for nociceptive neurons. The skin of both rat and mouse receives major projections from primary sensory afferents that bind the plant lectin isolectin B4 (IB4). The majority of IB4-positive neurons are known to be heat-responsive nociceptors. Previous studies suggested that, unlike rat, mouse IB4-positive cutaneous afferents did not express TRPV1 immunoreactivity. Here, multiple antisera were used to confirm that mouse and rat have different distributions of TRPV1 and that TRPV1 immunoreactivity is absent in heat-sensitive nociceptors. Intracellular recording in TRPV1 Ϫ/Ϫ mice was then used to confirm that TRPV1 was not required for detecting noxious heat. TRPV1 Ϫ/Ϫ mice had more heatsensitive neurons, and these neurons had normal temperature thresholds and response properties. Moreover, in TRPV1 Ϫ/Ϫ mice, 82% of heat-responsive neurons did not express immunoreactivity for TRPV2, another putative noxious heat channel.

Most, if not all, nociceptor sensory neurons are dependent on nerve growth factor (NGF) during early embryonic development. A large subpopulation of these sensory neurons loses NGF dependency between embryonic day 16 and postnatal day 14 and become responsive to glial cell line-derived growth factor (GDNF), a member of the transforming growth factor beta (TGF-beta) family. To examine the survival and phenotypic effects of GDNF on sensory neurons in vivo, we generated transgenic mice that overexpress GDNF in the skin. GDNF-overexpresser mice had increased numbers of small unmyelinated sensory neurons that express the tyrosine kinase receptor Ret and bind the plant isolectin B4 (IB4). Surprisingly, in wild-type and transgenic mice, few ( approximately 2%) IB4-positive neurons expressed the vanilloid receptor VR1, a heat-sensitive receptor expressed by many IB4-positive neurons of the rat. Thus, in mouse, GDNF-dependent IB4-positive neurons must use a non-VR1 heat receptor. In addition, the behavior of GDNF-overexpresser animals to noxious heat or mechanical stimuli was indistinguishable from wild-type animals, indicating that, on a behavioral level, peripherally applied GDNF does not alter the sensitivity of the somatosensory system.

In the present study, a murine ex-vivo somatosensory system preparation was used to determine the response characteristics of cutaneous sensory neurons staining positively for TRPV1 or TRPV2. TRPV1 immunostaining was found exclusively (11/11) in a specific set of mechanically insensitive unmyelinated (C) nociceptors that responded to heating of their receptive fields. No cutaneous Cfibers that responded to both mechanical and heat stimuli stained positively for TRPV1 (0/62). The relationship between TRPV2 and heat transduction characteristics was not as clear, as few unmyelinated or myelinated fibers that responded to heat contained TRPV2. TRPV2 was found most frequently in mechanically sensitive myelinated fibers, including both low threshold and high threshold mechanoreceptors (nociceptors). While TRPV2 was found in only 1 of 6 myelinated polymodal nociceptors, it was found in a majority (10/16) of myelinated mechanical nociceptors. Thus, while the in vivo role of TRPV1 as a heat sensitive channel in cutaneous sensory neurons is clearly defined, the role of TRPV2 in cutaneous neurons remains unknown. These results also suggest that TRPV1 may be essential for heat transduction in a specific subset of mechanically insensitive cutaneous nociceptors, and that this subset may constitute a discrete heat input pathway for inflammation-induced thermal pain.Perspective-The distinct subset of murine cutaneous nociceptors containing TRPV1 has many attributes in common with mechanically insensitive C-fibers in humans that are believed to play a role in pathological pain states. Therefore these murine fibers provide a clinically relevant animal model for further study of this group of cutaneous nociceptors.

Summary The perception of touch, including the direction of stimulus movement across the skin, begins with activation of low-threshold mechanosensory neurons (LTMRs) that innervate the skin. Here, we show that murine Aδ-LTMRs are preferentially tuned to deflection of body hairs in the caudal-to-rostral direction. This tuning property is explained by the finding that Aδ-LTMR lanceolate endings around hair follicles are polarized; they are concentrated on the caudal (downward) side of each hair follicle. The neurotrophic factor BDNF is synthesized in epithelial cells on the caudal, but not rostral, side of hair follicles, in close proximity to Aδ-LTMR lanceolate endings, which express TrkB. Moreover, ablation of BDNF in hair follicle epithelial cells disrupts polarization of Aδ-LTMR lanceolate endings and results in randomization of Aδ-LTMR responses to hair deflection. Thus, BDNF-TrkB signaling directs polarization of Aδ-LTMR lanceolate endings, which underlies direction-selective responsiveness of Aδ-LTMRs to hair deflection.

Neurons classified as nociceptors are dependent on nerve growth factor (NGF) during embryonic development, but a large subpopulation lose this dependence during embryonic and postnatal times and become responsive to the transforming growth factor ␤ family member, glial cell line-derived growth factor (GDNF). To elucidate the functional properties of GDNF-dependent nociceptors and distinguish them from nociceptors that retain NGF dependence, the cellular and physiologic properties of sensory neurons of wild-type and transgenic mice that overexpress GDNF in the skin (GDNF-OE) were analyzed using a skin, nerve, dorsal root ganglion, and spinal cord preparation, immunolabeling, and reverse transcriptase-PCR assays. Although an increase in peripheral conduction velocity of C-fibers in GDNF-OE mice was measured, other electrophysiological properties, including resting membrane potential and somal action potentials, were unchanged. We also show that isolectin B4 (IB4)-positive neurons, many of which are responsive to GDNF, exhibited significantly lower thresholds to mechanical stimulation relative to wild-type neurons. However, no change was observed in heat thresholds for the same population of cells. The increase in mechanical sensitivity was found to correlate with significant increases in acid-sensing ion channels 2A and 2B and transient receptor potential channel A1, which are thought to contribute to detection of mechanical stimuli. These data indicate that enhanced expression of GDNF in the skin can change mechanical sensitivity of IB4-positive nociceptive afferents and that this may occur through enhanced expression of specific types of channel proteins.