Profound alteration in cutaneous primary afferent activity produced by inflammatory mediators.

Inflammatory pain is thought to arise from increased transmission from nociceptors and recruitment of 'silent' afferents. To evaluate inflammation-induced changes, mice expressing GCaMP3 in cutaneous sensory neurons were generated and neuronal responses to mechanical stimulation in vivo before and after subcutaneous infusion of an 'inflammatory soup' (IS) were imaged in an unanesthetized preparation. Infusion of IS rapidly altered mechanical responsiveness in the majority of neurons. Surprisingly, more cells lost, rather than gained, sensitivity and 'silent' afferents that were mechanically insensitive and gained mechanosensitivity after IS exposure were rare. However, the number of formerly 'silent' afferents that became mechanosensitive was increased five fold when the skin was heated briefly prior to infusion of IS. These findings suggest that pain arising from inflamed skin reflects a dramatic shift in the balance of sensory input, where gains and losses in neuronal populations results in novel output that is ultimately interpreted by the CNS as pain.

Genetic Identification of an Expansive Mechanoreceptor Sensitive to Skin Stroking.

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 Aß field-LTMRs, identified 50 years ago but largely elusive thereafter. Remarkably, while Aß 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 Aß field-LTMRs form up to 180 circumferential endings, making them the most anatomically expansive LTMR identified to date. Thus, Aß 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.

The cellular and molecular basis of direction selectivity of Aδ-LTMRs.

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.

Delta opioid receptors presynaptically regulate cutaneous mechanosensory neuron input to the spinal cord dorsal horn.

Cutaneous mechanosensory neurons detect mechanical stimuli that generate touch and pain sensation. Although opioids are generally associated only with the control of pain, here we report that the opioid system in fact broadly regulates cutaneous mechanosensation, including touch. This function is predominantly subserved by the delta opioid receptor (DOR), which is expressed by myelinated mechanoreceptors that form Meissner corpuscles, Merkel cell-neurite complexes, and circumferential hair follicle endings. These afferents also include a small population of CGRP-expressing myelinated nociceptors that we now identify as the somatosensory neurons that coexpress mu and delta opioid receptors. We further demonstrate that DOR activation at the central terminals of myelinated mechanoreceptors depresses synaptic input to the spinal dorsal horn, via the inhibition of voltage-gated calcium channels. Collectively our results uncover a molecular mechanism by which opioids modulate cutaneous mechanosensation and provide a rationale for targeting DOR to alleviate injury-induced mechanical hypersensitivity.

The functional organization of cutaneous low-threshold mechanosensory neurons.

Innocuous touch of the skin is detected by distinct populations of neurons, the low-threshold mechanoreceptors (LTMRs), which are classified as Aß-, 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 Aß-, 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 Aß-, Aδ-, and C-LTMRs are integrated within dorsal horn LTMR columns and processed into outputs that underlie the perception of myriad touch sensations.

Early postnatal loss of heat sensitivity among cutaneous myelinated nociceptors in Swiss-Webster mice.

Cutaneous myelinated nociceptors are known to exhibit considerable heterogeneity in their response to noxious heat. In the present experiments, we studied heat sensitivity among myelinated nociceptors during early postnatal life to determine whether this heterogeneity is correlated with other physiological and anatomical properties. A total of 129 cutaneous myelinated nociceptors were recorded intracellularly and characterized using mechanical and thermal skin stimuli in ex vivo preparations from neonatal Swiss-Webster (SW) mice across postnatal ages P2-P10; physiologically identified cells were iontophoretically labeled with neurobiotin for analyses of dorsal horn terminations from heat-sensitive and heat-insensitive cells. Our results show that heat sensitivity is not strictly correlated with other physiological or anatomical properties, most notably mechanical threshold or laminar termination patterns, of myelinated nociceptors at these ages. Further, we found a marked decline in the number of heat-sensitive myelinated mechanonociceptors (A-mechanoheat nociceptors [AMHs]) during this early postnatal period. Indeed, 68% of myelinated nociceptors were AMHs between P2 and P5, whereas this percentage dropped to 36% between P6 and P10. Multiple independent lines of evidence suggest that this decrease reflects a change in phenotype in a subset of myelinated nociceptors that lose sensitivity to noxious heat in early postnatal life. Interestingly, evidence was also obtained for a significant strain difference since the early transient excess in the number of AMHs in P2-P5 SW neonates was not present in similarly aged neonates from the C57Bl/6 strain. Potential mechanisms underlying these postnatal changes in AMH number are discussed.

Injury-induced mechanical hypersensitivity requires C-low threshold mechanoreceptors.

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 glutamate, 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 (also known as SLC17A8). 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 injury. Because the different VGLUT isoforms generally have a non-redundant pattern of expression, we used Vglut3 knockout mice to assess the role of VGLUT3(+) primary afferents in the behavioural 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). The analysis of Vglut3(-/-) mice now indicates a critical role for C-LTMRs in the mechanical hypersensitivity caused by injury.

Identity of myelinated cutaneous sensory neurons projecting to nocireceptive laminae following nerve injury in adult mice.

It is widely thought that, after peripheral injury, some low-threshold mechanoreceptive (LTMR) afferents "sprout" into pain-specific laminae (I-II) of the dorsal horn and are responsible for chronic pain states such as mechanical allodynia. Although recent studies have questioned this hypothesis, they fail to account for a series of compelling results from single-fiber analyses showing extensive projections from large-diameter myelinated afferents into nocireceptive layers after nerve injury. Here we show that, in the thoracic spinal cord of naïve adult mouse, all myelinated nociceptors gave rise to terminal projections throughout the superficial dorsal horn laminae (I-II). Most (70%) of these fibers had large-diameter axons with recurving flame-shaped central arbors that projected throughout the dorsal horn laminae I-V. This morphology was reminiscent of that attributed to sprouted LTMRs described in previous studies. After peripheral nerve axotomy, we found that LTMR afferents with narrow, uninflected somal action potentials did not sprout into superficial laminae of the dorsal horn. Only myelinated noiceptive afferents with broad, inflected somal action potentials were found to give rise to recurving collaterals and project into superficial "pain-specific" laminae after axotomy. We conclude that the previously undocumented central morphology of large, myelinated cutaneous nociceptors may very well account for the morphological findings previously thought to require sprouting of LTMRs.

Myelinated skin sensory neurons project extensively throughout adult mouse substantia gelatinosa.

The substantia gelatinosa (SG) of the dorsal horn of the spinal cord is a recipient zone for unmyelinated sensory neurons in adults. Recent studies of the central anatomy of physiologically identified skin sensory neurons in neonatal mice have shown that this region also receives substantial inputs from a variety of myelinated afferents. The present experiments were performed to determine whether these neonatal inputs represent a transient phenotype that retracts from the SG. Studies were conducted in an in vivo spinal cord preparation from adult mice; thoracic levels were targeted to facilitate comparisons with previous in vitro findings. We show that the SG continues to receive substantial projections from myelinated skin sensory neurons throughout life. A large population of myelinated nociceptors conducting in the upper A delta and low A beta range maintained extensive projections throughout all areas of the SG well into adulthood; the latter gave rise to dorsally recurving "flame"-shaped arbors extending into the marginal layer that were identical to afferents described in neonates and after nerve injury in adult rats. Furthermore, exquisitely sensitive down hair follicle afferents projected throughout the inner half of the SG (i.e., lamina IIi) and sent dense clusters of terminals well into the outer SG (IIo), where they intermingled with those of unmyelinated nociceptors. Arguments are presented that the SG likely plays a predominant role in tactile processing under normal conditions, but that this role switches rapidly to nociceptive-only during environmental exigencies imposed by temperature extremes.

TRPV1 unlike TRPV2 is restricted to a subset of mechanically insensitive cutaneous nociceptors responding to heat.

UNLABELLED: 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 C-fibers 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). Although TRPV2 was found in only 1 of 6 myelinated polymodal nociceptors, it was found in a majority (10/16) of myelinated mechanical nociceptors. Thus, whereas 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.

Central and peripheral anatomy of slowly adapting type I low-threshold mechanoreceptors innervating trunk skin of neonatal mice.

Despite intensive study, our understanding of the neuronal structures responsible for transducing the broad spectrum of environmental energies that impinge upon the skin has rested on inference and conjecture. This major shortcoming motivated the development of ex vivo somatosensory system preparations in neonatal mice in the hope that their small size might allow the peripheral terminals of physiologically identified sensory neurons to be labeled intracellularly for direct study. The present report describes the first such study of the peripheral terminals of four slowly adapting type I low-threshold mechanoreceptors (SAIs) that innervated the back skin of neonatal mice. In addition, this report includes information on the central anatomy of the same SAI afferents that were identified peripherally with both physiological and anatomical means, providing an essentially complete view of the central and peripheral morphology of individual SAI afferents in situ. Our findings reveal that SAIs in neonates are strikingly adult-like in all major respects. Afferents were exquisitely sensitive to mechanical stimuli and exhibited a distinctly irregular, slowly adapting discharge to stimulation of 1-4 punctate receptive fields in the skin. Their central collaterals formed transversely oriented and largely nonoverlapping arborizations limited to regions of the dorsal horn corresponding to laminae III-V. Their peripheral arborizations were restricted entirely within miniaturized touch domes, where they gave rise to expanded disc-like endings in close apposition to putative Merkel cells in basal epidermis. These findings therefore provide the first direct confirmation of the functional morphology of this physiologically unique afferent class.

Physiological properties of mouse skin sensory neurons recorded intracellularly in vivo: temperature effects on somal membrane properties.

Recent combined analyses of the structural, functional, and molecular attributes of individual skin sensory neurons using semi-intact in vitro preparations from mice have provided a wealth of novel insights into nociceptor biology. How these findings translate to more natural conditions nevertheless remains unresolved. Toward this end, intracellular recordings were obtained from 362 physiologically identified dorsal root ganglion (DRG) neurons in a new in vivo mouse preparation developed for combined structure/function analyses of individual skin sensory neurons. Recordings were conducted at thoracic levels in adult decorticate mice for comparison with in vitro findings from the same trunk region. In all, 270 neurons were recorded at DRG temperatures tightly regulated at normal core values to establish a baseline and 137 skin sensory neurons were included in detailed analyses of somal properties for comparisons with similar data obtained under reduced temperatures mirroring in vitro conditions. Recovery of Neurobiotin-labeled central projections was crucial for verifying perceived afferent identity of certain neurons. Further, profound temperature dependency was seen across diverse physiological properties. Indeed, the broad, inflected somal spikes normally viewed as diagnostic of myelinated nociceptors were found to be a product of reduced temperatures and were not present at normal core values. Moreover, greater complexity was observed peripherally in the mechanical and thermal sensitivity profile of nociceptive and nonnociceptive populations than that seen under in vitro conditions. This novel in vivo preparation therefore holds considerable promise for future analyses of nociceptor function and plasticity in normal and transgenic models of pain mechanisms.

Glial cell-line-derived neurotrophic factor expression in skin alters the mechanical sensitivity of cutaneous nociceptors.

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 beta 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.

Nociceptors lacking TRPV1 and TRPV2 have normal heat responses.

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(-/-) mice was then used to confirm that TRPV1 was not required for detecting noxious heat. TRPV1(-/-) mice had more heat-sensitive neurons, and these neurons had normal temperature thresholds and response properties. Moreover, in TRPV1(-/-) mice, 82% of heat-responsive neurons did not express immunoreactivity for TRPV2, another putative noxious heat channel.

NT3 expressed in skin causes enhancement of SA1 sensory neurons that leads to postnatal enhancement of Merkel cells.

To determine the role of NT3 in the postnatal maturation of Merkel cell (MC) sensory neurite complexes (touch domes), we examined the development of their neural and end-organ components in wild-type and transgenic mice that overexpress NT3 (NT3-OE). Touch domes are sensory complexes of the skin that contain specialized MCs innervated by slowly adapting type 1 (SA1) neurons. Touch domes are dependent on NT3 and, though formed in newborn mice that lack NT3, are severely depleted during postnatal maturation. Mice that overexpress NT3 in the skin have larger touch domes characterized by enhanced neural innervation and MC number. In this study, we asked how this NT3-mediated enhancement occurs, whether through stimulatory effects of NT3 on the SA1 neuron, or the MC, or both. The innervation density and number of MCs associated with each touch dome were measured in wild-type and transgenic animals at postnatal times. In newborn NT3-OE mice, touch dome innervation was enhanced. Surprisingly, however, the number of MCs was lower in newborn NT3-OE animals than in wild-type littermates, and equivalent numbers were not reached until postnatal day 8 (PN8). Not until the PN12 and PN16 time points did MCs increase in NT3-OE mice. To examine the neural dependence of MCs in NT3-OE mice, touch domes were chronically denervated by resecting dorsal cutaneous nerves. Both wild-type and NT3-OE animals showed similar depletion in the number of MCs associated with touch domes. These data indicate that NT3 is not a survival factor for MCs and that the NT3-mediated enhancement of MC number is indirect and neurally dependent.

Widespread projections from myelinated nociceptors throughout the substantia gelatinosa provide novel insights into neonatal hypersensitivity.

Skin sensory neurons have long been thought to undergo major changes in anatomy and physiology over the first few weeks of postnatal life. Low-threshold mechanoreceptors (LTMRs) are believed to project extensively throughout superficial dorsal horn laminas initially and provide the afferent limb for hyperactive nocifensive reflexes. However, our recent studies revealed that neonatal LTMRs do not project into "pain-specific" regions; instead, they exhibit adult-like anatomy shortly after birth. We sought to determine whether the same might be true for myelinated high-threshold mechanoreceptors (HTMRs). We used an intact, ex vivo somatosensory system preparation from neonatal mice to allow intrasomal recording and neurobiotin labeling of individual sensory neurons characterized via natural skin stimuli. Neonatal HTMRs displayed a number of key hallmarks of their adult counterparts; relative to LTMRs, they exhibited broader, inflected somal spikes and higher mechanical thresholds and/or responded in an increasingly vigorous manner to incrementally graded forces in a manner capable of encoding stimulus intensity. Two types were discerned on the basis of central anatomy: one subset projected to superficial laminas (I/II); the other gave rise to diffuse, dorsally recurving collateral arbors extending throughout the entire dorsal horn (I-V). The latter represent a novel cutaneous afferent morphology that persists in older animals. These studies reveal that inputs from myelinated afferents to superficial pain-specific laminas in neonates arise from HTMRs and not LTMRs as commonly thought. This frequently overlooked population is in a position, therefore, to contribute substantially to paradoxical nocifensive behaviors in neonates and various pain states in adults.

Glial cell line-derived neurotrophic factor is a survival factor for isolectin B4-positive, but not vanilloid receptor 1-positive, neurons in the mouse.

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.