C. Jeffery Woodbury

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 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.

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.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.

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.

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.

On the problem of lamination in the superficial dorsal horn of mammals: A reappraisal of the substantia gelatinosa in postnatal life

Although it is one of the most distinctive and earliest recognized features in the spinal cord, the substantia gelatinosa (SG) remains among the most enigmatic of central nervous system regions. The present neuroanatomical studies employed transganglionic transport of horseradish peroxidase conjugates of choleragenoid (B‐HRP) and the B4 isolectin of Bandeiraea simplicifolia (IB4‐HRP) on opposite sides to compare the projection patterns of myelinated and unmyelinated cutaneous primary afferents, respectively, within the superficial dorsal horn of the spinal cord in postnatal mice, from shortly after birth to adulthood. Putative unmyelinated afferents labeled with IB4‐HRP gave rise to a dense sheet of terminal‐like labeling restricted to the outer half of the SG. In contrast, myelinated inputs labeled with B‐HRP gave rise to a similarly dense sheet of terminal‐like labeling that occupied the inner half of the SG. This adult organization, with two dense sheets of terminal labeling in the superficial dorsal horn, was clearly evident shortly after birth using these markers, prior to the emergence of the SG. Furthermore, the location of the SG proper varied considerably within the dorsoventral plane of the dorsal horn according to mediolateral and segmental locations, a finding that was also seen in comparative studies in rat and cat. These findings caution against equating the SG in particular, and the superficial dorsal horn in general, with nociceptive processing; at minimum, the SG subserves a clear duality of function, with only a thin portion of its outermost aspect devoted to pain. J. Comp. Neurol. 417:88–102, 2000. ©2000 Wiley‐Liss, Inc.

Central anatomy of individual rapidly adapting low-threshold mechanoreceptors innervating the “hairy” skin of newborn mice: Early maturation of hair follicle afferents

Adult skin sensory neurons exhibit characteristic projection patterns in the dorsal horn of the spinal gray matter that are tightly correlated with modality. However, little is known about how these patterns come about during the ontogeny of the distinct subclasses of skin sensory neurons. To this end, we have developed an intact ex vivo somatosensory system preparation in neonatal mice, allowing single, physiologically identified cutaneous afferents to be iontophoretically injected with Neurobiotin for subsequent histological analyses. The present report, centered on rapidly adapting mechanoreceptors, represents the first study of the central projections of identified skin sensory neurons in neonatal animals. Cutaneous afferents exhibiting rapidly adapting responses to sustained natural stimuli were encountered as early as recordings were made. Well‐stained representatives of coarse (tylotrich and guard) and fine‐diameter (down) hair follicle afferents, along with a putative Pacinian corpuscle afferent, were recovered from 2–7‐day‐old neonates. All were characterized by narrow, uninflected somal action potentials and generally low mechanical thresholds, and many could be activated via deflection of recently erupted hairs. The central collaterals of hair follicle afferents formed recurrent, flame‐shaped arbors that were essentially miniaturized replicas of their adult counterparts, with identical laminar terminations. The terminal arbors of down hair afferents, previously undescribed in rodents, were distinct and consistently occupied a more superficial position than tylotrich and guard hair afferents. Nevertheless, the former extended no higher than the middle of the incipient substantia gelatinosa, leaving a clear gap more dorsally. In all major respects, therefore, hair follicle afferents display the same laminar specificity in neonates as they do in adults. The widely held misperception that their collaterals extend exuberant projections into pain‐specific regions of the dorsal horn during early postnatal life is shown to have multiple, deep‐rooted underpinnings. J. Comp. Neurol. 436:304–323, 2001.

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.

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. J. Comp. Neurol. 508:500–509, 2008.

Comprehensive phenotyping of sensory neurons using an ex vivo somatosensory system

In adults, primary sensory neurons exhibit pronounced diversity in many phenotypic traits including peripheral response properties, somal spike shape, neurochemical content, and laminar distribution of projections in the spinal dorsal horn. While these traits are correlated with sensory submodality type, single traits are not sufficient to accurately characterize individual neurons. We have recently developed a novel mouse ex vivo somatosensory preparation that allows the examination of multiple traits for individual cutaneous sensory neurons. Here we describe the results from our initial studies of adult and neonatal mice employing this preparation. Adult mice were anesthetized and perfused with chilled oxygenated artificial cerebrospinal fluid (aCSF). The spinal cord, dorsal root ganglia (DRGs), several attached dorsal cutaneous nerves (DCNs), and skin were isolated and placed in a chamber with a circulating bath of oxygenated aCSF. Sensory neuron somata were impaled, their cutaneous mechanical response properties were determined, and then one soma/ganglion was injected with Neurobiotin (NB). Spinal cord sections containing the cells central projections were reacted with horseradish peroxidase-conjugated avidin and visualized using diaminobenzidene. DRG sections were reacted with primary antisera for calcitonin gene-related peptide (CGRP), and the NB-stained somata were visualized using avidin-FITC. In these initial studies, neonatal cutaneous sensory neurons are found to be virtually miniature replicas of the their adult counterparts. These findings challenge long-held beliefs that the laminar distribution of the central projections and somal spike properties of cutaneous low-threshold mechanoreceptors (LTMRs) undergo delayed maturation during the first 2-3 weeks of postnatal development.

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. J. Comp. Neurol. 505:547–561, 2007.