H. Richard Koerber

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.

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.

Glial Cell Line-Derived Neurotrophic Factor Family Members Sensitize Nociceptors In Vitro and Produce Thermal Hyperalgesia In Vivo

Nerve growth factor (NGF) has been implicated as an effector of inflammatory pain because it sensitizes primary afferents to noxious thermal, mechanical, and chemical [e.g., capsaicin, a transient receptor potential vanilloid receptor 1 (TRPV1) agonist] stimuli and because NGF levels increase during inflammation. Here, we report the ability of glial cell line-derived neurotrophic factor (GDNF) family members artemin, neurturin and GDNF to potentiate TRPV1 signaling and to induce behavioral hyperalgesia. Analysis of capsaicin-evoked Ca2+ transients in dissociated mouse dorsal root ganglion (DRG) neurons revealed that a 7 min exposure to GDNF, neurturin, or artemin potentiated TRPV1 function at doses 10–100 times lower than NGF. Moreover, GDNF family members induced capsaicin responses in a subset of neurons that were previously insensitive to capsaicin. Using reverse transcriptase-PCR, we found that artemin mRNA was profoundly upregulated in response to inflammation induced by hindpaw injection of complete Freund’s adjuvant (CFA): artemin expression increased 10-fold 1 d after CFA injection, whereas NGF expression doubled by day 7. No increase was seen in neurturin or GDNF. A corresponding increase in mRNA for the artemin coreceptor GFRα3 (for GDNF family receptor α) was seen in DRG, and GFRα3 immunoreactivity was widely colocalized with TRPV1 in epidermal afferents. Finally, hindpaw injection of artemin, neurturin, GDNF, or NGF produced acute thermal hyperalgesia that lasted up to 4 h; combined injection of artemin and NGF produced hyperalgesia that lasted for 6 d. These results indicate that GDNF family members regulate the sensitivity of thermal nociceptors and implicate artemin in particular as an important effector in inflammatory hyperalgesia.

Artemin Overexpression in Skin Enhances Expression of TRPV1 and TRPA1 in Cutaneous Sensory Neurons and Leads to Behavioral Sensitivity to Heat and Cold

Artemin, a neuronal survival factor in the glial cell line-derived neurotrophic factor family, binds the glycosylphosphatidylinositol-anchored protein GFRα3 and the receptor tyrosine kinase Ret. Expression of the GFRα3 receptor is primarily restricted to the peripheral nervous system and is found in a subpopulation of nociceptive sensory neurons of the dorsal root ganglia (DRGs) that coexpress the Ret and TrkA receptor tyrosine kinases and the thermosensitive channel TRPV1. To determine how artemin affects sensory neuron properties, transgenic mice that overexpress artemin in skin keratinocytes (ART-OE mice) were analyzed. Expression of artemin caused a 20.5% increase in DRG neuron number and increased the level of mRNA encoding GFRα3, TrkA, TRPV1, and the putative noxious cold-detecting channel TRPA1. Nearly all GFRα3-positive neurons expressed TRPV1 immunoreactivity, and most of these neurons were also positive for TRPA1. Interestingly, acid-sensing ion channel (ASIC) 1, 2a, 2b, and 3 mRNAs were decreased in the DRG, and this reduction was strongest in females. Analysis of sensory neuron physiological properties using an ex vivo preparation showed that cutaneous C-fiber nociceptors of ART-OE mice had reduced heat thresholds and increased firing rates in response to a heat ramp. No change in mechanical threshold was detected. Behavioral testing of ART-OE mice showed that they had increased sensitivity to both heat and noxious cold. These results indicate that the level of artemin in the skin modulates gene expression and response properties of afferents that project to the skin and that these changes lead to behavioral sensitivity to both hot and cold stimuli.

Genetic identification of C fibres that detect massage-like stroking of hairy skin in vivo

Stroking of the skin produces pleasant sensations that can occur during social interactions with conspecifics, such as grooming. Despite numerous physiological studies (reviewed in ref. 2), molecularly defined sensory neurons that detect pleasant stroking of hairy skin in vivo have not been reported. Previously, we identified a rare population of unmyelinated sensory neurons in mice that express the G-protein-coupled receptor MRGPRB4 (refs 5, 6). These neurons exclusively innervate hairy skin with large terminal arborizations that resemble the receptive fields of C-tactile (CT) afferents in humans. Unlike other molecularly defined mechanosensory C-fibre subtypes, MRGPRB4+ neurons could not be detectably activated by sensory stimulation of the skin ex vivo. Therefore, we developed a preparation for calcium imaging in the spinal projections of these neurons during stimulation of the periphery in intact mice. Here we show that MRGPRB4+ neurons are activated by massage-like stroking of hairy skin, but not by noxious punctate mechanical stimulation. By contrast, a different population of C fibres expressing MRGPRD was activated by pinching but not by stroking, consistent with previous physiological and behavioural data. Pharmacogenetic activation of Mrgprb4-expressing neurons in freely behaving mice promoted conditioned place preference, indicating that such activation is positively reinforcing and/or anxiolytic. These data open the way to understanding the function of MRGPRB4 neurons during natural behaviours, and provide a general approach to the functional characterization of genetically identified subsets of somatosensory neurons in vivo.

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.

Dynorphin Acts as a Neuromodulator to Inhibit Itch in the Dorsal Horn of the Spinal Cord

Summary Menthol and other counterstimuli relieve itch, resulting in an antipruritic state that persists for minutes to hours. However, the neural basis for this effect is unclear, and the underlying neuromodulatory mechanisms are unknown. Previous studies revealed that Bhlhb5−/− mice, which lack a specific population of spinal inhibitory interneurons (B5-I neurons), develop pathological itch. Here we characterize B5-I neurons and show that they belong to a neurochemically distinct subset. We provide cause-and-effect evidence that B5-I neurons inhibit itch and show that dynorphin, which is released from B5-I neurons, is a key neuromodulator of pruritus. Finally, we show that B5-I neurons are innervated by menthol-, capsaicin-, and mustard oil-responsive sensory neurons and are required for the inhibition of itch by menthol. These findings provide a cellular basis for the inhibition of itch by chemical counterstimuli and suggest that kappa opioids may be a broadly effective therapy for pathological itch.

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 β 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.

Sox11 transcription factor modulates peripheral nerve regeneration in adult mice

The ability of adult peripheral sensory neurons to undergo functional and anatomical recovery following nerve injury is due in part to successful activation of transcriptional regulatory pathways. Previous in vitro evidence had suggested that the transcription factor Sox11, a HMG-domain containing protein that is highly expressed in developing sensory neurons, is an important component of this regenerative transcriptional control program. To further test the role of Sox11 in an in vivo system, we developed a new approach to specifically target small interfering RNAs (siRNAs) conjugated to the membrane permeable molecule Penetratin to injured sensory afferents. Injection of Sox11 siRNAs into the mouse saphenous nerve caused a transient knockdown of Sox11 mRNA that transiently inhibited in vivo regeneration. Electron microscopic level analysis of Sox11 RNAi-injected nerves showed that regeneration of myelinated and unmyelinated axons was inhibited. Nearly all neurons in ganglia of crushed nerves that were Sox11 immunopositive showed colabeling for the stress and injury-associated activating transcription factor 3 (ATF3). In addition, treatment with Sox11 siRNAs in vitro and in vivo caused a transcriptional and translational level reduction in ATF3 expression. These anatomical and expression data support an intrinsic role for Sox11 in events that underlie successful regeneration following peripheral nerve injury.

Mrgprd Enhances Excitability in Specific Populations of Cutaneous Murine Polymodal Nociceptors

The Mas-related G protein-coupled receptor D (Mrgprd) is selectively expressed in nonpeptidergic nociceptors that innervate the outer layers of mammalian skin. The function of Mrgprd in nociceptive neurons and the physiologically relevant somatosensory stimuli that activate Mrgprd-expressing (Mrgprd+) neurons are currently unknown. To address these issues, we studied three Mrgprd knock-in mouse lines using an ex vivo somatosensory preparation to examine the role of the Mrgprd receptor and Mrgprd+ afferents in cutaneous somatosensation. In mouse hairy skin, Mrgprd, as marked by expression of green fluorescent protein reporters, was expressed predominantly in the population of nonpeptidergic, TRPV1-negative, C-polymodal nociceptors. In mice lacking Mrgprd, this population of nociceptors exhibited decreased sensitivity to cold, heat, and mechanical stimuli. Additionally, in vitro patch-clamp studies were performed on cultured dorsal root ganglion neurons from Mrgprd −/− and Mrgprd +/− mice. These studies revealed a higher rheobase in neurons from Mrgprd −/− mice than from Mrgprd +/− mice. Furthermore, the application of the Mrgprd ligand β-alanine significantly reduced the rheobase and increased the firing rate in neurons from Mrgprd +/− mice but was without effect in neurons from Mrgprd −/− mice. Our results demonstrate that Mrgprd influences the excitability of polymodal nonpeptidergic nociceptors to mechanical and thermal stimuli.