D-hair Receptors Research Articles

Specialized mechanoreceptor systems in rodent glabrous skin.

Key points An ex vivo preparation was developed to record from single sensory fibres innervating the glabrous skin of the mouse forepaw.The density of mechanoreceptor innervation of the forepaw glabrous skin was found to be three times higher than that of hindpaw glabrous skin.Rapidly adapting mechanoreceptors that innervate Meissner's corpuscles were severalfold more responsive to slowly moving stimuli in the forepaw compared to those innervating hindpaw skin.We found a distinct group of small hairs in the centre of the mouse hindpaw glabrous skin that were exclusively innervated by directionally sensitive D‐hair receptors.The directional sensitivity, but not the end‐organ anatomy, were the opposite to D‐hair receptors in the hairy skin.Glabrous skin hairs in the hindpaw are not ubiquitous in rodents, but occur in African and North American species that diverged more than 65 million years ago. Rodents use their forepaws to actively interact with their tactile environment. Studies on the physiology and anatomy of glabrous skin that makes up the majority of the forepaw are almost non‐existent in the mouse. Here we developed a preparation to record from single sensory fibres of the forepaw and compared anatomical and physiological receptor properties to those of the hindpaw glabrous and hairy skin. We found that the mouse forepaw skin is equipped with a very high density of mechanoreceptors; >3 times more than hindpaw glabrous skin. In addition, rapidly adapting mechanoreceptors that innervate Meissner's corpuscles of the forepaw were severalfold more sensitive to slowly moving mechanical stimuli compared to their counterparts in the hindpaw glabrous skin. All other mechanoreceptor types as well as myelinated nociceptors had physiological properties that were invariant regardless of which skin area they occupied. We discovered a novel D‐hair receptor innervating a small group of hairs in the middle of the hindpaw glabrous skin in mice. These glabrous skin D‐hair receptors were direction sensitive albeit with an orientation sensitivity opposite to that described for hairy skin D‐hair receptors. Glabrous skin hairs do not occur in all rodents, but are present in North American and African rodent species that diverged more than 65 million years ago. The function of these specialized hairs is unknown, but they are nevertheless evolutionarily very ancient. Our study reveals novel physiological specializations of mechanoreceptors in the glabrous skin that likely evolved to facilitate tactile exploration.

Genetic Tracing of Cav3.2 T-Type Calcium Channel Expression in the Peripheral Nervous System.

Characterizing the distinct functions of the T-type ion channel subunits Cav3.1, 3.2 or 3.3 has proven difficult due to their highly conserved amino-acid sequences and the lack of pharmacological blockers specific for each subunit. To precisely determine the expression pattern of the Cav3.2 channel in the nervous system we generated two knock-in mouse strains that express EGFP or Cre recombinase under the control of the Cav3.2 gene promoter. We show that in the brains of these animals, the Cav3.2 channel is predominantly expressed in the dentate gyrus of the hippocampus. In the peripheral nervous system, the activation of the promoter starts at E9.5 in neural crest cells that will give rise to dorsal root ganglia (DRG) neurons, but not sympathetic neurons. As development progresses the number of DRG cells expressing the Cav3.2 channel reaches around 7% of the DRG at E16.5, and remains constant until E18.5. Characterization of sensory neuron subpopulations at E18.5 showed that EGFP+ cells are a heterogeneous population consisting mainly of TrkB+ and TrkC+ cells, while only a small percentage of DRG cells were TrkA+. Genetic tracing of the sensory nerve end-organ innervation of the skin showed that the activity of the Cav3.2 channel promoter in sensory progenitors marks many mechanoreceptor and nociceptor endings, but spares slowly adapting mechanoreceptors with endings associated with Merkel cells. Our genetic analysis reveals for the first time that progenitors that express the Cav3.2 T-type calcium channel, defines a sensory specific lineage that populates a large proportion of the DRG. Using our Cav3.2-Cre mice together with AAV viruses containing a conditional fluorescent reporter (tdTomato) we could also show that Cre expression is largely restricted to two functionally distinct sensory neuron types in the adult ganglia. Cav3.2 positive neurons innervating the skin were found to only form lanceolate endings on hair follicles and are probably identical to D-hair receptors. A second population of nociceptive sensory neurons expressing the Cav3.2 gene was found to be positive for the calcitonin-gene related peptide but these neurons are deep tissue nociceptors that do not innervate the skin.

Stomatin‐domain protein interactions with acid‐sensing ion channels modulate nociceptor mechanosensitivity

Acid-sensing ion channels (ASICs) and their interaction partners of the stomatin family have all been implicated in sensory transduction. Single gene deletion of asic3, asic2, stomatin, or stoml3 all result in deficits in the mechanosensitivity of distinct cutaneous afferents in the mouse. Here, we generated asic3−/−:stomatin−/−, asic3−/−:stoml3−/− and asic2−/−:stomatin−/− double mutant mice to characterize the functional consequences of stomatin–ASIC protein interactions on sensory afferent mechanosensitivity. The absence of ASIC3 led to a clear increase in mechanosensitivity in rapidly adapting mechanoreceptors (RAMs) and a decrease in the mechanosensitivity in both Aδ- and C-fibre nociceptors. The increased mechanosensitivity of RAMs could be accounted for by a loss of adaptation which could be mimicked by local application of APETx2 a toxin that specifically blocks ASIC3. There is a substantial loss of mechanosensitivity in stoml3−/− mice in which ∼35% of the myelinated fibres lack a mechanosensitive receptive field and this phenotype was found to be identical in asic3−/−:stoml3−/− mutant mice. However, Aδ-nociceptors showed much reduced mechanosensitivity in asic3−/−:stoml3−/− mutant mice compared to asic3−/− controls. Interestingly, in asic2−/−:stomatin−/− mutant mice many Aδ-nociceptors completely lost their mechanosensitivity which was not observed in asic2−/− or stomatin−/− mice. Examination of stomatin−/−:stoml3−/− mutant mice indicated that a stomatin/STOML3 interaction is unlikely to account for the greater Aδ-nociceptor deficits in double mutant mice. A key finding from these studies is that the loss of stomatin or STOML3 in asic3−/− or asic2−/− mutant mice markedly exacerbates deficits in the mechanosensitivity of nociceptors without affecting mechanoreceptor function.

Mice mechanoreceptors are modulated by Cav3.2 T‐type calcium channels

Mechanoreceptors are sensory receptors that respond to mechanical displacement and provide the senses of touch, pressure, vibration and proprioception. Mice have three types of low-threshold mechanoreceptors: slowly adapting mechanoreceptors (SAMs), rapidly adapting mechanoreceptors (RAMs) and D-hair receptors (Driscoll & Tavernarakis, 2000). D-hair receptors are the end-terminal of type Aδ fibre axons and present a high sensitivity and ultra-rapid temporal detection of slowly moving stimuli. The mRNA expression for the T-type voltage-gated calcium channel Cav3.2 is the only marker defined for mechanoreceptors and found almost exclusively in D-hair receptors. The Cav3.2 gene encodes one of the three low-voltage-activated (LVA) Ca2+ channels (Cav3.1, Cav3.2 and Cav3.3) and are all expressed by sensory neurons in the DRG (Iftinca et al. 2007). Previous published studies proposed that Cav3.2 channel has a primary role in nociception (Bourinet et al. 2005). However, another research group that used miberfradil, a drug that blocks all T-type channels, suggested that the mechanosensitivity of D-hair receptors is reduced by blockade of LVA channels (Shin et al. 2003). For this reason the specific physiological properties of the Cav3.2 channel has not been accurately described. In a recent issue of The Journal of Physiology, Wang & Lewin (2011) conducted a study with the purpose of demonstrating the role of the T-type Cav3.2 channels. In this study, Cav3.2−/− mutant mice were used to directly address the role of this channel in sensory transduction of cutaneous mechanoreceptors and nociceptors. Mice were produced by gene targeting in mouse ES cells; the targeting vector was designed to delete the IS5 region of the murine Cacna1H gene, which resulted in deletion of exon 6, corresponding to amino acid residues 216 to 267. This method was helpful in examining the mechanosensitivity of nociceptors and mechanoreceptors in normal Cav3.2 mice and in Cav3.2−/− mutant mice, proving that nociceptors sensitivity was not altered when the Cav3.2 gene was suppressed; on the other hand, D-hair receptor sensibility was drastically impaired in their ability to encode the properties of moving stimuli, losing their high sensitivity and ability to ensure rapid temporal detection to skin movement. In addition the functional properties of other mechanoreceptors and nociceptors were unaffected by the suppression of Cav3.2 channels. Why is this important? These new data provide conclusive results of the specific function of the T-type Cav3.2 channel, solving the previous controversy about its physiological function and proving that this specific gene is responsible for providing the functional and distinctive properties of D-hair receptors. Thus, this study encloses the specific activity of this gene, being now considered not only a high specific marker of D-hair receptors but also indispensable to maintain their high sensitivity in the detection of skin deflection. The expression of Cav3.2 in D-hair mechanoreceptors may serve to provide highly accurate temporal information about rapidly changing stimuli. This information helps humans to make fast and precise judgments about the speed and direction of tactile stimuli, a characteristic that can be considered vital for survival and functionality. This is the first time that the regulation for temporal coding in mouse mechanoreceptors from Cav3.2 T-type channels has been proved. Nevertheless, results from this study showed that there exists a possibility that a minor sub-population of nociceptors is dependent on the presence of Cav3.2 channels. The research revealed no certainty that such a population exists, and it is highly unlikely that this kind of population could be identified. Further studies will be needed to ascertain if this is indeed a possibility.

The Cav3.2 T‐type calcium channel regulates temporal coding in mouse mechanoreceptors

In mammals there are three types of low-voltage-activated (LVA) calcium channels,Cav3.1, Cav3.2 and Cav3.3, which all give rise to T-type Ca2+currents. T-type Ca2+currents have long been known to be highly enriched in a sub-population of medium-sized sensory neurones in the dorsal root ganglia (DRG). However, the identity of the T-type-rich sensory neurones has remained controversial and the precise physiological role of the Cav3.2 calcium channel in these sensory neurones has not been directly addressed. Here we show, using Cav3.2−/− mutant mice,that these channels are essential for the normal temporal coding of moving stimuli by specialized skin mechanoreceptors called D-hair receptors.We show that D-hair receptors from Cav3.2−/− fire approximately 50% fewer spikes in response to ramp-and-hold displacement stimuli compared to wild type receptors. The reduced sensitivity of D-hair receptors in Cav3.2−/− mice is chiefly due to an increase in the mechanical threshold and a substantial temporal delay in the onset of high-frequency firing to moving stimuli.We examined the receptive properties of other cutaneous mechano receptors and Aδ- and C-fibre nociceptors in Cav3.2−/− mice, but found no alteration in their mechanosensitivity compared to Cav3.2+/+mice. However, C-fibre nociceptors recorded in Cav3.2−/− mutant mice displayed a small but statistically significant reduction in their spiking rate during noxious heat ramps when compared to C-fibres in control mice. The T-type calcium channel Cav3.2 is thus not only a highly specific marker of D-hair receptors but is also required to maintain their high sensitivity and above all to ensure ultra rapid temporal detection of skin movement.

Stomatin and Sensory Neuron Mechanotransduction

Somatic sensory neurons of the dorsal root ganglia are necessary for a large part of our mechanosensory experience. However, we only have a good knowledge of the molecules required for mechanotransduction in simple invertebrates such as the nematode Caenorhabiditis elegans. In C. elegans, a number of so-called mec genes have been isolated that are required for the transduction of body touch. One such gene, mec-2 codes for an integral membrane protein of the stomatin family, a large group of genes with a stomatin homology domain. Using stomatin null mutant mice, we have tested the hypothesis that the founding member of this family, stomatin might play a role in the transduction of mechanical stimuli by primary sensory neurons. We used the in vitro mouse skin nerve preparation to record from a large population of low- and high-threshold mechanoreceptors with myelinated A-fiber (n = 553) and unmyelinated C-fiber (n = 157) axons. One subtype of mechanoreceptor, the d-hair receptor, which is a rapidly adapting mechanoreceptor, had reduced sensitivity to mechanical stimulation in the absence of stomatin. Other cutaneous mechanoreceptors, including nociceptive C-fibers were not affected by the absence of a functional stomatin protein. Patch-clamp analysis of presumptive D-hair receptor mechanoreceptive neurons, which were identified by a characteristic rosette morphology in culture, showed no change in membrane excitability in the absence of the stomatin protein. We conclude that stomatin is required for normal mechanotransduction in a subpopulation of vertebrate sensory neurons.

Channeling Specific Sensations

Shin et al. determined that Ca v 3.2 channels, a type of T-type calcium channels, were specifically expressed in D-hair receptors, a distinct class of sensory neuron. Sensory neurons with their cell bodies in the dorsal root ganglia (DRG) mediate the sensations of cutaneous touch and pain. Different tactile sensations are recognized by specific types of sensory neurons with distinct physiological responses; the molecular distinctions among different classes of sensory neurons, however, remain unclear. Shin et al. used a combination of DNA microarray analysis, suppressive subtractive hybridization, and quantitative real-time polymerase chain reaction to identify genes whose expression was specifically reduced in neurotrophin 4 (NT-4) knockout mice ( NT-4 −/− ), which lose D-hair receptor neurons during development but have no other sensory deficits. The authors used in situ hybridization on the candidate genes thus identified to search for genes whose expression was restricted to a particular sensory neuronal subtype and was lost in the mutant mice with an appropriate time course. Two genes were identified by these criteria: The gene encoding the receptor for NT-4 and the gene encoding the Ca v 3.2 calcium channel. Electrophysiological and pharmacologic analysis indicated that the Ca v 3.2 calcium channel was required for D-hair receptor function. Identification of a particular class of channel as essential to the function of particular class of sensory neurons provides a key step toward determining the molecular mechanisms underlying the individual properties of the different types of neurons mediating different modalities of somatosensory response. J. B. Shin, C. Martinez-Salgado, P. A. Heppenstall, G. R. Lewin, A T-type calcium channel required for normal function of a mammalian mechanoreceptor. Nat. Neurosci . 6 , 724-730 (2003). [Online Journal]

Neurotrophin-4: A Survival Factor for Adult Sensory Neurons

The nerve growth factor (NGF) family of neurotrophins provides a substantial part of the normal trophic support for sensory neurons during development [1]. Although these neurotrophins, which include Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), and Neurotrophin-4 (NT-4), continue to be expressed into adulthood [2, 3], there is little evidence that they are survival factors for adult neurons [4]. Here we have examined the age-dependent neurotrophic requirements of a specialized type of mechanoreceptive neuron, called a D-hair receptor, in the dorsal root ganglion (DRG). Studies using knockout mice have demonstrated that the survival of D-hair receptors is dependent upon both NT-3 and NT-4 [5–7]. Here, we show that the time period when D-hair receptors require these two neurotrophins is different. Survival of D-hair receptors depends on NT-3 early in postnatal development and NT-4 later in the mature animal. The age-dependent loss of D-hair neurons in older NT-4 knockout mice was accompanied by a large reduction (78%) in neurons positive for the NT-4 receptor (trkB) together with neuronal apoptosis in the DRG. This is the first evidence that sensory neurons have a physiological requirement for a single neurotrophin for their continued survival in the adult.

Neurotrophin 4 Is Required for the Survival of a Subclass of Hair Follicle Receptors

Neurotrophin-4 (NT4) is the most recently discovered neurotrophic factor in mammals and, functionally, the least well understood. Here, we used mice that lack NT4 to determine whether NT4 is required for the survival of functionally identified subclasses of cutaneous sensory neurons. By using three independent methods of histological and electrophysiological analysis, we show that NT4 is specifically required for the survival of down hair (D-hair) receptors that innervate a subpopulation of hair follicles. All other functionally distinct types of afferents neurons innervating hairy skin were not affected in their survival or in their function. Previous studies have shown that BDNF is required for the mechanical sensitivity of slowly adapting (SA) mechanoreceptors but not for the postnatal survival of myelinated cutaneous afferent fibers. In contrast, the receptive properties of SA mechanoreceptors were not impaired in animals lacking NT4. Consequently, these data show that the two trkB ligands, NT4 and BDNF, have distinct and nonoverlapping roles in supporting cutaneous sensory neurons. Whereas NT4 is required for the survival of D-hair receptors, BDNF supports the mechanical function of SA fibers.

The Low-Affinity Neurotrophin Receptor p75 Regulates the Function But Not the Selective Survival of Specific Subpopulations of Sensory Neurons

Mice with a targeted deletion of the low-affinity neurotrophin receptor p75 (p75-/-) exhibit a 50% loss of large- and small-diameter sensory neurons in the dorsal root ganglion. Using neurophysiological recording techniques, we now show that p75 is not required for the survival of specific, functionally defined subpopulations of sensory neurons. Rather, p75-/- mice exhibit losses of neurons that subserve nociceptive as well as non-nociceptive functions. The receptive properties of large myelinated afferent fibers were normal in p75-/- mice. However, the receptive properties of subpopulations of afferent fibers with thin myelinated or unmyelinated axons were strikingly impaired in mice lacking p75. Furthermore, the presence of p75 is required for normal mechanotransduction in C fibers and D-hair receptors and normal heat sensitivity in A-fiber nociceptors.

Specific Subtypes of Cutaneous Mechanoreceptors Require Neurotrophin-3 Following Peripheral Target Innervation

Neurotrophin-3 (NT-3) is required for the development of most sensory neurons of the dorsal root ganglia. Using electrophysiological techniques in mice with null mutations of the NT-3 gene, we show that two functionally specific subsets of cutaneous afferents differentially require this factor: D-hair receptors and slowly adapting mechanoreceptors; other cutaneous receptors were unaffected. Merkel cells, which are the end organs of slowly adapting mechanoreceptors, are virtually absent in 14-day-old homozygous mutants and are severely reduced in adult NT-3 heterozygous animals. This loss of Merkel cells, together with their innervation, happens in the first postnatal weeks of life, in contrast to muscle spindles and afferents, which are never formed in the absence of NT-3. Thus, NT-3 is essential for the maintenance of specific cutaneous afferents known to subserve fine tactile discrimination in humans.