Burst gap code predictions for tactile frequency are valid across the range of perceived frequencies attributed to two distinct tactile channels.

Perceived frequency of vibrotactile stimuli can be divided into two distinctive cutaneous sensations-flutter (<60 Hz) and vibratory hum (>60 Hz), mediated by two different tactile afferent types [fast adapting type I (FA1) and fast adapting type II (FA2), respectively]. We recently demonstrated a novel form of neural coding in the human tactile system, where frequency perception of stimulus pulses grouped into periodic bursts in the flutter range depended on the duration of the silent gap between bursts, rather than the periodicity or mean impulse rate. Here, we investigated whether this interburst interval could also explain the perceived frequency of electrocutaneous pulse patterns delivered at frequencies above the flutter range. At stimulus rates of 50 to 190 pulses/s, the burst gap model correctly predicted the perceived frequency. This shows that the burst gap code represents a general coding strategy that spans the range of frequencies traditionally attributed to two different tactile channels.NEW & NOTEWORTHY We present evidence for a generalized frequency processing strategy on tactile afferent inputs that is shared across a broad range of frequencies extending beyond the flutter range, supporting the notion that spike timing has an important role in shaping tactile perception.

Identification of zebrafish magnetoreceptor and cryptochrome homologs.

Magnetoreception is essential for magnetic orientation in animal migration. The molecular basis for magnetoreception has recently been elucidated in fruitfly as complexes between the magnetic receptor magnetoreceptor (MagR) and its ligand cryptochrome (Cry). MagR and Cry are present in the animal kingdom. However, it is unknown whether they perform a conserved role in diverse animals. Here we report the identification and expression of zebrafish MagR and Cry homologs towards understanding their roles in lower vertebrates. A single magr gene and 7 cry genes are present in the zebrafish genome. Zebrafish has four cry1 genes (cry1aa, cry1ab, cry1ba and cry1bb) homologous to human CRY1 and a single ortholog of human CRY2 as well as 2 cry-like genes (cry4 and cry5). By RT-PCR, magr exhibited a high level of ubiquitous RNA expression in embryos and adult organs, whereas cry genes displayed differential embryonic and adult expression. Importantly, magr depletion did not produce apparent abnormalities in organogenesis. Taken together, magr and cry2 exist as a single copy gene, whereas cry1 exists as multiple gene duplicates in zebrafish. Our result suggests that magr may play a dispensable role in organogenesis and predicts a possibility to generate magr mutants for analyzing its role in zebrafish.

Multiplexed Population Coding of Stimulus Properties by Leech Mechanosensory Cells.

Sensory coding has long been discussed in terms of a dichotomy between spike timing and rate coding. However, recent studies found that in primate mechanoperception and other sensory systems, spike rates and timing of cell populations complement each other. They simultaneously carry information about different stimulus properties in a multiplexed way. Here, we present evidence for multiplexed encoding of tactile skin stimulation in the tiny population of leech mechanoreceptors, consisting of only 10 cells of two types with overlapping receptive fields. Each mechanoreceptor neuron of the leech varies spike count and response latency to both touch intensity and location, leading to ambiguous responses to different stimuli. Nevertheless, three different stimulus estimation techniques consistently reveal that the neuronal population allows reliable decoding of both stimulus properties. For the two mechanoreceptor types, the transient responses of T (touch) cells and the sustained responses of P (pressure) cells, the relative timing of the first spikes of two mechanoreceptors encodes stimulus location, whereas summed spike counts represent touch intensity. Differences between the cell types become evident in responses to combined stimulus properties. The best estimation performance for stimulus location is obtained from the relative first spike timing of the faster and temporally more precise T cells. Simultaneously, the sustained responses of P cells indicate touch intensity by summed spike counts and stimulus duration by the duration of spike responses. The striking similarities of these results with previous findings on primate mechanosensory afferents suggest multiplexed population coding as a general principle of somatosensation. SIGNIFICANCE STATEMENT: Multiplexing, the simultaneous encoding of different stimulus properties by distinct neuronal response features, has recently been suggested as a mechanism used in several sensory systems, including primate somatosensation. While a rigorous experimental verification of the multiplexing hypothesis is difficult to accomplish in a complex vertebrate system, it is feasible for a small population of individually characterized leech neurons. Monitoring the responses of all four mechanoreceptors innervating a patch of skin revealed striking similarities between touch encoding in the primate and the leech: summed spike counts represent stimulus intensity, whereas relative timing of first spikes encodes stimulus location. These findings suggest that multiplexed population coding is a general mechanism of touch encoding common to species as different as man and worm.

Mechanoreceptors in the Anterior Horn of the Equine Medial Meniscus: an Immunohistochemical Approach.

Lameness due to stifle and especially meniscal lesions is frequent in equine species. In humans, mechanoreceptors involved in proprioceptive function are well studied. Given the high incidence of meniscal injuries in horses, and the lack of information concerning them in equine menisci, our objective was to study these corpuscles in six healthy anterior horns of the equine medial meniscus, which is the most common localisation reported for equine meniscal injuries. Immunohistochemical stainings were performed using antibodies against high molecular weight neurofilaments and glial fibrillary acidic proteins. From a purely fundamental point of view, our work highlights for the first time the presence of Ruffini, Pacini and Golgi corpuscles in equine meniscus. They were found, isolated or in clusters and always located at the vicinity of blood vessels, at the level of the anterior horn of the equine medial meniscus. This morphological approach could serve as a basis for clinical studies, to evaluate the impact of these corpuscles on the poor sportive prognosis in equine meniscal tears.

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.

Single low-threshold afferents innervating the skin of the human foot modulate ongoing muscle activity in the upper limbs.

We have shown for the first time that single cutaneous afferents in the foot dorsum have significant reflex coupling to motoneurons supplying muscles in the upper limb, particularly posterior deltoid and triceps brachii. These observations strengthen what we know from whole nerve stimulation, that skin on the foot and ankle can contribute to the modulation of interlimb muscles in distant innervation territories. The current work provides evidence of the mechanism behind the reflex, where one single skin afferent can evoke a reflex response, rather than a population. Nineteen of forty-one (46%) single cutaneous afferents isolated in the dorsum or plantar surface of the foot elicited a significant modulation of muscle activity in the upper limb. Identification of single afferents in this reflex indicates the strength of the connection and, ultimately, the importance of foot skin in interlimb coordination. The median response magnitude was 2.29% of background EMG, and the size of the evoked response did not significantly differ among the four mechanoreceptor classes (P > 0.1). Interestingly, although the distribution of afferents types did not differ across the foot dorsum, there was a significantly greater coupling response from receptors located on the medial aspect of the foot dorsum (P < 0.01). Furthermore, the most consistent coupling with upper limb muscles was demonstrated by type I afferents (fast and slowly adapting). This work contributes to the current literature on receptor specificity, supporting the view that individual classes of cutaneous afferents may subserve specific roles in kinesthesia, reflexes, and tactile perception.

Cooling reduces the cutaneous afferent firing response to vibratory stimuli in glabrous skin of the human foot sole.

Skin on the foot sole plays an important role in postural control. Cooling the skin of the foot is often used to induce anesthesia to determine the role of skin in motor and balance control. The effect of cooling on the four classes of mechanoreceptor in the skin is largely unknown, and thus the aim of the present study was to characterize the effects of cooling on individual skin receptors in the foot sole. Such insight will better isolate individual receptor contributions to balance control. Using microneurography, we recorded 39 single nerve afferents innervating mechanoreceptors in the skin of the foot sole in humans. Afferents were identified as fast-adapting (FA) or slowly adapting (SA) type I or II (FA I n = 16, FA II n = 7, SA I n = 6, SA II n = 11). Receptor response to vibration was compared before and after cooling of the receptive field (2-20 min). Overall, firing response was abolished in 30% of all receptors, and this was equally distributed across receptor type (P = 0.69). Longer cooling times were more likely to reduce firing response below 50% of baseline; however, some afferent responses were abolished with shorter cooling times (2-5 min). Skin temperature was not a reliable indicator of the level of receptor activation and often became uncoupled from receptor response levels, suggesting caution in the use of this parameter as an indicator of anesthesia. When cooled, receptors preferentially coded lower frequencies in response to vibration. In response to a sustained indentation, SA receptors responded more like FA receptors, primarily coding "on-off" events.

Asymmetric response properties of rapidly adapting mechanoreceptive fibers in the rat glabrous skin.

Previous histological and neurophysiological studies have shown that the innervation density of rapidly adapting (RA) mechanoreceptive fibers increases towards the fingertip. Since the psychophysical detection threshold depends on the contribution of several RA fibers, a high innervation density would imply lower thresholds. However, our previous human study showed that psychophysical detection thresholds for the Non-Pacinian I channel mediated by RA fibers do not improve towards the fingertip. By recording single-unit spike activity from rat RA fibers, here we tested the hypothesis that the responsiveness of RA fibers is asymmetric in the proximo-distal axis which may counterbalance the effects of innervation density. RA fibers (n = 32) innervating the digital glabrous skin of rat hind paw were stimulated with 40-Hz sinusoidal mechanical bursts at five different stimulus locations relative to the receptive field (RF) center (two distal, one RF center, two proximal). Different contactor sizes (area: 0.39, 1.63, 2.96 mm²) were used. Rate-intensity functions were constructed based on average firing rates, and the absolute spike threshold and the entrainment threshold were obtained for each RA fiber. Thresholds for proximal stimulus locations were found to be significantly higher than those for distal stimulus locations, which suggests that the mechanical stimulus is transmitted better towards the proximal direction. The effect of contactor size was not significant. Mechanical impedance of the rat digital glabrous skin was further measured and a lumped-parameter model was proposed to interpret the relationship between the asymmetric response properties of RA fibers and the mechanical properties of the skin.

Fentanyl decreases discharges of C and A nociceptors to suprathreshold mechanical stimulation in chronic inflammation.

An essential component of mechanical hyperalgesia resulting from tissue injury is an enhanced excitability of nociceptive neurons, termed mechanical sensitization. Local application of opioids to inflamed rat paws attenuates mechanical hyperalgesia and reduces electrical excitability of C-fiber nociceptors in acute injury. Here, we examined the effects of the opioid receptor agonist fentanyl on the mechanical coding properties of not only C- but also A-fiber nociceptors innervating the rat hind paw in a model of chronic pain, i.e., 4 days after Freund's complete adjuvant-induced inflammation. The peripheral mechanosensitive terminals of C-fibers (n = 143), A-fibers (n = 79), and low-threshold mechanoreceptors (n = 25) were characterized using the in vitro skin-nerve preparation from the saphenous nerve. Although mechanical activation thresholds were not changed, discharges to suprathreshold mechanical stimuli were elevated significantly in both A- and C-fiber nociceptors from inflamed tissue. In addition, the proportion of nociceptors as well as the frequency of spontaneous discharges in A (14% vs. 0%)- and C (28% vs. 8%)-fibers were increased in inflamed compared with normal tissue. Fentanyl inhibited responses to suprathreshold stimuli in a significantly higher proportion of not only C (36% vs. 7%)- but also A (41% vs. 8%)-fibers in inflamed tissue in a naloxone-reversible and concentration-dependent manner. Our results demonstrate that mechanical sensitization persists in chronic inflammation, in correlation with behavioral hyperalgesia. Opioid sensitivity of both A- and C-fibers is markedly augmented. This is consistent with an upregulation or enhanced functionality of opioid receptors located at the peripheral terminals of sensitized nociceptors.

Projection of non-peptidergic afferents to mouse tooth pulp.

A large proportion of pulpal nociceptors are known to contain neuropeptides such as CGRP. However, the projection of non-peptidergic nociceptors to tooth pulp is controversial. Recently, the non- peptidergic subset of nociceptors has been implicated in mechanical pain in the skin. Since mechanical irritation of pulpal nociceptors is critical for evoking tooth pain under pathophysiological conditions, we investigated whether the non-peptidergic afferents project to tooth pulp as potential mechanotransducing afferents. For clear visualization of the non-peptidergic afferents, we took advantage of a recently generated knock-in mouse model in which an axonal tracer, farnesylated green fluorescence protein (GFP), is expressed from the locus of a sensory neuron-specific gene, Mrgprd. In the trigeminal ganglia (TG), we demonstrated that GFP is exclusively expressed in afferents binding to isolectin B4 (IB4), a neurochemical marker of non-peptidergic nociceptors, but is rarely co-localized with CGRP. Retrograde labeling of pulpal afferents demonstrated that a low proportion of pulpal afferents was co-localized with GFP. Immunohistochemical detection of the axonal tracer revealed that GFP-positive afferent terminals were densely projected into the tooth pulp. These results provide convincing evidence that non-peptidergic nociceptors are projected into the tooth pulp and suggest a potential role for these afferents in tooth pain.

Developmental differences in peripheral glabrous skin mechanosensory nerve receptive field and intracellular electrophysiologic properties: phenotypic characterization in infant and juvenile rats.

Developmental differences in peripheral neuron characteristics and functionality exist. Direct measurement of active and passive electrophysiologic and receptive field characteristics of single mechanosensitive neurons in glabrous skin was performed and phenotypic characterization of fiber subtypes was applied to analyze developmental differences in peripheral mechanosensitive afferents. After Institutional approval, male Sprague-Dawley infant (P7: postnatal day 7) and juvenile (P28) rats were anesthetized and single cell intracellular electrophysiology was performed in the dorsal root ganglion (DRG) soma of mechanosensitive cells with receptive field (RF) in the glabrous skin of the hindpaw. Passive and active electrical properties of the cells and RF size and characteristics determined. Fiber subtype classification was performed and developmental differences in fiber subtype properties analyzed. RF size was smaller at P7 for both low and high threshold mechanoreceptor (LTMR and HTMR) with no differences between A- and C-HTMR (AHTMR and CHTMR). The RF size was also correlated to anatomic location on glabrous skin, toes having smaller RF. Conduction velocity (CV) was adequate at P28 for AHTMR and CHTMR classification, but not at P7. Only width of the action potential at half height (D50) was significantly different between HTMR at P7, while D50, CV and amplitude of the AP were significant for HTMR at P28. RF size is determined in part by the RF distribution of the peripheral neuron. Developmental differences in RF size occur with larger RF sizes occurring in younger animals. This is consistent with RF size differences determined by measuring RF in the spinal cord, except the peripheral RF is much smaller, more refined, and in some cases pinpoint. Developmental differences make CV alone unreliable for neuron classification. Utilizing integration of all measured parameters allows classification of neurons into subtypes even at the younger ages. This will prove important in understanding changes that occur in the peripheral sensory afferents in the face of ongoing development and injury early in life.

An immunohistochemical study of mechanoreceptors in lumbar spine intervertebral discs.

There are limited data concerning mechanoreceptors in normal human lumbar intervertebral discs. The aim of our study was to determine the types of mechanoreceptors in the two lower intervertebral discs in normal adult cadaveric donors and to review the literature. Twenty-five lumbar (L4-5 and L5-S1) intervertebral discs were retrieved from 15 fresh cadavers. We utilized immunoreactivity against the S-100 protein to localize specialized nerve endings. Immunoreactivity showed receptors in 92% of discs. The most frequent type had morphology resembling the Ruffini type receptor (88%), followed by the Golgi type. Free nerve fibers were frequently present. All neural structures were found in the superficial layers of the annulus fibrosus, in longitudinal ligaments, or between these two. The anterior part of the L5-S1 disc had a greater frequency of encapsulated receptors than the other parts (p=0.022), which may be correlated with the high shear forces to which the lumbosacral junction is subjected.

Species-specific behavioral patterns correlate with differences in synaptic connections between homologous mechanosensory neurons.

We characterized the behavioral responses of two leech species, Hirudo verbana and Erpobdella obscura, to mechanical skin stimulation and examined the interactions between the pressure mechanosensory neurons (P cells) that innervate the skin. To quantify behavioral responses, we stimulated both intact leeches and isolated body wall preparations from the two species. In response to mechanical stimulation, Hirudo showed local bending behavior, in which the body wall shortened only on the side of the stimulation. Erpobdella, in contrast, contracted both sides of the body in response to touch. To investigate the neuronal basis for this behavioral difference, we studied the interactions between P cells. Each midbody ganglion has four P cells; each cell innervates a different quadrant of the body wall. Consistent with local bending, activating any one P cell in Hirudo elicited polysynaptic inhibitory potentials in the other P cells. In contrast, the P cells in Erpobdella had excitatory polysynaptic connections, consistent with the segment-wide contraction observed in this species. In addition, activating individual P cells caused asymmetrical body wall contractions in Hirudo and symmetrical body wall contractions in Erpobdella. These results suggest that the different behavioral responses in Erpobdella and Hirudo are partly mediated by interactions among mechanosensory cells.