Aδ Nociceptors Research Articles

The role of the endocannabinoid 2-arachidonoylglycerol in the in vivo spinal oxytocin-induced antinociception in male rats

Oxytocin receptor (OTR) activation at the spinal level produces antinociception. Some data suggest that central OTR activation enhances social interaction via an increase of endocannabinoids (eCB), but we do not know if this could occur at the spinal level, modulating pain transmission. Considering that oxytocin via OTR stimulates diacylglycerol formation, a key intermediate in synthesizing 2-arachidonylglycerol (2-AG), an eCB molecule, we sought to test the role of the eCB system on the spinal oxytocin-induced antinociception. Behavioral and electrophysiological experiments were conducted in naïve and formalin-treated (to induce long-term mechanical hypersensitivity) male Wistar rats. Intrathecal RHC 80267 injections, an inhibitor of the enzyme diacylglycerol lipase (thus, decreasing 2-AG formation), produces transient mechanical hypersensitivity, an effect unaltered by oxytocin but reversed by gabapentin. Similarly, in in vivo extracellular recordings of naïve spinal wide dynamic range cells, juxtacellular picoinjection of RHC 80267 increases the firing of nociceptive Aδ-, C-fibers, and post-discharge, an effect unaltered by oxytocin. Interestingly, in sensitized rats, oxytocin picoinjection reverses the RHC 80627-induced hyperactivity of Aδ-fibers (but not C- or post-discharge activity). In contrast, a sub-effective dose of JZL184 (a monoacylglycerol lipase inhibitor, thus favoring 2-AG levels), which does not have per se an antinociceptive effect in the formalin-induced hypernociception, the oxytocin-induced antinociception is boosted. Similarly, electrophysiological experiments suggest that juxtacellular JZL184 diminishes the neuronal firing of nociceptive fibers, and co-injection with oxytocin prolongs and enhances the antinociceptive effect. These data may imply that 2-AG formation may play a role in the spinal antinociception induced by oxytocin.

Effects of remifentanil on the noxiously stimulated somatosensory evoked potentials recorded at the spinal cord in dogs and cats

This study assessed the somatosensory evoked potentials (SEPs) in dogs and cats to compare the effect of remifentanil on the action potentials evoked by peripheral noxious stimulation in the spinal cord. Five healthy dogs and five healthy cats underwent general anaesthesia induced with propofol and maintained with isoflurane. Each animals received all dosage of a constant-rate infusion of remifentanil at 0 (control), 0.25, 0.5, 1.0 or 2.0 μg/kg/min. The hair of the dorsal foot of a hind limb was clipped and an intraepidermal stimulation electrode that could selectively stimulate the nociceptive Aδ and C fibres was attached. An electrical stimulus was generated by a portable peripheral nerve testing device. The evoked potentials were recorded by two needle electrodes inserted subcutaneously in the dorsal midline between the lumbar vertebra: L3-L4 and L4-L5. Bimodal waveforms were obtained by electrical stimulation in control dogs and cats. The inhibitory effect of remifentanil was evaluated by comparing the changes in the N1P2 and P2N2 amplitudes. The N1P2 amplitude was depressed by remifentanil in a dose-dependent manner in dogs, but it showed no remifentanil-induced changes in cats. While the P2N2 amplitude was also depressed in a dose-dependent manner in dogs, it showed milder remifentanil-induced effects in cats. The N1P2 and P2N2 amplitudes observed herein are assumed to represent the evoked potentials derived from the Aδ and C fibres, respectively. Thus, the inhibitory effect of remifentanil on nociceptive transmission at the spinal cord was much weaker in cats, especially for transmissions possibly derived from Aδ fibres.

Investigations into an overlooked early component of painful nociceptive withdrawal reflex responses in humans.

The role of pain as a warning system necessitates a rapid transmission of information from the periphery for the execution of appropriate motor responses. The nociceptive withdrawal reflex (NWR) is a physiological response to protect the limb from a painful stimulus and is often considered an objective measure of spinal nociceptive excitability. The NWR is commonly defined by its latency in the presumed Aδ-fiber range consistent with the canonical view that "fast pain" is signaled by Aδ nociceptors. We recently demonstrated that human skin is equipped with ultrafast (Aβ range) nociceptors. Here, we investigated the short-latency component of the reflex and explored the relationship between reflex latency and pain perception. We revisited our earlier work on NWR measurements in which, following convention, only reflex responses in the presumed Aδ range were considered. In our current analysis, we expanded the time window to search for shorter latency responses and compared those with pain ratings. In both cohorts, we found an abundance of recordings with short-latency reflex responses. In nearly 90% of successful recordings, only single reflex responses (not dual) were seen which allowed us to compare pain ratings based on reflex latencies. We found that shorter latency reflexes were just as painful as those in the conventional latency range. We found a preponderance of short-latency painful reflex responses. Based on this finding, we suggest that short-latency responses must be considered in future studies. Whether these are signaled by the ultrafast nociceptors remains to be determined.

Acupuncture-Induced Changes in Nociception, Measured by Pain-Related Evoked Potentials: A Pilot Mechanistic Study.

Objective: The nociceptive system has been implicated in acupuncture analgesia, although acupuncture's precise mechanism of action remains unknown. Electric pain-related evoked potentials (PREPs) have emerged as an effective and reliable electrophysiologic method for evaluation of the human nociceptive system by electric stimulation of nociceptive Aδ and C fibers. This pilot mechanistic study aims to assess the feasibility of using advanced PREP techniques together with electroacupuncture and to use PREPs to characterize acupuncture's effect on nociception. Methods: Seven healthy volunteers underwent a previously designed electroacupuncture protocol using acupoints in the legs bilaterally, which has been demonstrated to induce systemic analgesia. Advanced PREP techniques involving tripolar stimulating electrode, varying interstimulus interval, and incorporating a cognitive task during PREPs were used. PREPs were assessed before electroacupuncture, during electroacupuncture, and 30 min after electroacupuncture. Subjective pain perception in response to the PREP-related electric pain stimuli delivered to the nondominant hand was assessed on the visual analog scale (VAS) at baseline, during electroacupuncture, and 30 min postelectroacupuncture. Results: Reliable PREP N1, P1, and N2 waves were obtained from all subjects at the following average latencies: N1 = 131.5 msec, P1 = 189.4 msec, and N2 = 231.1 msec. Electroacupuncture caused a significant reduction in PREP N1P1 wave amplitudes from 25.6 to 15.4 μV (p = 0.006) and electric pain perception on the VAS-from 2.86 to 2.14 (p = 0.008), compared to baseline. These effects were sustained at 30 min postacupuncture with N1P1 wave amplitude 17.2 μV (p = 0.030) and VAS 2.28 (p = 0.030), compared to baseline. Conclusions: Electroacupuncture causes significant changes in objective nociception, measured by PREP N1P1 wave amplitudes, and in subjective nociception, measured by the VAS, and these effects are sustained for 30 min after electroacupuncture. Planned future studies will involve chronic pain populations and will aim to assess acupuncture's longer term analgesic effects.

Event-related potentials evoked by skin puncture reflect activation of Aβ fibers: comparison with intraepidermal and transcutaneous electrical stimulations.

BackgroundRecently, event-related potentials (ERPs) evoked by skin puncture, commonly used for blood sampling, have received attention as a pain assessment tool in neonates. However, their latency appears to be far shorter than the latency of ERPs evoked by intraepidermal electrical stimulation (IES), which selectively activates nociceptive Aδ and C fibers. To clarify this important issue, we examined whether ERPs evoked by skin puncture appropriately reflect central nociceptive processing, as is the case with IES.MethodsIn Experiment 1, we recorded evoked potentials to the click sound produced by a lance device (click-only), lance stimulation with the click sound (click+lance), or lance stimulation with white noise (WN+lance) in eight healthy adults to investigate the effect of the click sound on the ERP evoked by skin puncture. In Experiment 2, we tested 18 heathy adults and recorded evoked potentials to shallow lance stimulation (SL) with a blade that did not reach the dermis (0.1 mm insertion depth); normal lance stimulation (CL) (1 mm depth); transcutaneous electrical stimulation (ES), which mainly activates Aβ fibers; and IES, which selectively activates Aδ fibers when low stimulation current intensities are applied. White noise was continuously presented during the experiments. The stimulations were applied to the hand dorsum. In the SL, the lance device did not touch the skin and the blade was inserted to a depth of 0.1 mm into the epidermis, where the free nerve endings of Aδ fibers are located, which minimized the tactile sensation caused by the device touching the skin and the activation of Aβ fibers by the blade reaching the dermis. In the CL, as in clinical use, the lance device touched the skin and the blade reached a depth of 1 mm from the skin surface, i.e., the depth of the dermis at which the Aβ fibers are located.ResultsThe ERP N2 latencies for click-only (122 ± 2.9 ms) and click+lance (121 ± 6.5 ms) were significantly shorter than that for WN+lance (154 ± 7.1 ms). The ERP P2 latency for click-only (191 ± 11.3 ms) was significantly shorter than those for click+lance (249 ± 18.6 ms) and WN+lance (253 ± 11.2 ms). This suggests that the click sound shortens the N2 latency of the ERP evoked by skin puncture. The ERP N2 latencies for SL, CL, ES, and IES were 146 ± 8.3, 149 ± 9.9, 148 ± 13.1, and 197 ± 21.2 ms, respectively. The ERP P2 latencies were 250 ± 18.2, 251 ± 14.1, 237 ± 26.3, and 294 ± 30.0 ms, respectively. The ERP latency for SL was significantly shorter than that for IES and was similar to that for ES. This suggests that the penetration force generated by the blade of the lance device activates the Aβ fibers, consequently shortening the ERP latency.ConclusionsLance ERP may reflect the activation of Aβ fibers rather than Aδ fibers. A pain index that correctly and reliably reflects nociceptive processing must be developed to improve pain assessment and management in neonates.

Ultrastructural Evidence for Oxytocin and Oxytocin Receptor at the Spinal Dorsal Horn: Mechanism of Nociception Modulation

Oxytocin is a hypothalamic neuropeptide involved in the inhibition of nociception transmission at spinal dorsal horn (SDH) level (the first station where the incoming peripheral signals is modulated). Electrophysiological, behavioral, and pharmacological data strongly support the role of this neuropeptide and its receptor (the oxytocin receptor, OTR) as a key endogenous molecule with analgesic properties. Briefly, current data showed that oxytocin release from the hypothalamus induces OTR activation at the SDH, inducing selective inhibition of the nociceptive Aδ- and C-fibers (probably peptidergic) activity, but not the activity of proprioceptive fibers (i.e. Aβ-fibers). The above inhibition could be a direct presynaptic mechanism, or a mechanism mediated by GABAergic interneurons. However, the exact anatomical localization of oxytocin and OTR remains unclear. In this context, the present study set out to analyze the role of OTRs, GABAergic cells and CGRP fibers in the SDH in rats by using electron microscopy. Ultrastructural analyses of the SDH tissue show that: (i) oxytocin and OTR are found in asymmetrical synapsis; (ii) OTR is found in GABAergic interneurons (near unmyelinated fibers), CGRPergic fibers and glial cells; (iii) whereas oxytocin is present in supraspinal descending projection fibers. These anatomical data strongly support the notion that oxytocin released at the SDH could presynaptically inhibit the nociceptive input from the peripheral primary afferent fibers. This inhibitory action could be direct or use a GABA interneuron. Furthermore, our findings that OTR is exhibited in glial tissue at the SDH requires further exploration in nociception assays.

Granger Causality-Based Pain Classification Using EEG Evoked by Electrical Stimulation Targeting Nociceptive Aδ and C Fibers

Classification of pain levels from evoked electroencephalography (eEEG) has been achieved for the purpose of objective assessment of pain. However, differentiation of acute and chronic pain resulting from the activation of nociceptive nerves, the $\text{A}\delta $ and C fibers, respectively, has remained unsolved, mainly due to the lack of effective features. Granger causality (GC) was applied to show the role of specific brain areas in the differentiation between pain and tactile sensation but yet was used to differentiate nociceptive fiber activations. The purpose of this study is two-fold: first, to develop pain perception classification systems using GC as features, and second, to identify causal connectivity for effective differentiation of nociceptive fiber activations. The eEEG responding to three stimulation waveforms, 1 Hz square waves, 5 Hz sine waves, both targeting C fibers, and 250 Hz sine waves, targeting $\text{A}\delta $ , were recorded. Three GC categories based on channel areas, frequency bands, and stimulation waveforms were computed from the eEEG and used as features to classify nociceptive fiber activations and pain levels. It was found that GCs in the alpha band produced the highest average classification accuracy of 99.71%, followed by the gamma band, with 98.66%. Meanwhile, the GC feature contributing most to differentiating $\text{A}\delta $ and C was found in the alpha band. The GC flow computed from the results of 5 Hz and 250 Hz stimulation had an important role in the prediction of mild pain and maximum pain, respectively. These results demonstrated the high potential of GC as features of pain-related information.

Attitudes toward Complementary and Alternative Medicine, Beliefs in Afterlife and Religiosity among Psychiatrists, Psychologists and Theologists.

The aim of our study was to investigate the differences in beliefs, attitudes toward CAM, beliefs in afterlife and religiosity among the sample of psychiatrists, psychologists, and theologists. Relationship among these constructs could have impact on the concept of mental health. Research was conducted in the Split urban area, Croatia, during 2017 on a sample of psychiatrists (n=51), psychologists (n=55), and theologists (n=25). Participants were presented a figure of the human body, which contained numbers identifying eight different regions of the body. Participants were asked to select which region best represents the location of the self, soul, and mind in the body. We used CAIMAQ (The Complementary, Alternative and Integrative Medicine Attitudes Questionnaire) which contains five subscales. The Afterdeath Beliefs Scale was used to measure the varieties of afterlife beliefs. Analyses showed that applied questionnaires have appropriate reliability and expected factor structure. The most frequent locations of the Soul were 9 (37%, Not located in any centralized region in the body) and 5 (31% chest), whereas Self and Mind were mostly located in the head (43% and 73%). Psychiatrists and psychologists have average scores on positive pole of CAIMAQ but did not differ significantly (p>0.05). There were statistical differences between theologists and psychologists/psychiatrists on two subscales: "nutritional counseling and dietary/food supplements can be effective in the treatment of pathology" and "attitudes toward a holistic understanding of the disease" (p<0.05). There were significant correlations between religion and three CAIMAQ subscales. Although they were mostly religious, psychiatrists and psychologists had a higher average score on Annihilation than theologists. They also did not believe in body resurrection and connection between behavior during life and after death. The results of our study could have impact on the concept of mental health and in the future must be deeper evaluated within qualitative research methodology.

A newly identified nociresponsive region in the transitional zone (TZ) in rat sensorimotor cortex

The primary somatosensory cortex (S1) comprises a number of functionally distinct regions, reflecting the diversity of somatosensory receptor submodalities innervating the body. In particular, two spatially and functionally distinct nociceptive regions have been described in primate S1 (Vierck et al., 2013; Whitsel et al., 2019). One region is located mostly in Brodmann cytoarchitectonic area 1, where a subset of neurons exhibit functional characteristics associated with myelinated Aδ nociceptors and perception of 1st/sharp, discriminative pain. The second region is located at the transition between S1 and primary motor cortex (M1) in area 3a, where neurons exhibit functional characteristics associated with unmyelinated C nociceptors and perception of 2nd/slow, burning pain. To test the hypothesis that in rats the transitional zone (TZ) – which is a dysgranular region at the transition between M1 and S1 – is the functional equivalent of the nociresponsive region of area 3a in primates, extracellular spike discharge activity was recorded from TZ neurons in rats under general isoflurane anesthesia. Thermonoxious stimuli were applied by lowering the contralateral forepaw or hindpaw into a 48–51 °C heated water bath for 5–10 s. Neurons in TZ were found to be minimally affected by non-noxious somatosensory stimuli, but highly responsive to thermonoxious skin stimuli in a slow temporal summation manner closely resembling that of nociresponsive neurons in primate area 3a. Selective inactivation of TZ by topical lidocaine application suppressed or delayed the nociceptive withdrawal reflex, suggesting that TZ exerts a tonic facilitatory influence over spinal cord neurons producing this reflex. In conclusion, TZ appears to be a rat homolog of the nociresponsive part of monkey area 3a. A possibility is considered that this region might be primarily engaged in autonomic aspects of nociception.

Descending inhibition selectively counteracts the capsaicin-induced facilitation of dorsal horn neurons activated by joint nociceptive afferents.

Previous studies from our laboratory showed that in the anesthetized cat, the intradermal injection of capsaicin in the hindpaw facilitated the intraspinal field potentials (IFPs) evoked by stimulation of the intermediate and high-threshold myelinated fibers in the posterior articular nerve (PAN). The capsaicin-induced facilitation was significantly reduced 3-4h after the injection, despite the persistence of hindpaw inflammation. Although this effect was attributed to an incremented descending inhibition acting on the spinal pathways, it was not clear if it was set in operation once the capsaicin-induced effects exceeded a certain threshold, or if it was continuously operating to keep the increased neuronal activation within manageable limits. To evaluate the changes in descending inhibition, we now examined the effects of successive reversible spinal blocks on the amplitude of the PAN IFPs evoked at different times after the intradermal injection of capsaicin. We found that after capsaicin the PAN IFPs recorded in laminae III-V by activation of high-threshold nociceptive Aδ myelinated fibers increased gradually during successive reversible spinal blocks, while the IFPs evoked by intermediate and low threshold proprioceptive Aβ afferents were only slightly affected. It is concluded that during the development of the central sensitization produced by capsaicin, there is a gradual increase of descending inhibition that tends to limit the nociceptive-induced facilitation, mainly by acting on the neuronal populations receiving inputs from the capsaicin-activated afferents without significantly affecting the information on joint angle transmitted by the low threshold afferents.

Characterizing the Short-Term Habituation of Event-Related Evoked Potentials.

Fast-rising sensory events evoke a series of functionally heterogeneous event-related potentials (ERPs). Stimulus repetition at 1 Hz induces a strong habituation of the largest ERP responses, the vertex waves (VWs). VWs are elicited by stimuli regardless of their modality, provided that they are salient and behaviorally relevant. In contrast, the effect of stimulus repetition on the earlier sensory components of ERPs has been less explored, and the few existing results are inconsistent. To characterize how the different ERP waves habituate over time, we recorded the responses elicited by 60 identical somatosensory stimuli (activating either non-nociceptive Aβ or nociceptive Aδ afferents), delivered at 1 Hz to healthy human participants. We show that the well-described spatiotemporal sequence of lateralized and vertex ERP components elicited by the first stimulus of the series is largely preserved in the smaller-amplitude, habituated response elicited by the last stimuli of the series. We also found that the earlier lateralized sensory wave habituates across the 60 trials following the same decay function of the VWs: this decay function is characterized by a large drop at the first stimulus repetition followed by smaller decreases at subsequent repetitions. Interestingly, the same decay functions described the habituation of ERPs elicited by repeated non-nociceptive and nociceptive stimuli. This study provides a neurophysiological characterization of the effect of prolonged and repeated stimulation on the main components of somatosensory ERPs. It also demonstrates that both lateralized waves and VWs are obligatory components of ERPs elicited by non-nociceptive and nociceptive stimuli.

Presynaptic Inhibition of Primary Nociceptive Signals to Dorsal Horn Lamina I Neurons by Dopamine

The dorsal horn of the spinal cord represents the first relay station in the pain pathway where primary nociceptive inputs are modulated by local circuits and by descending signals before being relayed to supraspinal nuclei. To determine whether dopamine can modulate primary nociceptive Aδ- and C-fiber signals, the effects of dopamine were tested on the excitatory postsynaptic currents (EPSCs) recorded from large lamina I neurons and from retrograde-labeled spinoparabrachial lamina I neurons upon stimulation of the L4/L5 dorsal root in horizontal spinal cord slices in vitro Dopamine inhibited the EPSCs in a dose-dependent manner, with substantial inhibition (33%) at 1 μm and maximum inhibition (∼70%) at 10-20 μm Dopamine reduced the frequency of miniature EPSCs recorded from large lamina I neurons, increased the paired pulse depression ratio of paired EPSCs, and induced similar inhibition of EPSCs after dialysis of large lamina I neurons with GDP-β-S, consistent with actions at presynaptic sites. Pharmacological experiments suggested that the inhibitory effects of dopamine were largely mediated by D4 receptors (53%). Similar inhibition (66%) by dopamine was observed on EPSCs recorded from ipsilateral large lamina I neurons 6 d after injection of complete Freund's adjuvant in the hindpaw, suggesting that dopamine downregulates primary nociceptive inputs to lamina I neurons during chronic inflammatory pain. We propose that presynaptic inhibition of primary nociceptive inputs to lamina I projection neurons is a mechanism whereby dopamine can inhibit incoming noxious stimuli to the dorsal horn of the spinal cord.SIGNIFICANCE STATEMENT Lamina I projection neurons represent the main output for the pain signals from the dorsal horn of the spinal cord to brainstem and thalamic nuclei. We found that dopamine inhibits the nociceptive Aδ- and C-fiber synaptic inputs to lamina I projection neurons via presynaptic actions. Similar inhibitory effects of dopamine on the EPSCs were observed in rats subjected to complete Freund's adjuvant to induce peripheral inflammation, suggesting that dopamine inhibits the synaptic inputs to lamina I neurons in the setting of injury. A better understanding of how primary nociceptive inputs to the dorsal horn of the spinal cord are modulated by descending monoaminergic signals may help in the development of new pharmacological strategies to selectively downregulate the output from lamina I projection neurons.

Somatotopic Representation of Second Pain in the Primary Somatosensory Cortex of Humans and Rodents.

There is now compelling evidence that selective stimulation of Aδ nociceptors eliciting first pain evokes robust responses in the primary somatosensory cortex (S1). In contrast, whether the C-fiber nociceptive input eliciting second pain has an organized projection to S1 remains an open question. Here, we recorded the electrocortical responses elicited by nociceptive-specific laser stimulation of the four limbs in 202 humans (both males and females, using EEG) and 12 freely moving rats (all males, using ECoG). Topographical analysis and source modeling revealed in both species, a clear gross somatotopy of the unmyelinated C-fiber input within the S1 contralateral to the stimulated side. In the human EEG, S1 activity could be isolated as an early-latency negative deflection (C-N1 wave peaking at 710–730 ms) after hand stimulation, but not after foot stimulation because of the spatiotemporal overlap with the subsequent large-amplitude supramodal vertex waves (C-N2/P2). In contrast, because of the across-species difference in the representation of the body surface within S1, S1 activity could be isolated in rat ECoG as a C-N1 after both forepaw and hindpaw stimulation. Finally, we observed a functional dissociation between the generators of the somatosensory-specific lateralized waves (C-N1) and those of the supramodal vertex waves (C-N2/P2), indicating that C-fiber unmyelinated input is processed in functionally distinct somatosensory and multimodal cortical areas. These findings demonstrated that C-fiber input conveys information about the spatial location of noxious stimulation across the body surface, a prerequisite for deploying an appropriate defensive motor repertoire.SIGNIFICANCE STATEMENT Unmyelinated C-fibers are the evolutionarily oldest peripheral afferents responding to noxious environmental stimuli. Whether C-fiber input conveys information about the spatial location of the noxious stimulation to the primary somatosensory cortex (S1) remains an open issue. In this study, C-fibers were activated by radiant heat stimuli delivered to different parts of the body in both humans and rodents while electrical brain activity was recorded. In both species, the C-fiber peripheral input projects to different parts of the contralateral S1, coherently with the representation of the body surface within this brain region. These findings demonstrate that C-fiber input conveys information about the spatial location of noxious stimulation across the body surface, a prerequisite for deploying an appropriate defensive motor repertoire.

Identification of a novel spinal nociceptive-motor gate control for Aδ pain stimuli in rats.

Physiological responses to nociceptive stimuli are initiated within tens of milliseconds, but the corresponding sub-second behavioral responses have not been adequately explored in awake, unrestrained animals. A detailed understanding of these responses is crucial for progress in pain neurobiology. Here, high-speed videography during nociceptive Aδ fiber stimulation demonstrated engagement of a multi-segmental motor program coincident with, or even preceding, withdrawal of the stimulated paw. The motor program included early head orientation and adjustments of the torso and un-stimulated paws. Moreover, we observed a remarkably potent gating mechanism when the animal was standing on its hindlimbs and which was partially dependent on the endogenous opioid system. These data reveal a profound, immediate and precise integration of nociceptive inputs with ongoing motor activities leading to the initiation of complex, yet behaviorally appropriate, response patterns and the mobilization of a new type of analgesic mechanism within this early temporal nociceptive window.

Mechanically sensitive Aδ nociceptors that innervate bone marrow respond to changes in intra-osseous pressure.

Sensory neurons that innervate the bone marrow provide the CNS with information about pain associated with bone disease and pathology, but little is known of their function. Here we use a novel in vivo bone-nerve electrophysiological preparation to study how they respond to noxious mechanical stimulation delivered by increasing intra-osseous pressure. We provide evidence that sensory neurons that innervate the bone marrow respond to high threshold noxious mechanical stimulation, have response properties consistent with a role in nociception, provide information about different features of an intra-osseous pressure stimulus and express the Piezo2 mechano-transducer molecule. Our findings show how some bone marrow nociceptors signal pain in bony diseases and pathologies that involve a mechanical disturbance or increased intra-osseous pressure, and that the Piezo2 mechano-transducer may be involved. Whilst the sensory neurons and nerve terminals that innervate bone marrow have a morphology and molecular phenotype consistent with a role in nociception, little is known about their physiology or the mechanisms that generate and maintain bone pain. In the present study, we provide evidence that Aδ nociceptors that innervate the bone marrow respond to high threshold noxious mechanical stimulation, exhibit fatigue in response to prior stimulation and in some cases can be sensitized by capsaicin. They can be classified on the basis of their response properties as either phasic-tonic units that appear to code for different intensities of intra-osseous pressure, or phasic units that code for the rate of change in intra-osseous pressure. Three different subclasses of mechanically sensitive Aδ units were observed: phasic units that were sensitized by capsaicin, phasic units that were not sensitized by capsaicin and phasic-tonic units (that were not sensitized by capsaicin). These could also, in part, be distinguished by differences in their thresholds for activation, mean discharge frequency, latency to peak activation and peak-to-peak action potential amplitude. The majority of small-diameter myelinated sensory neurons projecting to the bone marrow expressed Piezo2. Our findings indicate that Aδ mechano-nociceptors are likely to play an important role in generating and maintaining pain in response to bony pathologies that involve a mechanical disturbance or increased intra-osseous pressure, and imply that Piezo2 signalling may be involved in mechano-transduction in these receptors.

The effect of heterotopic noxious conditioning stimulation on Aδ-, C- and Aβ-fibre brain responses in humans.

Human studies have shown that heterotopic nociceptive conditioning stimulation (HNCS) applied to a given body location reduces the percept and brain responses elicited by noxious test stimuli delivered at a remote body location. It remains unclear to what extent this effect of HNCS relies on the spinal-bulbar-spinal loop mediating the effect of diffuse noxious inhibitory controls (DNICs) described in animals, and/or on top-down cortical mechanisms modulating nociception. Importantly, some studies have examined the effects of HNCS on the brain responses to nociceptive input conveyed by Aδ-fibres. In contrast, no studies have explored the effects of HNCS on the responses to selective nociceptive C-fibre input and non-nociceptive Aβ-fibre input. In this study, we measured the intensity of perception and event-related potentials (ERPs) to stimuli activating Aδ-, C- and Aβ-fibres, before, during and after HNCS, obtained by immersing one foot in painful cold water. We observed that (i) the perceived intensity of nociceptive Aδ- and C-stimuli was reduced during HNCS, and (ii) the ERPs elicited by Aδ- and Aβ- and C-stimuli were also reduced during HNCS. Importantly, because Aβ-ERPs are related to primary afferents that ascend directly through the dorsal columns without being relayed at spinal level, the modulation of these responses may not be explained by an influence of descending projections modulating the transmission of nociceptive input at spinal level. Therefore, our results indicate that, in humans, HNCS should be used with caution as a direct measure of DNIC-related mechanisms.

Laser-evoked potentials mediated by mechano-insensitive nociceptors in human skin.

Laser-evoked potentials (LEP) were assessed after peripheral nerve block of the lateral femoral cutaneous nerve (LFCN) in healthy volunteers from partially anesthetized skin areas to differentially stimulate mechano-insensitive nociceptors. An ultrasound-guided nerve block of the LFCN was performed in 12 healthy male subjects with Ropivacain 1%. After 30min, the nerve block induced significantly larger anesthetic areas to mechanical stimuli than to electrical stimuli revealing an area of differential sensitivity. LEPs, reaction times and pain ratings were recorded in response to the laser stimuli of (1) completely anesthetic skin, (2) mechano-insensitive, but electrically excitable skin ('differential sensitivity'), (3) normal skin. LEP latencies in the area of differential sensitivity were increased compared to unaffected skin (228±8.5ms, vs. 181±3.6ms, p<0.01) and LEP amplitudes were reduced (14.8±1.2μV vs. 24.6±1.7μV, p<0.01). Correspondingly, psychophysically assessed response latencies in the differentially anesthetic skin were increased (649ms vs. 427ms, p<0.01) and pain ratings reduced (1.5/10 vs. 5/10 NRS, p<0.01). The increase in LEP latency suggests that mechano-insensitive heat-sensitive Aδ nociceptors (MIA, type II) have a slower conduction velocity or higher utilization time than mechano-sensitive type II Aδ nociceptors. Moreover, widely branched, slowly conducting and mechano-insensitive branches of Aδ nociceptors can explain our finding. LEPs in the differentially anesthetized skin provide specific information about a mechanically insensitive but heat-sensitive subpopulation of Aδ nociceptors. These findings support the concept thatA-fibre nociceptors exhibit a similar degree of modality specificity as C-fibre nociceptors.

Selective innervation of NK1 receptor–lacking lamina I spinoparabrachial neurons by presumed nonpeptidergic Aδ nociceptors in the rat

Fine myelinated (Aδ) nociceptors are responsible for fast, well-localised pain, but relatively little is known about their postsynaptic targets in the spinal cord, and therefore about their roles in the neuronal circuits that process nociceptive information. Here we show that transganglionically transported cholera toxin B subunit (CTb) labels a distinct set of afferents in lamina I that are likely to correspond to Aδ nociceptors, and that most of these lack neuropeptides. The vast majority of lamina I projection neurons can be retrogradely labelled from the lateral parabrachial area, and these can be divided into 2 major groups based on expression of the neurokinin 1 receptor (NK1r). We show that CTb-labelled afferents form contacts on 43% of the spinoparabrachial lamina I neurons that lack the NK1r, but on a significantly smaller proportion (26%) of those that express the receptor. We also confirm with electron microscopy that these contacts are associated with synapses. Among the spinoparabrachial neurons that received contacts from CTb-labelled axons, contact density was considerably higher on NK1r-lacking cells than on those with the NK1r. By comparing the density of CTb contacts with those from other types of glutamatergic bouton, we estimate that nonpeptidergic Aδ nociceptors may provide over half of the excitatory synapses on some NK1r-lacking spinoparabrachial cells. These results provide further evidence that synaptic inputs to dorsal horn projection neurons are organised in a specific way. Taken together with previous studies, they suggest that both NK1r+ and NK1r-lacking lamina I projection neurons are directly innervated by Aδ nociceptive afferents.

P416: Sensory-motor integration after selective stimulation of intraepidermal Aδ nociceptors: results from normal cases and patients with polyneuropathy

P416: Sensory-motor integration after selective stimulation of intraepidermal Aδ nociceptors: results from normal cases and patients with polyneuropathy

Evaluation of nociceptive Aδ- and C-fiber dysfunction with lidocaine using intraepidermal electrical stimulation

ObjectiveTo investigate whether intraepidermal electrical stimulation (IES) can evaluate nociceptive Aδ- and C-fiber dysfunctions of an experimental model of small-fiber neuropathy (SFN) with transdermal lidocaine. MethodsLidocaine tape or placebo was applied to the dorsum of the feet in 14 healthy subjects. Reaction time (RT), sensory threshold, and evoked potentials (EPs) were measured using IES before, and 30 and 60min after lidocaine/placebo application. ResultsAll subjects felt pricking sensations following Aδ-fiber stimulation, and light painful sensations such as pricking, tingling, or burning following C-fiber stimulation using IES. RT was divided bimodally between Aδ- and C-fiber stimulations. At 30min, lidocaine increased the sensory threshold and decreased the amplitude of EPs in both fiber stimulations. At 60min, lidocaine’s effects were greater for C fibers than for Aδ fibers. The sensory threshold and amplitude of EPs were unchanged among placebo sessions. ConclusionsIES demonstrated differential effects of transdermal lidocaine on nociceptive Aδ and C fibers, and elucidated the pathophysiology of the experimental model of SFN. SignificanceIES has advantages in terms of cost, convenience, and invasiveness. It may have potential for a clinical tool to elucidate the pathophysiology of patients with SFN, including the differences between Aδ and C fibers.