Mechanosensitive Receptive Fields Research Articles

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.

Scratching inhibits serotonin-evoked responses of rat dorsal horn neurons in a site- and state-dependent manner

Scratching inhibits pruritogen-evoked responses of neurons in the superficial dorsal horn, implicating a spinal site for scratch inhibition of itch. We investigated if scratching differentially affects neurons depending on whether they are activated by itchy vs. painful stimuli, and if the degree of inhibition depends on the relative location of scratching. We recorded from rat lumbar dorsal horn neurons responsive to intradermal (id) microinjection of serotonin (5-hydroxytryptamine, 5-HT). During the response to 5-HT, scratch stimuli (3mm, 300mN, 2Hz, 20s) were delivered at the injection site within the mechanosensitive receptive field (on-site), or 4–30mm away, outside of the receptive field (off-site). During off-site scratching, 5-HT-evoked firing was significantly attenuated followed by recovery. On-site scratching excited neurons, followed by a significant post-scratch decrease in 5-HT-evoked firing. Most neurons additionally responded to mustard oil (allyl isothiocyanate). Off-site scratching had no effect, while on-site scratching excited the neurons. These results indicate that scratching exerts a state-dependent inhibitory effect on responses of spinal neurons to pruritic but not algesic stimuli. Moreover, on-site scratching first excited neurons followed by inhibition, while off-site scratching immediately evoked the inhibition of pruritogen-evoked activity. This accounts for the suppression of itch by scratching at a distance from the site of the itchy stimulus.

Extracranial projections of meningeal afferents and their impact on meningeal nociception and headache

Headaches can be evoked by activation of meningeal nociceptors, but an involvement of pericranial tissues is debated. We aimed to examine a possible extracranial innervation by meningeal afferents in the rat. For in vivo neuronal tracing, dextran amines were applied to the periosteum underlying the temporal muscle. Labeling was observed 2days later in the parietal dura mater, trigeminal ganglion, and spinal trigeminal nucleus with confocal and electron microscopy. In the hemisected rat head, extracellular recordings were made from meningeal nerve fibers. Release of calcitonin gene-related peptide (CGRP) from the cranial dura mater during noxious stimulation of pericranial muscles was quantified. In vivo capsaicin was injected into the temporal muscle while meningeal blood flow was recorded. In the parietal dura mater, labeled C- and Aδ fibers ramified extensively, accompanied the middle meningeal artery, and passed through the spinosus nerve into the maxillary and mandibular, but not the ophthalmic division of the trigeminal ganglion. Some fibers could be traced into the ipsilateral spinal trigeminal nucleus. Electrophysiological recordings revealed afferent fibers with mechanosensitive receptive fields both in the dura mater and in the parietal periosteum. Noxious stimulation of the temporal muscle caused CGRP release from the dura mater and elevated meningeal blood flow. Collaterals of meningeal nerve fibers project through the skull, forming functional connections between extra- and intracranial tissues. This finding offers a new explanation of how noxious stimulation of pericranial tissues can directly influence meningeal nociception associated with headache generation and why manual therapies of pericranial muscles may be useful in headaches.

Skin incision-induced receptive field responses of mechanosensitive peripheral neurons are developmentally regulated in the rat

Maturation of the nervous system results in changes in both central and peripheral processing. To better understand responses to injury in the young, developmental differences in the acute response to incision were investigated in both tactile and nociceptive myelinated peripheral mechanosensitive afferent neurons in vivo. Neuronal intrasomal recordings were performed in juvenile and infant rats in 34 L5 dorsal root ganglia, and each neuron was phenotypically defined. Neurons had a mechanosensitive receptive field in the glabrous skin on the plantar surface of the hind paw, which was characterized at baseline and for up to 45 min after incision. Fundamental maturational differences in the effect of incision were clear: in high-threshold nociceptive mechanoreceptors, the mechanical threshold decreased immediately and the receptive field size increased rapidly in juvenile rats but not in infant rats. Additionally, a divergence in changes in the instantaneous response frequency of tactile afferents occurred between the two ages. These differences may help explain maturational differences in responses to peripheral injury and suggest that differences in central nervous system responses may be partially mitigated by spatially confined and frequency-dependent differences resulting from tactile and nociceptive mechanosensitive input.

S1799 Specialized Spinal Afferent Neurons to Detect Mucosal Shear in Guinea-Pig Colon

Background and aims. Extrinsic sensory neurons to the gut mediate both sensation and gastrointestinal reflexes important for gut function in health and disease. Different classes of afferents which convey various types of information about the gut wall and contents can be reliably identified by characterizing the morphology of their physiologically-identified peripheral axons. “Muscular mucosal” afferents have been identified on the basis of their responses to both colorectal distension and to light mucosal stroking. Methods. Extracellular recording from colonic nerves in isolated preparations of guinea pig distal colon were combined with biotinamide filling In Vitro. Results. When all layers of the colon were present in the preparation, a class of afferents could be strongly activated by light von Frey hairs (0.1 10 mN, maximum instantaneous firing: 83+/-18 Hz, n=6). The same units could also be activated by circumferential distension with constant loads (10 100mN) evoking firing of 52+/-30 Hz (n=10). Both types of activity were preserved in preparations where the muscularis externa had been removed prior to recording, suggesting that transduction sites were located in the submucosa or mucosa. Surprisingly, afferents of this class also fired in concert with spontaneous contractions of preparations, measured under isotonic conditions. The action potentials of these units were significantly larger and of shorter duration than action potentials from mechanonociceptors axons recorded from the same nerve trunks (341+/-146 vs 139+/-34μV P<0.01, 0.4+/-0.1 vs 1.1+/-0.8 ms, P<0.05, n=9). None of 9 “muscular mucosal” axons recorded responded to focal application of capsaicin (0.3 μM) and their mechanosensitivity persisted in calcium-free solution (with raised [Mg++]). This suggests that they are intrinsically mechanosensitive rather than relying on release of mediators from other cells. Biotinamide dye filling revealed fine branching axons running in the subepithelial plexus in regions corresponding closely to mechanosensitive receptive fields of these units (n=4). Conclusions. We conclude that “muscular mucosal” afferents comprise a discrete and distinctive class of spinal afferent innervation, well suited to detect physiological levels of mucosal shear in the distal colon of the guinea-pig.

S1800 Characterization of a Reproducible Gastric Pain Model Using Oral Capsaicin Titration: A Placebo-Controlled, Double-Blind Study

Background and aims. Extrinsic sensory neurons to the gut mediate both sensation and gastrointestinal reflexes important for gut function in health and disease. Different classes of afferents which convey various types of information about the gut wall and contents can be reliably identified by characterizing the morphology of their physiologically-identified peripheral axons. “Muscular mucosal” afferents have been identified on the basis of their responses to both colorectal distension and to light mucosal stroking. Methods. Extracellular recording from colonic nerves in isolated preparations of guinea pig distal colon were combined with biotinamide filling In Vitro. Results. When all layers of the colon were present in the preparation, a class of afferents could be strongly activated by light von Frey hairs (0.1 10 mN, maximum instantaneous firing: 83+/-18 Hz, n=6). The same units could also be activated by circumferential distension with constant loads (10 100mN) evoking firing of 52+/-30 Hz (n=10). Both types of activity were preserved in preparations where the muscularis externa had been removed prior to recording, suggesting that transduction sites were located in the submucosa or mucosa. Surprisingly, afferents of this class also fired in concert with spontaneous contractions of preparations, measured under isotonic conditions. The action potentials of these units were significantly larger and of shorter duration than action potentials from mechanonociceptors axons recorded from the same nerve trunks (341+/-146 vs 139+/-34μV P<0.01, 0.4+/-0.1 vs 1.1+/-0.8 ms, P<0.05, n=9). None of 9 “muscular mucosal” axons recorded responded to focal application of capsaicin (0.3 μM) and their mechanosensitivity persisted in calcium-free solution (with raised [Mg++]). This suggests that they are intrinsically mechanosensitive rather than relying on release of mediators from other cells. Biotinamide dye filling revealed fine branching axons running in the subepithelial plexus in regions corresponding closely to mechanosensitive receptive fields of these units (n=4). Conclusions. We conclude that “muscular mucosal” afferents comprise a discrete and distinctive class of spinal afferent innervation, well suited to detect physiological levels of mucosal shear in the distal colon of the guinea-pig.

S1803 Early Life Esophageal Acid Exposure Accentuates the Expression of Nr1 and PSD-95 in ACC Neurons in Response to Acid Rechallenge in Adulthood

Background and aims. Extrinsic sensory neurons to the gut mediate both sensation and gastrointestinal reflexes important for gut function in health and disease. Different classes of afferents which convey various types of information about the gut wall and contents can be reliably identified by characterizing the morphology of their physiologically-identified peripheral axons. “Muscular mucosal” afferents have been identified on the basis of their responses to both colorectal distension and to light mucosal stroking. Methods. Extracellular recording from colonic nerves in isolated preparations of guinea pig distal colon were combined with biotinamide filling In Vitro. Results. When all layers of the colon were present in the preparation, a class of afferents could be strongly activated by light von Frey hairs (0.1 10 mN, maximum instantaneous firing: 83+/-18 Hz, n=6). The same units could also be activated by circumferential distension with constant loads (10 100mN) evoking firing of 52+/-30 Hz (n=10). Both types of activity were preserved in preparations where the muscularis externa had been removed prior to recording, suggesting that transduction sites were located in the submucosa or mucosa. Surprisingly, afferents of this class also fired in concert with spontaneous contractions of preparations, measured under isotonic conditions. The action potentials of these units were significantly larger and of shorter duration than action potentials from mechanonociceptors axons recorded from the same nerve trunks (341+/-146 vs 139+/-34μV P<0.01, 0.4+/-0.1 vs 1.1+/-0.8 ms, P<0.05, n=9). None of 9 “muscular mucosal” axons recorded responded to focal application of capsaicin (0.3 μM) and their mechanosensitivity persisted in calcium-free solution (with raised [Mg++]). This suggests that they are intrinsically mechanosensitive rather than relying on release of mediators from other cells. Biotinamide dye filling revealed fine branching axons running in the subepithelial plexus in regions corresponding closely to mechanosensitive receptive fields of these units (n=4). Conclusions. We conclude that “muscular mucosal” afferents comprise a discrete and distinctive class of spinal afferent innervation, well suited to detect physiological levels of mucosal shear in the distal colon of the guinea-pig.

A novel preparation to study rat pancreatic spinal and vagal mechanosensitive afferents in vitro

The management of pancreatic pain is a significant clinical problem so understanding of how sensory signals are generated in pancreatic tissue is fundamental. We aimed to characterize mechanosensitive and chemosensitive properties of pancreatic spinal and vagal afferents in vitro. Spinal and vagal afferent preparations from Sprague-Dawley rats were established incorporating the left splanchnic nerve or vagus nerves respectively. The common bile duct was cannulated for distension of the pancreatic duct with fluid. Nerve discharge evoked by blunt probing, duct distension or electrical stimulation was obtained from teased nerve bundles using standard extra-cellular recording. Discharge from 197 spinal afferent bundles was recorded, of which 57% displayed spontaneous activity. Blunt probing revealed 61 mechanosensitive receptive fields which were associated primarily with arteries/blood vessels (33/61) and the parenchyma (22/61). All mechanosensitive responses were slowly adapting, with 33% continuing to discharge after termination of the stimulus and 60% displaying a response threshold <10 g. Application of chemical mediators (bradykinin, histamine, 5-hydroxytryptamine, cholecystokinin octapeptide) evoked a response from 31/57 units, with 33% excitatory and 23% inhibitory. Spontaneous discharge was recorded from 72% of 135 vagal bundles. Mechanosensitive receptive fields were not identified in the pancreas but were evident in adjacent organs. No spinal or vagal afferent response to duct distension was obtained. In conclusion, pancreatic mechanosensitive spinal afferents are common, in contrast to pancreatic mechanosensitive vagal afferents indicating that pancreatic sensory innervation is predominantly spinal. Chemosensitive spinal afferent nerve endings are present in the pancreas and respond to a variety of inflammatory and physiological mediators.

ACTIVATION OF RAT PANCREAS MECHANOSENSITIVE SPINAL AFFERENTS IN A NOVEL IN VITRO PREPARATION

Few studies have recorded afferent nerve discharge from the pancreas and the role of afferent nerves in pancreatic physiology and pathophysiology is very poorly understood. Aims: To map and characterise mechanosensitive afferents in the normal rat pancreas. Methods: A preparation consisting of the pancreas, attached spleen and duodenum, and including the pancreatic duct (PD), common bile duct (CBD) and the sympathetic chain was obtained from anaesthetised Sprague Dawley rats (n = 9). The CBD was cannulated. The preparation was placed in a 2 chamber organ bath. Extracellular nerve recordings were obtained from fibers teased from the sympathetic chain adjacent to the coeliac ganglia. Nerve discharge evoked by blunt probing, CBD/PD distension (44 mmHg), or electrical field stimulation (EFS; n = 3; 20 V, 0.5 ms duration) of pancreas was recorded. Results: Discharge from 137 fibers was recorded, of which 50% displayed spontaneous activity. Blunt probing evoked a discharge in 20% of fibers whereas EFS evoked discharge in 11/42 fibers, of which 3 receptive fields responded to blunt probing. Based on conduction velocity measurements (0.36 ± 0.06 m/s; n = 16 fibers), recordings were from C fibers. Mechanosensitive receptive fields were associated with arteries/blood vessels in the pancreas (16/32), pancreas near lymph nodes (2/32), pancreatic tissue (12/32), CBD (1/32) and duodenal mesentery (1/32). Non-mechanosensitive receptive fields were located in pancreatic tissue (5/11), arteries/blood vessels (5/11) or CBD/PD (1/11). CBD/PD distension did not evoke any discharge. Conclusion: Mechanosensitive and non-mechanosensitive afferent nerves are associated with several structures in the pancreas. Their role in physiological and pathophysiological signaling remains to be established. Supported by the FMC Foundation.

The sensory mechanotransduction ion channel ASIC2 (acid sensitive ion channel 2) is regulated by neurotrophin availability

Almost all sensory neurons of the dorsal root ganglia have a mechanosensitive receptive field in the periphery. We have shown that the sensitivity to mechanical stimuli of a subset of sensory neurons that are slowly adapting mechanoreceptors (SAM) is strongly dependent on the availability of brain-derived neurotrophic factor (BDNF). Here we have investigated whether the ASIC2 sodium channel, recently shown by us to be necessary for normal SAM sensitivity, might be regulated by BDNF and thus partially account for the down-regulation of SAM sensitivity seen in BDNF deficient mice. We show that the mRNA for ASIC2 channels is reduced in the DRG of BDNF deficient mice indicating that BDNF might maintain its expression in vivo. We also made short-term cultures of sensory neurons from adult BDNF deficient mice and used a specific antibody to detect the presence of ASIC2 channels in different classes of sensory neurons. We observed that the channel protein was dramatically down-regulated selectively in medium and large diameter neurons and this expression could be rescued in a dose and time dependent manner by addition of BDNF to the culture (10–100 ng/ml). Drugs that block new transcription or protein synthesis also prevented the rescue effects of BDNF. We observed that ASIC2 channels were down-regulated in sensory neurons taken from neurotrophin-4 and neurotrophin-3 deficient mice; these effects might be due to a selective loss of neurons that normally express large amounts of ASIC2 channels. In summary, our data identify the ASIC2 channel as a target of BDNF signaling in vivo and suggest that the functional down-regulation of sensory mechanotransduction in BDNF deficient mice is in part due to loss of ASIC2 expression.

An electrophysiologic study of mechanoreceptors in the sacroiliac joint and adjacent tissues.

The somatosensory afferent units in the sacroiliac joint of an animal model were investigated using an electrophysiologic technique. To identify the mechanosensitive receptive fields in the sacroiliac joint, and to determine their distribution and characteristics. The sacroiliac joint is considered to be a source of lower back pain. Although there have been clinical studies on the diagnosis of sacroiliac joint pain, no satisfactory diagnostic method other than joint blocks has been reported. It still is not clear whether the sacroiliac joints actually transmit pain to the central nervous system. The sensory innervation of the sacroiliac joint has not been fully characterized neurophysiologically. Experiments were performed on 10 adult cats weighing 2.6 to 4 kg. The animals were anesthetized with intravenous sodium pentobarbital. An L4-L7 laminectomy was performed. The L4-L6 dorsal roots were cut at their proximal ends, split, and draped over a bipolar recording electrode. Glass probes were used to search the sacroiliac joint and adjacent tissues for mechanosensitive units. When units were identified, they were stimulated electrically to obtain conduction velocities and by Semmes-Weinstein monofilaments to determine mechanical thresholds. In the sacroiliac joint and adjacent muscles, 29 discrete mechanosensitive units were identified. Of these 29 units, 26 were found in the posterior sacroiliac ligament and the remaining 3 in the adjacent muscles. Also, 16 units (55%) were identified in the proximal third of the sacroiliac joint. Conduction velocities of the units ranged from 3.1 to 22 m/second (average, 9.2 m/second), and 26 units were group III. Mechanical thresholds of the units ranged from 4.6 to 164.3 g (average, 69.7 g). Whereas 28 units (96.6%) had thresholds higher than 7 g, one unit (3.4%) had a threshold lower than 7 g. Group III units with mechanical thresholds higher than 7 g may serve as nociceptors, and units with thresholds of lower than 7 g may serve as proprioceptors. The current study showed that most of the units in the sacroiliac joint were high-threshold group III units that perhaps had a nociceptive function. This result suggests that the sacroiliac joint may be a source of lower back pain in humans. This study also showed that the sacroiliac joint has little proprioceptive function.

Mechanosensitive afferent units in the lumbar posterior longitudinal ligament.

The mechanosensitive afferent units in the lumbar posterior longitudinal ligament were investigated in an animal model using an electrophysiologic technique. The objectives of this study were to identify the mechanosensitive receptive fields in the lumbar posterior longitudinal ligament and to investigate their distribution and characteristics. The lumbar posterior longitudinal ligament has a nerve network originating from the sinuvertebral nerve. These fibers are thin, and most of their terminals are free nerve endings. Some immunohistochemical studies have indicated that they are immunoreactive to calcitonin gene-related peptide and/or substance P, suggesting a nociceptive function. Most of these studies investigated morphologic aspects, and there have been few studies employing electrophysiologic techniques to examine mechanosensitive units. We used 13 adult cats. They were anesthetized and then laminectomy was performed. The L5 and L6 dorsal rootlets were draped over a recording electrode. To investigate the receptive fields in the posterior longitudinal ligament, afferent impulses were evoked by mechanical stimulation with a glass probe. When the receptive fields were located, they were stimulated electrically to obtain conduction velocity and were stimulated with a set of 17 nylon filaments to determine their mechanical thresholds. Thirteen units were identified in the lumbar posterior longitudinal ligament. The majority of the units were located around the intervertebral disc level of the posterior longitudinal ligament. The mean mechanical threshold was 47.04 +/- 15.25 g. According to the conduction velocities of the units, 12 units were classified into Group III (0.5-2.5 m/sec) and one unit into Group IV (2.5-20 m/sec). Mechanosensitive units classified into Group III or Group IV and with a high mechanical threshold (>7.0 g) were thought to act as nociceptive units. All units identified in this study satisfied these criteria. Our result suggests that afferent fibers from the lumbar posterior longitudinal ligament have a principally nociceptive function.

Superficial Dorsal Horn Neurons Identified by Intracutaneous Histamine: Chemonociceptive Responses and Modulation by Morphine

We have investigated whether neurons in superficial laminae of the spinal dorsal horn respond to intracutaneous (ic) delivery of histamine and other irritant chemicals, and thus might be involved in signaling sensations of itch or chemogenic pain. Single-unit recordings were made from superficial lumbar dorsal horn neurons in pentobarbital sodium-anesthetized rats. Chemoresponsive units were identified using ic microinjection of histamine (3%, 1 microl) into the hindpaw as a search stimulus. All superficial units so identified [9 nociceptive-specific (NS), 26 wide-dynamic-range (WDR)] responded to subsequent ic histamine. A comparison group of histamine-responsive deep dorsal horn neurons (n = 16) was similarly identified. The mean histamine-evoked discharge decayed to 50% of the maximal rate significantly more slowly for the superficial (92.2 s +/- 65.5, mean +/- SD) compared with deep dorsal horn neurons (28. 2 s +/- 11.6). In addition to responding to histamine, most superficial dorsal horn neurons were also excited by ic nicotine (22/25 units), capsaicin (21/22), topical mustard oil (5/6), noxious heat (26/30), and noxious and/or innocuous mechanical stimuli (except for 1 unit that did not have a mechanosensitive receptive field). Application of a brief noxious heat stimulus during the response to ic histamine evoked an additive response in all but two cases, followed by transient depression of firing in 11/20 units. Intrathecal (IT) administration of morphine had mixed effects on superficial dorsal horn neuronal responses to ic histamine and noxious heat. Low morphine concentrations (100 nM to 1 microM) facilitated histamine-evoked responses (to >130% of control) in 9/24 units, depressed the responses (by >70%) in 11/24, and had no effect in 4. Naloxone reversed morphine-induced effects in some but not all cases. A higher morphine concentration (10 microM) had a largely depressant, naloxone-reversible effect on histamine responses. Responses of the same superficial neurons to noxious heat were facilitated (15/25), reduced (8/25), or unaffected (2/25) by low morphine concentrations and were depressed by the higher morphine concentration. In contrast, deep dorsal horn neuronal responses to both histamine and noxious heat were primarily depressed by low concentrations of morphine in a naloxone-reversible manner. These results indicate that superficial dorsal horn neurons respond to both pruritic and algesic chemical stimuli and thus might participate in transmitting sensations of itch and/or chemogenic pain. The facilitation of superficial neuronal responses to histamine by low concentrations of morphine, coupled with inhibition of deep dorsal horn neurons, might underlie the development of pruritus that is often observed after epidural morphine.

Activation of spinal wide dynamic range neurons by intracutaneous microinjection of nicotine.

Nicotine evokes pain in the skin and oral mucosa and excites a subpopulation of cutaneous nociceptors, but little is known about the central transmission of chemogenic pain. We have investigated the responses of lumbar spinal wide dynamic range (WDR)-type dorsal horn neurons to intracutaneous (ic) microinjection of nicotine in pentobarbital-anesthetized rats. Nearly all (97%) units responded to nicotine microinjected ic (1 microl) into the low-threshold region of the hind-paw mechanosensitive receptive field in a concentration-related manner (0.01-10%). Responses to repeated injections of 10% nicotine exhibited tachyphylaxis at 5-, 10-, and 15-min interstimulus intervals. Significant tachyphylaxis was not seen with 1% nicotine. All nicotine-responsive units tested (n = 30) also responded to ic histamine (1 microl, 3%) and did not exhibit tachyphylaxis to repeated histamine. However, there was significant cross-tachyphylaxis of nicotine to histamine. Thus 5 min after ic nicotine, histamine-evoked responses were attenuated significantly compared with the initial histamine-evoked response prior to nicotine, with partial recovery over the ensuing 15 min. Neuronal excitation by ic nicotine was not mediated by histamine H1 receptors because ic injection of the H1 receptor antagonist, cetirizine, had no effect on ic nicotine-evoked responses, whereas it significantly attenuated ic histamine-evoked responses in the same neurons. The lowest-threshold portion of cutaneous receptive fields showed a significant expansion in area at 20 min after ic nicotine 10%, indicative of sensitization. Responses to 1% nicotine were significantly reduced after ic injection of the nicotinic antagonist, mecamylamine (0.1% ic), with no recovery over the ensuing 40-60 min. These data indicate that nicotine ic excites spinal WDR neurons, partly via neuronal nicotinic acetylcholine receptors that are presumably expressed in cutaneous nociceptor terminals. Repeated injections of high concentrations of nicotine led to tachyphylaxis and cross-tachyphylaxis with histamine, possibly relevant to peripheral analgesic effects of nicotine.

Behavioral manifestations of neuropathic pain and mechanical allodynia, and changes in spinal dorsal horn neurons, following L4–L6 dorsal root constriction in rats

We investigated behavioral symptoms of neuropathic pain, and associated changes in dorsal horn neurons, in a rat model involving loose ligation of lumbar dorsal roots. The L4–L6 dorsal roots were exposed unilaterally and loosely constricted central to the respective ganglia with one (1-ligation) or two (2-ligation) silk 7-O ligatures. In control groups the dorsal roots were exposed but not ligated (sham-operated), or sutures were placed lengthwise between the dorsal roots (suture control). There was a significant reduction in mechanical withdrawal threshold on the operated side in both 1- and 2-ligation groups which began at 3 days, peaked at 2–5 week, and gradually recovered. A delayed threshold reduction was also seen on the non-operated side. Immediately post-surgery there was a significant increase (hypoalgesia) in thermal paw withdrawal latency (Hargreaves test) in 1- and 2-ligation groups on the operated (but not non-operated) side that recovered after 1 week. Significantly less weight was borne by the operated limb 1–5 weeks post-operatively in 1- and 2-ligation groups. The force of hind limb withdrawals elicited by graded noxious heat pulses (38–52°C) was significantly lower 1 week post-surgery on the operated side (1-ligation group) followed by recovery. Withdrawal forces were higher 5–9 week post-surgery on the non-operated side in 1- and 2-ligation groups. We found no evidence of cold allodynia. Neither sham-operated nor suture controls showed any signs of allodynia or hyperalgesia. Following behavioral testing, rats were anesthetized with halothane for single-unit recordings from lumbar wide dynamic range-type (WDR) neurons. At 22 week post-surgery, the mean area of mechanosensitive receptive fields was significantly larger for units on the operated side in 1- and 2-ligation groups compared with those on the non-operated side or with those from sham-operated rats. Mean stimulus-response functions to graded noxious heat pulses (38–52°C, 5 s) were not significantly different between operated and non-operated sides for 1- or 2-ligation groups, or compared with the 22-week sham-operated group. At 5 week post-surgery, the mean area of cutaneous receptive fields, and stimulus-response functions to graded noxious heat, were not significantly different between units recorded on operated versus non-operated sides, or compared with units from 5-week sham-operated rats. Spontaneous unit activity was significantly higher on the operated versus non-operated side in the 2-ligation (22-week) and sham (5-week) groups. Enlarged cutaneous receptive fields of dorsal horn neurons may contribute to mechanical allodynia associated with dorsal root constriction. However, the slow (>5 week) development of receptive field enlargement does not match the rapid development of allodynia. The lack of effect of dorsal root constriction on thermal sensitivity of dorsal horn units ipsilaterally corresponds to the lack of marked thermal hyperalgesia observed behaviorally.

Spinal NMDA receptor involvement in expansion of dorsal horn neuronal receptive field area produced by intracutaneous histamine.

Histamine elicits the sensation of itch at the site of skin application as well as alloknesis (itch elicited by innocuous mechanical stimuli) in a surrounding area in humans and expansion of the low-threshold mechanosensitive receptive field area of spinal wide dynamic range (WDR)-type dorsal horn neurons in rats. We presently tested if the histamine-evoked expansion of neuronal receptive field area depends on a spinal N-methyl-D-aspartate (NMDA) receptor-mediated process. In pentobarbital sodium-anesthetized rats, mechanical receptive field areas of single WDR-type dorsal horn neurons were mapped with graded von Frey filaments before and 10 min after intracutaneous (ic) microinjection of histamine (1 microl; 1, 3, or 10%) at a low-threshold site within the receptive field. Intracutaneous microinjection of histamine evoked dose-related increases in firing rate, as well as a dose-dependent expansion in mean receptive field area 10 min after 3 and 10%, but not 1%, histamine doses. When a noncompetitive or competitive NMDA receptor antagonist dizocilpine [MK-801; D(-)-2-amino-5-phosphonovalerate (APV), respectively; 1 microM] was first applied topically to the surface of the spinal cord, there was no significant change in mean receptive field area after ic microinjection of 10% histamine. The mean neuronal response to histamine in the presence of spinal MK-801 or APV was not significantly different from the mean response to histamine in the absence of these drugs. These results suggest that spinal NMDA receptors are involved in histamine-induced expansion of mechanical receptive field area, a neural event possibly involved in the development of alloknesis.

Effects of chemical stimulation of masseter muscle nociceptors on trigeminal motoneuron and interneuron activities during fictive mastication in the rabbit

An electrophysiological study was carried out in sixteen decerebrate and paralyzed New Zealand rabbits to determine how a bolus injection of a nociceptor stimulant (hypertonic saline, 5%) into the masseter muscle influences the activity of the trigeminal motor circuitry during fictive jaw movements. Hypodermic needles connected to a syringe held in a computer- controlled infusion pump were inserted into the anterior deep layer of either the right or the left masseter. Twenty-three infusions of 50, 70 or 80 μl saline were made in fourteen animals at constant rates over 1 min. Eight control infusions of normal saline (0.9%) were made in a subpopulation of five animals in an identical manner. Fictive jaw movements were evoked before and after the infusions by repetitive electrical stimulation of the cortico-bulbar tract. Effects were assessed by extracellular microelectrode recordings made from the digastric motoneuron pool and from putative last-order interneurons in the oral subnucleus of the spinal trigeminal tract and adjacent structures. In comparison with pre-infusion control cycles, nociceptor stimulation caused significant slowing of the rhythm and a reduction of the area of the digastric motoneuron bursts in the majority of the animals (12/14). The decrease in cycle frequency was due almost entirely to a lengthening of the time between the digastric bursts. Changes usually began 1–2 min after the infusion and returned to pre-infusion values within 10–15 min. No significant effects were seen when isotonic saline was applied. Recordings were obtained from nine interneurons, eight of which had low threshold mechanosensitive receptive fields. One neuron was, in addition, excited by pinch. Eight were not active in the absence of motor activity and this did not change when hypertonic saline was applied. However, once fictive movements began, all started to fire rhythmic bursts of spikes. In five cases, there was a significant post-infusion increase in spike frequency, and three showed decreases. Seven showed significant post-infusion changes in mean phase and/or concentration of their firing within the movement cycle. Changes in the preferred phase of interneuronal firing were significantly correlated to changes in the phase of offset of the digastric burst. The present results provide evidence that the stimulation of nociceptors in a muscle slows the frequency of rhythmical movements in the absence of sensory feedback. They confirm that infusions into one muscle affect the output of its antagonist. The results also suggest that neurons in the oral subnucleus of the spinal trigeminal tract and adjacent reticular formation appear to participate in programming these changes in motor output.

Responses of rat sacral spinal neurons to mechanical and noxious thermal stimulation of the tail.

In this study we investigated the receptive field properties, responses to mechanical and thermal stimuli, and sensitivity to systemic administration of pentobarbital sodium and morphine of single neurons recorded in the sacral spinal cords of pentobarbital-anesthetized rats. Fifty-three neurons responded to innocuous mechanical stimulation of the tail. Of 45 neurons that were additionally tested with noxious thermal stimulation, 62% responded and were classified as wide-dynamic-range or multireceptive neurons. Recording sites were located mainly in the middle layers of the S2-S4 dorsal horn. Mechanosensitive receptive fields on the tail varied widely in size (range 0.14-35 cm2, mean 10.33 cm2) and form, and were in nearly all cases bilateral. Most neurons responded with a high-frequency discharge followed by a more slowly adapting response to pressure stimuli delivered with von Frey hairs. Responses (maximal frequency and total number of impulses) increased in a graded manner to pressure stimuli ranging from 1.2 to 447 g. For neurons responsive to noxious heating of the tail, responses increased in a linear manner over the range of 38-54 degrees C and often leveled off at higher temperatures. Of nine neurons tested with both graded von Frey and noxious heat stimuli, mean responses (maximal frequency and total number of impulses) evoked by the strongest pressure stimuli were larger than those evoked by the most intense heat stimuli, but the difference was not statistically significant. Responses to repeated 48 degrees C stimuli were significantly attenuated within 8 min after systemic administration of morphine (1 or 2 mg/kg ip), reaching maximal suppression (to 37.3%; N = 13) after 18 min, with recovery following systemic naloxone. After morphine (1 and 2 mg/kg ip), the slope of the population stimulus-response function for noxious heat was reduced (51.8%), and the threshold was increased (by 4 degrees C). Responses to noxious heat were significantly depressed (to a mean of 54%; N = 10) by supplemental administration of pentobarbital (mean 17 mg/kg over 5 min). On the basis of similarities between the present data and previous behavioral measures of tail flick stimulus-response functions and their modulation, it is suggested that some of the present neurons might function as interneurons in the tail flick reflex arc.

H2O2 sensitivity of afferent splanchnic C fiber units in vitro.

1. Single-unit impulse activity evoked by transient, focal application of hydrogen peroxide (H2O2) to identified visceral receptive fields has been characterized in an in vitro rat splanchnic nerve-mesentery preparation. In addition to H2O2 responsiveness, units were characterized in terms of sensitivity to mechanical stimuli, warming, and bradykinin. 2. Mesenteric receptive fields of single splanchnic afferent C fibers in vitro were located with the use of warm (approximately 45 degrees C saline) or mechanical search stimuli. After delimitation of the warm-sensitive and/or mechanosensitive receptive field, units were tested for responsiveness to transient, focal application of H2O2. Microliter volumes (usually 1 microliter) of H2O2 (88-880 mM) evoked responses in 25 of 42 (60%) units with identified warm-sensitive and/or mechanosensitive receptive fields, and in an additional 10 units for which H2O2 was the only effective stimulus. 3. Tachyphylaxis to repeated H2O2 stimulation was observed with interstimulus intervals <30 min, but did not indicate irreversible inactivation of the terminal, because 1) during this period warm and mechanical stimuli elicited responses equal to or greater than those before H2O2 treatment, and 2) H2O2 sensitivity was restored after units were allowed to recover. 4. Eight units unresponsive to an initial dose of H2O2 responded vigorously to a repeated application at the same site, suggesting a potentiating effect of prior H2O2 exposure. 5. Sixty-two percent (8 of 13) of H2O2-responsive units, but no (0 of 6) H2O2-unresponsive units responded to transient, focal bradykinin (9-90 nM) application. 6. An indirect mode of H2O2-evoked afferent excitation in some units was suggested by several observations, including the prolonged (up to 8 min) duration of the response of some units to transient H2O2 application, and the occasionally long (>2 min) response latencies to focal application of H2O2 to defined receptive fields. 7. Excitation of splanchnic neurons by H2O2 may be relevant to the modulation of reactive oxygen species production by immunocompetent cells, because sensory neuropeptides contained in these afferent fibers are known to influence the respiratory burst of macrophages and neutrophils.

Chapter 10 The possible role of substance P in eliciting and modulating deep somatic pain

This chapter focuses on the results and discussions concerning the involvement of substance P (SP) in the mediation of deep somatic pain (from muscle, fascia, tendon, and joint). SP-related events in skeletal muscle and spinal cord are addressed. The physiological function of the SP- and calcitonin gene-related peptide (CGRP)-ir fibres that exhibited an increase in numerical density is unknown. If the effect is present also in nociceptors, pain sensations from an inflamed muscle could be due not only to an increased discharge activity in these endings but also to a recruitment of active nociceptive endings. This latter effect could lead to an enhancement of pain because of an increased spatial summation at the first synapses in the spinal cord. The chapter presents only the neurones that had no mechanosensitive receptive fields (RFs) in the skin. No information is available concerning a possible relationship between functional properties and type of SP effect. It is likely, however, that such a relationship exists, because in nociceptive neurones the only SP effect observed is an increase in RF size or number of RFs. Whether all nociceptive neurones react to SP in this way is unknown.