Proceedings of Neuropeptides 2004, the XIV European Neuropeptides Club meeting.
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Animals↗
Biomedical subjects
R F Schmidt
Publications and source records attributed to R F Schmidt.
Elastoviscous substances with analgesic effects on joint pain reduce stretch-activated ion channel activity in vitro.
Activation by noxious mechanical stimuli of sensory nerve fibres that signal joint pain takes place through stretch-activated ion channels, which open in response to increased membrane tension. It has been suggested that the analgesic effect of hyaluronan solutions used for intra-articular treatment of joint pain in humans are mediated by a reduction of the sensitivity of mechanosensory ion channels of nociceptive nerve terminals. We have investigated whether cross-linked hyaluronan solutions (hylans) of different elastoviscosities modify the response characteristics of stretch-activated ion channels of Xenopus laevis oocytes. Patch-clamp recordings on intact oocytes and in excised membrane patches (outside-out and inside-out configurations) were performed in Barth's solution (control condition) and after exposure to hylans of different elastoviscosities. For mechanical stimulation, monitored suction was applied through the microelectrode and the activity of stretch-activated channels was recorded. The activity of stretch-activated channels was significantly reduced in the presence of high elastoviscous hylan A (0.8% polymer content, molecular weight 6M) and of a mixture of hylan A (90% by weight) and hylan B (10% by weight), 0.9% total polymer content, a clinically used hylan product. In contrast, solutions of hylan A with the same chemical composition but reduced elastoviscosity (0.8% polymer content, molecular weight 96000) were found ineffective. It is concluded that stretch-activated channels have a decreased mechanical sensitivity in the presence of elastoviscous solutions of hylan, but not in the presence of non-elastoviscous solutions of hylan of the same concentration. These data suggest that the analgesic effects of intra-articular injections of elastoviscous solutions of hylans are due to a reduction of the sensitivity to mechanical forces of stretch-activated channels present in the membrane of joint mechanonociceptors.
Analgesics↗
Three-dimensional reconstruction of scleral cold thermoreceptors of the cat eye.
Sensory endings that respond to local cooling were identified electrophysiologically in the cat's sclera. Functionally identified scleral thermal fibers were then used to analyze the structural characteristics of cold receptor endings. Four Adelta units sensitive to controlled cooling of their scleral receptive fields were recorded. The receptive areas were mapped, demarcated with pins and examined electron microscopically using extensive three-dimensional reconstructions. The supporting tissue within the receptive areas of cold units consisted of dense collageneous tissue with a small number of blood vessels that were either veins or capillaries. Adelta nerve fibers were found within these tissue blocks presumably corresponding with cold sensitive fibers. Small nerves and single nerve fibers devoid of a perineurium were found in all parts of the tissue, only occasionally passing a blood vessel. The terminal portions showed axonal swellings all along the unmyelinated segment filled with mitochondria, glycogen particles, and some vesicles. About 30% of the terminal axonal membrane is not covered by Schwann cells. In the unmyelinated distal portion, the mitochondrial content ranged from 0.012 to 0.038 microm(3) mitochondrial volume per microm(2) nerve fiber membrane. In comparison with sensory endings in the cat's knee joint, cold receptors in the cat sclera showed many similarities in their three-dimensional structure with polymodal nociceptor endings of the knee joint but contain less mitochondria. This suggests that cold sensory endings do not require specialized cellular processes for the transduction of cold stimuli, as is the case for multimodal transduction and sensitization in the terminal portion of polymodal nociceptors.
Animals↗
No evidence for bradykinin B1 receptors in rat dorsal root ganglion neurons.
Bradykinin receptors are believed to contribute to hyperalgesia under conditions of neuropathic pain. Using calcium imaging we investigated responses to B1 and B2 agonists on isolated rat dorsal root ganglion neurons. No response to the B1 agonist was detected, whereas 12% of neurons responded to the B2 agonist. Northern blot analysis confirmed the lack of B1 receptor expression in dorsal root ganglia, as B1 mRNA was neither detected under normal conditions nor after nerve injury. In the calcium imaging experiments, agonists were applied with an elevated superfusion flow rate to avoid tachyphylaxis to the drug. Normal external solution applied at this flow rate constituted a mechanical stimulus causing a response in some neurons. Thus, in comparable set-ups mechanosensitivity has first to be tested to avoid masking effects.
Animals↗
Cortical projection of the rat knee joint innervation and its processing in the somatosensory areas SI and SII.
In recent years the rat knee joint has become an important model for the study of nociception of deep tissues. In contrast to the cortical processing of superficial pain, the knowledge about the processing of deep pain evoked by noxious stimuli in tissues such as tendons, bone, and joint is sparse. To obtain a basis for further functional studies, the projections of the knee joint in the cerebral cortex were determined. Cortical surface potentials evoked by electrical stimulation of the posterior articular nerve were recorded by a platinum ball electrode. Evoked activity was found in the primary somatosensory area SI in an area of about 3 x 3 mm on the contralateral side. Its center was located about 3 mm caudal to the bregma and about 3 mm lateral to the superior sagittal sinus. A small projection in SII was found on the lateral side of the cortex about 6 mm lateral from SI. This area had a size of about 1 x 1 mm, and the amplitudes of the potentials were smaller but had similar latencies to those in SI. An additional projection with small potentials and longer latencies was observed in SI on the ipsilateral side. Cooling of the contralateral SI revealed deprivation of the ipsilateral evoked potentials in SI whereas the potentials in SII remained unchanged. These data indicate that information from the knee joint is processed in parallel in SI and SII on the contralateral side and that there is an additional serial processing in SI on the ipsilateral side.
Afferent Pathways↗
Histamine excites groups III and IV afferents from the cat knee joint depending on their resting activity.
The effect of histamine on the sensory activity of primary afferents was studied in normal and acutely inflamed cat knee joints. A subpopulation of groups III and IV articular afferents could be activated by close-arterial bolus injections of histamine: units with a high resting activity (about 100/min) were particular sensitive to histamine and were excited even by 3.3 fg histamine. The lower the resting discharges of groups III and IV units both from normal and acutely inflamed joints, the higher the dose of histamine (up to 3.3 or 33 microg) necessary to excite the nerve fibres. Thirty-seven of 39 units without any resting activity were completely insensitive to histamine. In contrast to its clear excitatory effect, histamine caused only minor changes in the responses to joint movements. Movement-evoked activity remained unchanged in 22 of 28 units, 1 unit was sensitized and 5 units showed reduced activity after histamine (3.3 microg). The present results support the notion that histamine may participate in the mediation of pain from injured or inflamed tissue. It is remarkable that histamine has a profound excitatory action on a proportion of both groups III and IV articular afferents without changing their sensitivity to joint movements.
Acute Disease↗
Sensitisation of group III articular afferents to mechanical stimuli by substance P.
OBJECTIVE: To examine the excitatory and sensitising effects of substance P (SP) on articular afferents in normal and acutely inflamed cat knee joints. MATERIALS AND METHODS: In anesthetised cats recordings were made from 15 group III (conduction velocity 2.5-20 m/s) and 25 group IV afferent units (conduction velocity < 2.5 m/s) of the medial articular nerve of normal and acutely inflamed knee joints. SP (10 and 100 microg) was administered close-arterially. RESULTS: SP at doses of 10 microg and 100 microg activated less than 50% of both group III and group IV units. The proportion of SP-positive units was significantly higher in inflamed (10 of 21) than in normal joints (2 of 18). SP induced activity in initially silent units or increased ongoing activity after latencies varying from 2 s to 5 min. The SP-evoked activity had an irregular pattern, a variable duration, and was not related to the dose injected. Bolus injections of SP (100 microg) sensitised group III articular afferents but not group IV units to noxious movements of the joint, regardless whether the units were from normal or acutely inflamed joints. The responses to innocuous movements were not influenced by SP. Group III units, initially not activated by any movement, displayed vigorous discharges to noxious movements after close-arterial SP. In 3 group III units tested, the SP-induced augmentation of responses to noxious movements was not mimicked by close-arterial injection of histamine (3.3 microg). CONCLUSIONS: It is concluded that SP contributes to the sensitisation of a subpopulation of high-threshold articular afferents. Thus this neuronal mediator released peripherally in response to an injury or acute inflammation causes considerable changes in the mechanosensitivity of this subpopulation of nociceptive joint afferents.
Animals↗
Participation of NK1 receptors in nociceptin-induced modulation of rat knee joint mechanosensitivity.
Nociceptin is known to act peripherally in the rat knee joint to modulate articular mechanosensitivity and it has been postulated that neurokinin-1 (NK1) receptors may be involved in this process. To test this hypothesis, single unit extracellular recordings were made from knee joint primary afferents in response to normal and extreme rotation of the joint. Afferent firing rate was assessed following close intraarterial injection of the NK1 antagonist RP67580 followed by administration of 20 nmol nociceptin. With both normal and hyper-rotation of the knee, nociceptin was unable to elicit its usual mechanomodulatory effect such that afferent activity was not significantly different from control (P = 0.7572 and P = 0.9182 for normal and hyper-rotation of the joint, respectively). These data indicate that nociceptin-induced mechanosensitivity changes in the rat knee depend upon NK1 receptor activation possibly through secondary release of substance P.
Action Potentials↗
Swelling-activated calcium signalling in cultured mouse primary sensory neurons.
The effects of hypo-osmotic membrane stretch on intracellular calcium concentration ([Ca(2+)](i)), cell volume and cellular excitability were investigated in cultured mouse primary sensory trigeminal neurons. Hypotonic solutions (15--45%) led to rapid cell swelling in all neurons. Swelling was accompanied by dose-dependent elevations in [Ca(2+)](i) in a large fraction of neurons. Responses could be classified into three categories. (i) In 57% of the neurons [Ca(2+)](i) responses had a slow rise time and were generally of small amplitude. (ii) In 21% of the neurons, responses had a faster rise and were larger in amplitude. (iii) The remaining cells (22%) did not show [Ca(2+)](i) responses to hypo-osmotic stretch. Slow and fast [Ca(2+)](i) changes were observed in trigeminal neurons of different sizes with variable responses to capsaicin (0.5 microM). The swelling-induced [Ca(2+)](i) responses were not abolished after depletion of intracellular Ca2+ stores with cyclopiazonic acid or preincubation in thapsigargin, but were suppressed in the absence of external Ca(2+). They were strongly attenuated by extracellular nickel and gadolinium. Hypotonic stimulation led to a decrease in input resistance and to membrane potential depolarization. Under voltage-clamp, the [Ca(2+)](i) elevation produced by hypotonic stimulation was accompanied by the development of an inward current and a conductance increase. The time course and amplitude of the [Ca(2+)](i) response to hypo-osmotic stimulation showed a close correlation with electrophysiological properties of the neurons. Fast [Ca(2+)](i) responses were characteristic of trigeminal neurons with short duration action potentials and marked inward rectification. These findings suggest that hypo-osmotic stimulation activates several Ca(2+)-influx pathways, including Gd(3+)-sensitive stretch-activated ion channels, in a large fraction of trigeminal ganglion neurons. Opening of voltage-gated Ca(2+) channels also contributes to the response. The pattern and rate of Ca(2+) influx may be correlated with functional subtypes of sensory neurons.
Animals↗
The neurokinin-1 receptor antagonist RP 67580 reduces the sensitization of primary afferents by substance P in the rat.
The inflammatory mediator substance P (SP) produces a variety of biological effects in several tissues by binding to the tachykinin receptor neurokinin 1 (NK1) and, to a lesser extent, by binding to the neurokinin 2 receptor (NK2). To assess the sensitizing effect of SP on articular afferent fibres the NK1receptor antagonist RP 67580 was applied in normal and acutely inflamed rat knee joints. Altogether 38 fine afferent nerve fibres from the rat knee with conduction velocities of 0.71-13.5 m/s were recorded as single units, during non-noxious and noxious joint rotations. SP, injected i.a. as a bolus close to the knee joint, was able to sensitize 45.5% (10 of 22) of the units recorded from normal joints and 33.3% (five of 15) of afferents from inflamed joints. The following i.a. application of RP 67580 in a range of 20-200 nmol antagonized in a dose-dependent manner the sensitizing effect of SP in a large proportion of slowly conducting articular afferents from normal (66.7%) and inflamed (46.2%) knee joints. Subsequent SP application enhanced the afferent sensitivity further. The electrophysiological results presented here further support the suggestion that the sensitization of afferents by SP in the rat knee joint is mediated mainly by the NK1 receptor, which is probably located on the primary afferents.
Acute Disease↗
Composition of the medial and posterior articular nerves of the mouse knee joint.
The number and the distribution of fiber size in the medial (MAN) and posterior (PAN) articular nerves of the mouse knee joint were studied by electron microscopy. The MAN contained 75 +/- 28 nerve fibers consisting of 63 +/- 24 unmyelinated and 12 +/- 6 myelinated fibers. The PAN was composed of 195 +/- 50 nerve fibers, namely 129 +/- 28 unmyelinated and 66 +/- 24 myelinated fibers. A skewed unimodal distribution of the unmyelinated nerve fiber diameters was seen in both nerves ranging from 0.1 to 1.2 microm with a maximum between 0.3 and 0.6 microm. The myelinated nerve fibers in the MAN ranged from 1 to 8 microm with a peak between 2 and 5 microm. In the PAN, their diameters ranged from 1 to 12 microm with a clearly visible peak at 4-5 microm and a plateau at 8-9 microm that may represent a second maximum. These data show that the knee joint innervation of the mouse is comparable to those of the cat and rat concerning the types of nerve fibers and the composition of the two nerves. However, in relation to the much smaller area of tissue to be innervated the total number of primary afferents is considerable smaller in the mouse.
Animals↗
Peripheral modulation of rat knee joint afferent mechanosensitivity by nociceptin/orphanin FQ.
The peripheral effects of nociceptin were examined in normal and acutely inflamed rat knee joints by analyzing single unit recordings from articular primary afferents in response to normal and extreme rotation of the knee. Bolus close intraarterial injection of nociceptin (0.01, 1 and 100 microM) caused a sensitization of normal and inflamed knee joint afferents in response to movements in the normal working range of the joint. When the joint was hyper-rotated, nociceptin had no significant effect on afferent discharge rate in normal knees, however, in inflamed joints the top dose of the neuropeptide caused a decrease in articular mechanosensitivity. These findings suggest that nociceptin seems to be involved in the control of peripheral nociceptive mechanisms, although the behaviour of the peptide is dependent upon the inflammatory status of the tissue.
Animals↗
Involvement of nitric oxide in the modulation of dural arterial blood flow in the rat.
1. Nitric oxide (NO) has been proposed to be a key molecule in the pathogenesis of migraine pain and other headaches that are linked to vascular disorders. Several lines of evidence indicate that the meningeal vascularization is crucially involved in the generation of these headaches. In an experimental model in the rat a dominating role of calcitonin gene-related peptide (CGRP) in causing neurogenic vasodilatation and increased blood flow has been shown. The aim of the present study was to clarify the role of NO in this model with regard to the meningeal blood flow. 2. The blood flow in and around the medial meningeal artery (dural arterial flow) was recorded in the exposed parietal dura mater encephali of barbiturate anaesthetized rats using laser Doppler flowmetry. Local electrical stimulation of the dura mater (pulses of 0.5 ms delivered at 7.5 - 17.5 V and 5 or 10 Hz for 30 s) caused temporary increases in dural arterial flow for about 1 min that reached peaks of 1.6 - 2.6 times the basal flow. The effects of NO synthase (NOS) inhibitors on the basal flow and the electrically evoked increases in flow were examined. 3. Systemic (i. v.) administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) at cumulative doses of 10 and 50 mg kg(-1) lowered the basal flow to 87 and 72%, respectively, of the control and reduced the evoked increases in blood flow to 82 and 44% on an average. Both these effects could partly be reversed by 300 mg kg(-1) L-arginine. The systemic arterial pressure was increased by L-NAME at both doses. Injection of the stereoisomer D-NAME at same doses did not change basal flow and evoked increases in flow. 4. 4. Topical application of L-NAME (10(-4) - 10(-2) M) was effective only at the highest concentration, which caused lowering of the basal blood flow to 78% of the control; the evoked increases in flow were not changed. Topical application of 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a specific inhibitor of the inducible NOS, at concentrations of 10(-4) - 10(-2) M lowered the basal flow to 89, 87.5 and 85%, respectively, but did not significantly change the evoked flow increases. Same concentrations of 7-nitroindazole monosodium salt (7-NINA), a specific inhibitor of the neuronal NOS, had no significant effects on basal flow and evoked increases in flow. 5. It is concluded that NO is involved in the maintenance of the basal level of dural arterial blood flow as well as in the electrically evoked flow increases, which have been shown to be mainly mediated by CGRP released from dural afferent fibres. The most important source of NO is probably the endothelium of dural arterial vessels. The synergistic effect of NO and CGRP on the stimulated blood flow may be in part due to a NO mediated facilitation of the CGRP release.
Animals↗
Somatostatin reduces the meningeal arterial blood flow in the rat.
The effect of somatostatin (SOM) on neurogenic increases in meningeal blood flow was examined in barbiturate anaesthetized rats. The parietal skull was trepanized and the blood flow in the medial meningeal artery was monitored using a laser Doppler flowmeter with needle probes. Electrical stimulation (pulses of 8-10 V at 5-10 Hz for 30 s) close to the superior sagittal sinus evoked reproducible increases in blood flow. These increases were reduced by topical applications of SOM at concentrations of 10(-5)-10(-3) M in a dose-dependent manner. The effect was most pronounced within 10 min after application of SOM followed by a recovery of the flow responses. We conclude that stimulus-evoked increases in dural arterial flow, which are most likely caused by afferent activation and can be regarded as an element of neurogenic inflammation, are reduced by anti-inflammatory peptides such as SOM.
Animals↗
Presynaptic inhibition in the vertebrate spinal cord revisited.
The present review examines the experimental evidence supporting the existence of central mechanisms able to modulate the synaptic effectiveness of sensory fibers ending in the spinal cord of vertebrates. The first section covers work on the mode of operation and the synaptic mechanisms of presynaptic inhibition, in particular of the presynaptic control involving axo-axonic synapses made by GABAergic interneurons with the terminal arborizations of the afferent fibers. This includes reviewing of the ionic mechanisms involved in the generation of primary afferent depolarization (PAD) by GABAergic synapses, the ultrastructural basis underlying the generation of PAD, the relationship between PAD and presynaptic inhibition, the conduction of action potentials in the terminal arborizations of the afferent fibers, and the modeling of the presynaptic inhibitory synapse. The second section of the review deals with the functional organization of presynaptic inhibition. This includes the segmental and descending presynaptic control of the synaptic effectiveness of group-I and group-II muscle afferents, the evidence dealing with the local character of PAD as well as the differential inhibition of PAD in selected collaterals of individual muscle-spindle afferents by cutaneous and descending inputs. This section also examines observations on the presynaptic modulation of large cutaneous afferents, including the modulation of the synaptic effectiveness of thin myelinated and unmyelinated cutaneous fibers and of visceral afferents, as well as the presynaptic control of the synaptic actions of interneurons and descending tract neurons. The third section deals with the changes in PAD occurring during sleep and fictive locomotion in higher vertebrates and with the changes of presynaptic inhibition in humans during the execution of a variety of voluntary movements. In the final section, we examine the non-synaptic presynaptic modulation of transmitter release, including the possibility that the intraspinal endings of primary afferents also release colocalized peptides in a similar way as in the periphery. The outcome of the studies presently reviewed is that intraspinal terminals of sensory fibers are not hard-wired conductors of the information generated in their peripheral sensory receptors, but dynamic systems that convey information that can be selectively addressed by central mechanisms to specific neuronal targets. This central control of information flow in peripheral afferents appears to play an important role in the generation of integrated movements and processing of sensory information, including nociceptive information.
Animals↗
Examination of colocalization of calcitonin gene-related peptide- and substance P-like immunoreactivity in the knee joint of the dog.
It is generally assumed that the majority of substance P (SP)-containing afferents are also immunoreactive for calcitonin gene-related peptide (CGRP). In order to determine whether this is also the case in articular afferents where the contents of these peptides are low, we carried out a double labeling study using Fast Blue (FB) as a retrograde tracer injected into the center of the knee joint cavity of the dog together with immunohistochemistry for SP and CGRP. After 7-36 days of survival, dorsal root ganglia (DRGs, L4-S1) were removed. Labeled cells were found mainly (94%) in L5 - 6 DRGs, and SP- and CGRP-like immunoreactivity was found in about 17 and 29% of FB-labeled cells, respectively. The coexistence of SP and CGRP was observed in 10.4% of articular afferents and only 62.7% of SP-positive articular neurons contained CGRP, a much lower ratio than in other afferents of the dog such as testicular afferents. Our data suggest that these peptides are not always released together and that they do not always work together in the joint under normal conditions.
Animals↗
Positron emission tomography reveals changes in global and regional cerebral blood flow during noxious stimulation of normal and inflamed elbow joints in anesthetized cats.
In cats the global (gCBF) as well as the regional cerebral blood flow (rCBF) and blood pressure were measured before, during, and after noxious inward and outward rotations of normal and inflamed elbow joints. The animals were anesthetized with halothane and immobilized by gallamine triethiodide. The gCBF as well as the rCBF were measured using positron emission tomography (PET) with a camera specifically designed for use in small animals. Slow intravenous bolus injections of 15O-labeled water were followed by 3-min acquisition of regional radioactivity starting at the time of injection. In all experiments the gCBF as well as the blood pressure were increased by noxious inward-outward rotations of the normal and of the inflamed joint, whereas the blood pressure and the rCBF remained unchanged during bolus injections under control conditions (without any joint movement). Movements of the inflamed joint evoked significantly greater increases in blood pressure and gCBF than corresponding ones of the normal joint. These increases in gCBF were paralleled by increases in rCBF along the complete anterior to posterior axis of the brain. Again, the increases in rCBF were larger, more extensive and more uniform following the stimulation of the inflamed joint relative to the results obtained with stimulation of the normal joint. No significant laterality was seen, but when an atlas-based region of interest (ROI) analysis was carried out and when the individual variations in rCBF were removed with two-way ANOVA, significant differences were disclosed in rCBF between the stimulated condition and the resting condition in a large number of brain regions. In particular, noxious rotation of the normal (right) elbow joint induced a significant increase in rCBF over the cerebral cortex and in the right thalamus and hippocampus. The same stimulation of the (left) inflamed joint induced a significant increase in rCBF throughout the brain; the biggest increase being over the right posterior cortex. It is concluded that under the conditions of the present experiments the generally accepted autoregulation of the cerebral blood flow is not fully functioning, and various factors that may be responsible for this failure (which obscures rCBF differences) are discussed. The more pronounced increases in rCBF when moving inflamed joints instead of normal ones is thought to be a direct consequence of the peripheral sensitization of the articular nociceptors and the consequent central hyperexcitability induced in the articular nociceptive pathways.
Anesthesia↗
Calcitonin gene-related peptide- and substance P-like immunoreactive fibers in the spermatic nerve and testis of the dog.
In order to determine if calcitonin gene-related peptide (CGRP) and substance P (SP) coexist in peripheral spermatic nerve fibers, we carried out a double-staining immunofluorescence study using confocal microscopy and fluorescence microscopy. CGRP- and SP-like immunoreactivity (LI) coexisted in the spermatic nerve trunk and in the single fibers running along the surface of the testis. The great majority of the SP-containing fibers also held CGRP-LI, although some fibers contained CGRP-LI without SP-LI. These observations are consistent with previous observations on testicular dorsal root ganglion neurons. Additionally, we carried out an immunogold silver staining for CGRP and found CGRP-containing nerve bundles, single nerve fibers and their nerve terminals. Some CGRP-containing nerve terminals were located very superficially in the tunica albuginea (<5 microm from the surface).
Animals↗