Synovial joints are complex sensory organs which provide continuous feedback regarding position sense and degree of limb movement. The transduction mechanisms which convert mechanical forces acting on the joint into an electrochemical signal which can then be transmitted to the central nervous system are not well understood. The present investigation examined the effect of the mechanogated ion channel blockers amiloride and gadolinium on knee joint mechanosensitivity. In deeply anaesthetised rats (sodium thiopental: 100–120 mg/kg, i.p.), single unit extracellular recordings were made from knee joint group III (Aδ) and group IV (C) primary afferents in response to mechanical rotation of the joint. Afferent firing rate was measured before and after topical application of either amiloride (0.1 mM, 1 mM) or gadolinium (250 μM) onto the receptive field of the sensory unit and recording was continued every 10 min up to a total of 50 min. With normal rotation of the knee, joint mechanosensitivity was significantly reduced by both amiloride (P<0.0001; n=10–21) and gadolinium (P=0.001; n=12) and this effect was sustained throughout the recording period. This investigation provides the first in vivo electrophysiological evidence that joint mechanotransduction involves the activation of amiloride and gadolinium-sensitive mechanogated ion channels. Future studies to determine the mechanogated ion channel subtypes present in joints and the modulation of their gating properties during inflammation may yield novel approaches for the control of arthritis pain.

Voltage-gated Ca2+ channels play an important role in the central processing of nociceptive information. Recently, it has been shown that L- and N-type voltage-gated Ca2+ channels are also present on peptidergic, fine afferent nerve fibers in the knee joint capsule. Therefore, the influence of specific blockers for L-type (verapamil) or N-type (ω-conotoxin GVIA) Ca2+ channels on the mechanosensitivity of slowly conducting afferents was tested in the rat knee joint. Topical application of 100 μM verapamil onto the receptive field reduced the mean response to knee joint rotation to 67±8% (SEM, n=12), obtained by outward rotations with a torque of 10 mNm above the mechanical threshold and compared with control movements. In the presence of 50 μM ω-conotoxin GVIA, the mean response decreased to 44±5% (n=12), a reduction that was also observed during rotations of other intensities. Simultaneous application of both substances further reduced the response to 25±11% (n=6). In additional experiments it was shown that L- and N-type voltage-gated Ca2+ channels do not influence activity-dependent changes of the mechanical excitability. In conclusion, the data of the present study indicate that voltage-gated Ca2+ channels may also be involved in the regulation of the mechanosensitivity of nociceptive nerve fiber endings.

The aim of the present study was to examine the effect of electrical saphenous nerve stimulation (14 V, 1-10 Hz) on the mechanosensitivity of rat knee joint afferents, The responses to passive joint rotations at defined torque were recorded from slowly conducting knee joint afferent nerve fibres (0.6 20.0 m/s). After repeated nerve stimulation with 1 Hz, the mechanosensitivity of about 79% of the units was significantly affected. The effects were most prominent at a torque close to the mechanical threshold. In about 46% of the examined nerve fibres a significant increase was obtained, whereas about 33% reduced their mechanosensitivity. The sensitisation was prevented by an application of 5 muM phentolamine, an alpha-adrenergic receptor blocker, together with a neuropeptide Y receptor blocker. An inhibition of N-type Ca2+ channels by an application of 1 muM omega-conotoxin GVIA caused comparable changes of the mechanosensitivity during the electrical stimulation. Electrical nerve stimulation with higher frequencies resulted in a further reduction of the mean response to joint rotations. After stimulation with 10 Hz, there was a nearly complete loss of mechanosensitivity.In conclusion, antidromic electrical nerve stimulation leads to a frequency dependent transient decrease of the mechanosensitivity. A sensitisation was only obtained at 1 Hz, but this effect may be based on the influence of sympathetic nerve fibres. (C) 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.

ABSTRACT The sensitivity of articular nociceptors can be influenced directly by drugs either applied systemically or locally, and this is a very promising way to exert analgesic actions. For instance, Pozo et al. reported in 19971 that an elastoviscous solution of hylan, a hyaluronan derivative, significantly reduced both the ongoing activity and the movement evoked responses of primary afferent units from inflamed knee joints of the cat. These results support the assumption that hylans injected into the knee joint cavity may act as an elastoviscous filter, reducing the transmission of stretch to the nociceptive terminals where the mechano-electrical transduction takes place. More recently we have extended these observation to the knee joint of the rat. So far, with no appreciable exception, the results from nociceptors with fine afferent fibers (A δ- and C-fibers) of the medial articular nerve of the rat correspond to those obtained in the cat. The mechanoprotective effects found in the knee joint of rat are in the same order of magnitude as those obtained by systemic application of peripherally acting analgesic drugs. This duplication allows a further generalization of the results obtained in the cat. In addition it has opened the possibility to test in a much more controlled and systematic manner the effects of hylans and other substances with different elastic and viscous properties (alone or in connection with various analgesic drugs) on the discharge characteristics of nociceptors innervating normal and acutely inflamed knee joints

In the search to make classical radioactive in situ hybridization (ISH) methods using 3′-tailed synthetic oligonucleotides less complex and more accessible to researchers without radiochemical facilities, various nonradioactive methods have been developed. Although many of these indirect in situ methods are sensitive and give excellent results on membrane blots and tissue sections, they may be more suited to qualitative rather than quantitative studies, as the detection procedure involves several amplification steps. For both qualitative and semiquantitative studies, alkaline phosphatase (AP) is the reporter of choice for labeling oligonucleotides. Although these probes offer many advantages over their non-radioactive adversaries, and radiolabeled oligonucleotides, they do have their limitations. For qualitative studies of gene expression, paraffin-embedded semi-thin sections offer excellent cellular resolution of the AP hybridization signal combined with preserved tissue morphology. After development of the colored AP reaction product, sections may be processed for immunocytochemistry allowing antigens and mRNA to be simultaneously visualized in the same tissue section.

Vergleichbar mit den Verhältnissen im peripheren Gewebe, wo eine Vielzahl an Substanzen die Erregbarkeit von Nozizeptoren beeinflusst, sind auch im Rückenmark verschiedene Transmitter, Peptide und Rezeptoren an der Verarbeitung nozizeptiver Informationen beteiligt. Für die spinale Analgesie beim Menschen können inzwischen einige spezifische Agonisten bzw. Antagonisten eingesetzt werden. Da bestimmte Transmitter und Neuropeptide nicht nur bei der Nozizeption sondern auch bei verschiedenen anderen physiologischen Vorgängen eine Rolle spielen, sind entsprechende Nebenwirkungen unausweichlich. Eine Möglichkeit, diese zu reduzieren oder sogar zu verhindern, könnte darin bestehen, mehrere Pharmaka mit einer jeweils geringeren Konzentration zu kombinieren und somit eine multimodale Analgesie einzusetzen.

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 mum with a maximum between 0.3 and 0.6 mum. The myelinated nerve fibers in the MAN ranged from 1 to 8 mum with a peak between 2 and 5 mum. In the PAN, their diameters ranged from 1 to 12 mum with a clearly visible peak at 4-5 mum and a plateau at 8-9 mum 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.

The afferent (excitability) and efferent functions (release of neuropeptides) of primary afferent nerve fibres are based on Ca(2+)-influx. The aim of the present study was to examine the presence of L- and N-type Ca(2+)-channels at sensory nerve fibres in the mouse knee joint capsule. Specific fluorescent labelled channel blockers and antisera against these channel subtypes were combined with an immunohistochemical staining for calcitonin gene-related peptide (CGRP), a neuropeptide that is widely distributed in primary afferents. There was a nearly complete colocalisation of CGRP immunoreactivity and the binding of omega-conotoxin GVIA (toxin VIA of Conus geographus or BODIPY-verapamil (BODIPY(R) FL verapamil, hydrochloride) demonstrating the presence of N-type and L-type Ca(2+)-channels, respectively. These data were further confirmed by identical results obtained after an immunohistochemical demonstration of the two channel subtypes at the peptidergic nerve fibres.

1. The aim of the present study was to examine the effects of the sympathetic co-transmitter Neuropeptide Y on primary afferent nerve fibres of the rat knee joint. The responses to passive joint rotations at defined torque were recorded from 41 slowly conducting afferent nerve fibres (0.9 - 18.8 m s(-1)) innervating the knee joint capsule. 2. About 70% of the joint afferents were significantly affected in their mechanosensitivity by topical application of Neuropeptide Y. Significant effects occurred at a concentration of 10 nM. 3. Decreased mechanosensitivity was observed in about 40% of nerve fibres, whereas 30% of the units increased the mechanosensitivity. In addition, in about 35% of the fibres resting activity was induced or increased. Neither the conduction velocity nor the mechanical threshold of the units correlated with the described effects of Neuropeptide Y. 4. NPY(13--36), a specific Y2-receptor agonist, only modulated the mechanosensitivity, with no effect on the resting activity. The effects on the mechanosensitivity were similar to Neuropeptide Y, i.e. increase and decrease of the response. 5. Studies with the Y1-agonist (Leu(31), Pro(34))-NPY showed that activation of the Y1-receptor predominantly resulted in an enhanced mechanosensitivity and an induction or increase of a resting activity. The opposite effect was observed by application of BIBP 3226 BS, a Y1-receptor antagonist. 6. In conclusion, these data indicate that Neuropeptide Y affects the excitability of sensory nerve fibre endings.

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.

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.

The aim of the present study was to examine the effect of galanin on group III and IV afferent nerve fibres (n = 53) innervating normal and acutely inflamed knee joints in rats. They responded to local mechanical stimulation, movements of the joint and i.a. injections of KCl close to the joint. Single i.a. bolus injections of galanin (0.1 mm, 0.2 mL) caused no direct responses of the units. In normal and acutely inflamed joints, about half of the units did not change the responses to knee joint rotation. A significant reduction of the responses to noxious movements was found in similar to 40% of the units reaching a mean value of 57% in normal joints and 70% in inflamed joints compared with control movements. In similar to 10% the responses increased to 143% in normal joints and 120% in inflamed joints. Injection of a galanin receptor antagonist (M35) doubled the responses to noxious movements in 36% of the units in normal joints and reduced it in 18% to 86% of the control movements, indicating a tonic release and influence on the mechanosensitivity of a proportion of primary afferents by galanin. In conclusion, these data further support the hypothesis that the mechanosensitivity of fine afferent nerve fibres is regulated by a mixture of different substances being released into the innervated tissue. Besides the action of several pro-inflammatory peptides there seems to exist a tonic inhibitory system.

In total 23 rat knee joint afferent units with conduction velocities of 1.2–17 m/s were recorded extracellularly during inward and outward joint rotations of defined torque using a newly developed torque-meter. There was not a linear relation between the torque and the obtained angle of the joint. The mechanical thresholds of the units ranged from 10 to 60 mNm. During 10 s of stimulation with 60 mNm they responded with 12–300 action potentials. Two mechanosensitive nerve fibres could not be activated via knee joint rotation at any torque at all. The units could be classified according to their response pattern during stimulation: (1) phasic-tonic response behaviour; (2) constant discharge rate; (3) delayed onset of activity. In conclusion, the novel torque-meter allows a precise characterisation of the mechanical threshold and the corresponding response of fine afferent joint units during knee joint rotation.

Recently it has been shown that neuropeptides may be involved in the regulation of the mechanosensitivity of fine afferent nerve fibres. Somatostatin seems to provide a tonic inhibition of the sensitivity of sensory endings in normal tissue. In the present study cyclo-somatostatin, a somatostatin antagonist, was used to test this hypothesis. In a normal joint, 50% (8/16) of the examined primary afferents significantly increased their responses to noxious joint movements after the cyclo-somatostatin application. In an acutely inflamed joint, however, only 9% (1/11) of the nerve fibres significantly increased the response to noxious movements. These data further support the hypothesis that the mechanosensitivity of articular afferents in a normal joint is also regulated by a balance of pro-inflammatory peptides and anti-inflammatory peptides such as somatostatin. In an inflamed joint, pro-inflammatory peptides predominate and together with other mediators, they may be involved in the sensitization of the peripheral nerve fibres.

Using gold-labelled somatostatin, somatostatin binding sites were predominantly found in laminae I–III, X and on motorneurones of the rat lumbar spinal cord. A comparison with immunohistochemical staining using antisera against somatostatin receptor sequences revealed that the marked binding in laminae I–III coincided with the presence of somatostatin receptor-like immunoreactivity for the receptor subtypes 1, 2 and 3. Binding sites on motorneurones were only paralleled by an immunoreaction for subtype 3. In lamina X, however, the lack of a positive immunoreaction indicates that in this part other subtypes may be present.

Recently we presented a method to label the neuropeptide substance P with a 1.4-nm gold particle covalently bound at the N-terminus that can be used for demonstrating its binding sites in histological sections. In this study we examined whether the peptides neuropeptide Y, somatostatin, calcitonin gene-related peptide and bradykinin can be labelled in the same way. Polyacrylamide gel electrophoresis revealed a reduction in mobility for peptide-gold conjugates over gold particles alone consistent with peptide binding. In cryostat sections of the rat lumbar spinal cord, the peptides showed a distinct binding pattern in the grey matter corresponding to data of studies using autoradiographic methods. Therefore, we conclude that this simple and fast method can be used for labelling peptides in general to demonstrate their binding sites in histological sections, provided the peptide binds by its C-terminus.

Neurotrophins mediate specific effects on sensory neurons through tyrosine kinase receptors. Most of these neurons also co-express the neurotrophin receptor p75 (p75NTR), but its function has remained obscure. We now show that nerve growth factor but not brain-derived neurotrophic factor or neurotrophin-3 selectively increases the expression of bradykinin binding sites on cultured dorsal root ganglion neurons from adult mouse via p75NTR. This up-regulation of bradykinin binding sites did not occur in neurons from mice lacking p75NTR or in neurons from wild-type mice treated with p75NTR-blocking antibody, indicating that tyrosine kinase receptors alone are not sufficient to trigger this physiological neuronal response. Thus, the interaction of nerve growth factor with p75NTR is an important factor contributing to chronic pain conditions.