Intra-axonal Transport Research Articles

Microtubule Dynamics Following Central and Peripheral Nervous System Axotomy.

Disturbance in the neuronal network leads to instability in the microtubule (MT) railroad of axons, causing hindrance in the intra-axonal transport and making it difficult to re-establish the broken network. Peripheral nervous system (PNS) neurons can stabilize their MTs, leading to the formation of regeneration-promoting structures called "growth cones". However, central nervous system (CNS) neurons lack this intrinsic reparative capability and, instead, form growth-incompetent structures called "retraction bulbs", which have a disarrayed MT network. It is evident from various studies that although axonal regeneration depends on both cell-extrinsic and cell-intrinsic factors, any therapy that aims at axonal regeneration ultimately converges onto MTs. Understanding the neuronal MT dynamics will help develop effective therapeutic strategies in diseases where the MT network gets disrupted, such as spinal cord injury, traumatic brain injury, multiple sclerosis, and amyotrophic lateral sclerosis. It is also essential to know the factors that aid or inhibit MT stabilization. In this review, we have discussed the MT dynamics postaxotomy in the CNS and PNS, and factors that can directly influence MT stability in various diseases.

Visualization of Intra-neuronal Motor Protein Transport through Upconversion Microscopy.

Cargo transport along axons, a physiological process mediated by motor proteins, is essential for neuronal function and survival. A current limitation in the study of axonal transport is the lack of a robust imaging technique with a high spatiotemporal resolution to visualize and quantify the movement of motor proteins in real-time and in different depth planes. Herein, we present a dynamic imaging technique that fully exploits the characteristics of upconversion nanoparticles. This technique can be used as a microscopic probe for the quantitative in situ tracking of retrograde transport neurons with single-particle resolution in multilayered cultures. This study may provide a powerful tool to reveal dynamic neuronal activity and intra-axonal transport function as well as any associated neurodegenerative diseases resulting from mutation or impairment in the axonal transport machinery.

The brainstem pathologies of Parkinson's disease and dementia with Lewy bodies.

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are among the human synucleinopathies, which show alpha-synuclein immunoreactive neuronal and/or glial aggregations and progressive neuronal loss in selected brain regions (eg, substantia nigra, ventral tegmental area, pedunculopontine nucleus). Despite several studies about brainstem pathologies in PD and DLB, there is currently no detailed information available regarding the presence of alpha-synuclein immunoreactive inclusions (i) in the cranial nerve, precerebellar, vestibular and oculomotor brainstem nuclei and (ii) in brainstem fiber tracts and oligodendroctyes. Therefore, we analyzed the inclusion pathologies in the brainstem nuclei (Lewy bodies, LB; Lewy neurites, LN; coiled bodies, CB) and fiber tracts (LN, CB) of PD and DLB patients. As reported in previous studies, LB and LN were most prevalent in the substantia nigra, ventral tegmental area, pedunculopontine and raphe nuclei, periaqueductal gray, locus coeruleus, parabrachial nuclei, reticular formation, prepositus hypoglossal, dorsal motor vagal and solitary nuclei. Additionally we were able to demonstrate LB and LN in all cranial nerve nuclei, premotor oculomotor, precerebellar and vestibular brainstem nuclei, as well as LN in all brainstem fiber tracts. CB were present in nearly all brainstem nuclei and brainstem fiber tracts containing LB and/or LN. These findings can contribute to a large variety of less well-explained PD and DLB symptoms (eg, gait and postural instability, impaired balance and postural reflexes, falls, ingestive and oculomotor dysfunctions) and point to the occurrence of disturbances of intra-axonal transport processes and transneuronal spread of the underlying pathological processes of PD and DLB along anatomical pathways.

Cryo Electron Tomography of Herpes Simplex Virus during Axonal Transport and Secondary Envelopment in Primary Neurons

During herpes simplex virus 1 (HSV1) egress in neurons, viral particles travel from the neuronal cell body along the axon towards the synapse. Whether HSV1 particles are transported as enveloped virions as proposed by the ‘married’ model or as non-enveloped capsids suggested by the ‘separate’ model is controversial. Specific viral proteins may form a recruitment platform for microtubule motors that catalyze such transport. However, their subviral location has remained elusive. Here we established a system to analyze herpesvirus egress by cryo electron tomography. At 16 h post infection, we observed intra-axonal transport of progeny HSV1 viral particles in dissociated hippocampal neurons by live-cell fluorescence microscopy. Cryo electron tomography of frozen-hydrated neurons revealed that most egressing capsids were transported independently of the viral envelope. Unexpectedly, we found not only DNA-containing capsids (cytosolic C-capsids), but also capsids lacking DNA (cytosolic A-/B-capsids) in mid-axon regions. Subvolume averaging revealed lower amounts of tegument on cytosolic A-/B-capsids than on C-capsids. Nevertheless, all capsid types underwent active axonal transport. Therefore, even few tegument proteins on the capsid vertices seemed to suffice for transport. Secondary envelopment of capsids was observed at axon terminals. On their luminal face, the enveloping vesicles were studded with typical glycoprotein-like spikes. Furthermore, we noted an accretion of tegument density at the concave cytosolic face of the vesicle membrane in close proximity to the capsids. Three-dimensional analysis revealed that these assembly sites lacked cytoskeletal elements, but that filamentous actin surrounded them and formed an assembly compartment. Our data support the ‘separate model’ for HSV1 egress, i.e. progeny herpes viruses being transported along axons as subassemblies and not as complete virions within transport vesicles.

Intraaxonal uptake and transport of marker proteins: Novel ways of tracing peripheral nerve regeneration

Disruption of peripheral nerve continuity occurs after accidents or in obstetrics. Brachial plexus palsy is accompanied by loss of function of the upper extremity. For optimal regeneration, surgical reconstruction should be performed within 6 months after injury. Postoperative monitoring is difficult because reliable diagnostic tools are not available. Here we present a hypothesis how to monitor peripheral nerve regeneration taking into account two intriguing features of nerve repair. First, the nerve itself with its numerous axons has been neglected as transportation pathway hitherto. Second, growth associated proteins are exclusively upregulated during peripheral nerve repair and development providing excellent target molecules. Contrast media could be designed using truncated forms of these proteins. Previous studies showed that peripheral nerves have the ability to incorporate proteins with a size up to 240 kDa. After wounding, growth associated proteins are transported intraaxonally and incorporated into growth cone membranes. We hypothesize that injection of contrast media close to nerve cell bodies or into the end organ of the injured nerve would lead to protein uptake and transport to the injury site. Sprouting of peripheral nerves could be monitored by magnetic resonance imaging or single photon emission computer tomography using adequate tracer-coupled proteins. By this new method, nerve repair could be monitored non-invasively, selectively and repeatedly. Less contrast media at lower price would be needed and only the injured part of the body would be exposed to the diagnostic apparatus. Aberrant nerve repair could be detected easily followed by surgical therapy within the critical time frame of sustained nerve regeneration.

Ultrastructural Analysis of Virion Formation and Anterograde Intraaxonal Transport of the Alphaherpesvirus Pseudorabies Virus in Primary Neurons

A hallmark of alphaherpesviruses is their capacity to be neuroinvasive and establish latent infections in neurons. After primary replication in epithelial cells at the periphery, entry into nerve endings occurs, followed by retrograde transport of nucleocapsids to the nucleus where viral transcription, genome replication, and nucleocapsid formation take place. Translocation of nucleocapsids to the cytoplasm is followed by axonal transport to infect synaptically linked neurons. Two modes of intraaxonal anterograde herpesvirus transport have been proposed: transport of complete, enveloped virions within vesicles ("married model"), and separate transport of capsids and envelopes ("subassembly model"). To assess this in detail for the alphaherpesvirus pseudorabies virus (PrV), we used high-resolution transmission electron microscopy of primary neuronal cultures from embryonic rat superior cervical ganglia after infection with wild-type and gB-deficient PrV. Our data show that intranuclear capsid maturation, nuclear egress and cytoplasmic secondary envelopment occur as in cultured nonpolarized cells (H. Granzow, F. Weiland, A. Jöns, B. G. Klupp, A. Karger, and T. C. Mettenleiter, J. Virol. 71:2072-2082, 1997). PrV virions were present in axons as enveloped particles within vesicles associated with microtubules and apparently leave the neuron by exocytosis primarily at the growth cone. Only a few nonenveloped nucleocapsids were found in the axon. The same picture was observed after infection by phenotypically complemented gB-deficient PrV, which is able to complete only a single round of replication. Our data thus support intraaxonal anterograde transport of enveloped PrV virions within vesicles following the "married model."

Directional Transneuronal Spread of α-Herpesvirus Infection

Most α-herpesviruses are pantropic, neuroinvasive pathogens that establish a reactivateable, latent infection in the PNS of their natural hosts. Various manifestations of herpes disease rely on extent and direction of the spread of infection between the surface epithelia and the nervous system components that innervate that surface. One aspect of such controlled spread of infection is the capacity for synaptically defined, transneuronal spread, a property that makes α-herpesviruses useful tools for determining the connectivity of neural circuits. The current understanding of intra-axonal transport and transneuronal spread of α-herpesviruses is reviewed, focusing on work with herpes simplex virus and pseudorabies virus, the available in vitro technology used to study viral transport and spread is evaluated and how certain viral mutants can be used to examine neural circuit architecture is described in this article.

Hypothesis of double polarization

Mutations in a large number of genes that encode ubiquitously expressed proteins have been found to selectively or predominantly cause neurological disorders. Speculation has been that impaired intra-axonal transport along the long-extended axons is responsible for this tissue specificity. However this hypothesis may be insufficient in that it does not account for the potential role of the glial cells that interact with axons. Both Schwann cells and oligodendrocytes are also long and polarized cells with extended membranes that concentrically wrap around the axon to form myelin. The resultant myelin is largely compacted, which prevents the transport of many intracellular materials or organelles from one end of the membrane to the other. However patent, non-compacted spaces in the myelin, such as Schmidt–Lanterman incisures or paranodal loops, are also long-extended cytoplasmic channels that extend from the cell body to the distal membranes and may also be subject to transport problems. The nervous system therefore distinguishes itself by having a double long-polarized cellular system. Both neurons and myelinating glia must transport materials long distances, making them vulnerable to a variety of insults; and both must interact with each other. Thus, vulnerability of the nervous system may derive from additive impairments not only in axonal transport, but also in glial transport and neuronal–glial interactions.

Changing distribution patterns of synaptophysin-immunoreactive structures in the human dorsal striatum of the fetal brain.

Within the striatum two compartments, matrix and patches, can be distinguished by differences in the expression of neuroactive substances, afferent and efferent connections and time of neurogenesis. The present study was done to demonstrate the pattern of synaptophysin (SYN) expression which is indicative of synaptogenesis in the human fetal striatum (15th-32nd weeks of gestation) with special reference to developmental changes. From the 15th to the 22nd gestational weeks an intense diffuse SYN immunolabelling of striatal patches is observed. In the matrix SYN-immunoreactive fiber bundles are seen until the 20th week. Thereafter, the matrix is nearly devoid of SYN-immunoreactive structures. From the 28th week of gestation the matrix contains diffuse SYN immunoreactivity which gradually becomes as intense as that of the patches. The latter, thus, can no longer be delineated in the 30th week. The results show that fibrous SYN immunolabelling most probably indicating intra-axonal transport of synaptic vesicles can only be observed during the first half of gestation. Moreover, it becomes obvious that the patch compartment can selectively be visualized by anti-SYN until the 28th week. This pattern may correspond to the early dopaminergic innervation from the substantia nigra which is known to reach the developing patches. From the 28th week a transition from patchy to diffuse immunolabelling is seen. The increase in matrix labelling may be due to the occurrence of new neuronal contacts. The changeover from patchy to homogeneous SYN immunolabelling takes place distinctly earlier than changes in the distribution of other neuroactive substances described before.

Destruction of locus coeruleus neuronal perikarya after injection of anti-dopamine-B-hydroxylase immunotoxin into the olfactory bulb of the rat

Saporin, a ribosome-inactivating protein, was coupled to a monoclonal antibody to dopamine-B-hydroxylase (DBH) and injected unilaterally into the olfactory bulb of rats. After 4–13 days survival, the rat brain was processed histologically and the locus coerulei (LC) examined with Nissl and anti-DBH staining. There were degenerating dendrites in surviving LC neurons on the side ipsilateral to the immunotoxin-injected olfactory bulb. The number of Nissl-positive LC neurons in a transverse section through the caudal one third of the LC was reduced from 116±10 to 50±8 neurons ( P<0.01, n=7) and the number of DBH-positive neurons in the more rostral LC sections was reduced from 13±2 to 5±1 ( P<0.05, n=4). Our results indicate that it is possible to lesion LC neurons via retrograde intraaxonal transport of saporin-anti-DBH immunotoxin from the olfactory bulb.

Efferents and Centrifugal Afferents of the Main and Accessory Olfactory Bulbs in the Snake Thamnophis sirtalis

This study reinvestigates the efferent projections of the main and accessory olfactory bulbs and describes for the first time the centrifugal projections to the main and accessory olfactory bulbs in the snake Thamnophis sirtalis, using the intraaxonal transport of the anterograde and retrograde tracer biotinylated dextran amine and the retrograde tracer horseradish peroxidase. The olfactory projection consists of three tracts: the lateral olfactory tract, which projects bilaterally to the lateral cortex and the rostral amygdala, crossing the midline through the stria medullaris-habenular commissure system; the intermediate olfactory tract, which projects ipsilaterally to the olfactory tubercle and contributes to the contralateral projection; and the medial olfactory tract, which projects ipsilaterally to the dorsomedial retrobulbar formation. The vomeronasal projection is formed by a single tract, the accessory olfactory tract, that projects ipsilaterally to the nucleus of the accessory olfactory tract, the medial amygdala and the nucleus sphericus. The centrifugal projections to the main and accessory olfactory bulb are composed of two components: one that arises in areas that receive the olfactory or vomeronasal input (neurons in the olfactory tubercle, retrobulbar formation and lateral cortex project to the main olfactory bulb; and neurons in the nucleus of the accessory olfactory tract, the medial amygdala and the nucleus sphericus project to the accessory olfactory bulb), and another that arises in areas not directly implicated in processing the chemosensory information (the nucleus of the diagonal band of Broca and the dorsal cortex). These data allow the recognition of the general pattern of organization of the reptilian olfactory and vomeronasal systems.

Loss of primary afferent nerve terminals in the brainstem after peripheral nerve transection: an anatomical study in monkeys.

In order to investigate the changes in the somatosensory organization that occur after a peripheral nerve injury, a purely sensory nerve (radial nerve - superficial branch) was divided in adult monkeys (Macaca fascicularis). The nerve ends were immediately rejoined by an epineural suturing technique. After 6-21 months the nerve investigated was exposed to an intra-axonal nerve tracer (horseradish peroxidase conjugate) in order to label the primary afferent terminals within the cuneate nucleus of the brainstem. The non-transected nerve on the contralateral side was similarly exposed and served as a control. Terminal labelling was seen throughout the cuneate nucleus, mainly in the middle of its rostro-caudal extension, and in this part it showed a patchy appearance superimposed on cell clusters within the pars rotunda. This pattern of distribution was seen both on the experimental and control sides. On the experimental side there was an obvious loss of terminal labelling within the terminal field as estimated using an image-analysing system: Compared with the contralateral side the median loss (peroxidase activity) was 83% and between 6 and 21 months only minor restoration of the terminal intensity was observed. These results in the primate confirm earlier results in the cat that transection and microsurgical repair of a sensory nerve causes a considerable loss of neurons capable of intraaxonal transport.

Tract-tracing in the nervous system of vertebrates using horseradish peroxidase and its conjugates: tracers, chromogens and stabilization for light and electron microscopy

Surprisingly the first generation of tract-tracing techniques based on intra-axonal transport, that is the methods utilizing the uptake and transport of horseradish peroxidase (HRP), still rank among the most widely used neuroanatomical tracing techniques. The success of these methods can be ascribed to several characteristics. They are fast and easy to implement. Complicated injection apparatus is unnecessary. The reaction products are visualized through simple histochemical reactions, and they are permanent or can be stabilized into permanency. Most usefully, the reaction products are visible in the light and electron microscopes. HRP (mol. wt. 44 kDa) is extracted from the roots of the horseradish plant ( Cochlearia armoracia L.). Uptake of HRP into cells occurs via a passive process of endocytosis [3]. Since lectins like wheat germ agglutinin (WGA) and bacterial toxin fragments (subunit B of cholera toxin (CTB)) [17, 32, 34]strongly induce active, receptor-mediated uptake mechanisms, conjugates of these substances with HRP have been successfully applied in sensitive tract-tracing [4, 8, 34, 35] HRP and its conjugates are transported both in anterograde and retrograde direction [19, 20]. Retrograde transport occurs in small vesicles that are incorporated in lysosome-like vacuoles and in the Golgi apparatus. These vesicles differ in membrane properties from the anterogradely transported HRP vesicles [3, 4, 17]. The retrogradely transported vesicles tend to fuse and thus accumulate HRP at high densities, facilitating the visibility of the final reaction product. The anterogradely transported HRP does not accumulate directly in lysosome-like bodies and is distributed diffusely and therefore often requires specific visualization methods [5, 8, 10, 12, 31]. HRP and WGA-HRP may therefore be used in anterograde and retrograde transneuronal (multineuron) transport studies [15, 19]. Even in fixed material, labeling through diffusion of HRP can provide details on neural connections [18]. Visualization of transported HRP is achieved by means of using the oxidative enzymatic activity of HRP to precipitate a chromogen according to the following reaction: HRP+H 2O 2⇄[HRP.H 2O 2] [HRP.H 2O 2]+(chromogen)H 2⇄HRP+2H 2O 2+chromogen precipitate The final reaction product may be soluble in buffer or ethanol and may require stabilization to prevent fading. In this protocol we discuss the widely used chromogens 3,3′-diaminobenzidine tetrahydrochloride (DAB), 3,3′,5,5′-tetramethylbenzidine (TMB), benzidine dihydrochloride (BDHC) and p-phenylenediamine dihydrochloride with pyrocatechol (PPD-PC). Other possible chromogens, not discussed here, are 4-chloro-1-naphthol (4C1N), 3-amino-9-ethylcarbazole (AEC) and o-phenylenediamine (OPD). The visualization of the reaction product can be further improved by intensification with metal salts. At the light microscopic level (LM) this intensification enables color differentiation between distinct markers [1, 14, 23]. In the present protocol we provide an up-to-date guideline for the application of HRP and its conjugates in tracing with special emphasis on electron microscopic (EM) visualization. Some modifications for stabilization and of metal intensification to enhance visibility are incorporated in conjunction with specific methods for multiple labeling in combined tract-tracing experiments.

Unforgettable tetanus

Unforgettable tetanus

The organization of acromiodeltoid and spinodeltoid motor nuclei in rat spinal cord

The retrograde intraaxonal transport of fluorescent dyes (10% Bisbenzimide or 2% Nuclear Yellow) was used to identify the motoneuron populations that control two prime movers of the rat shoulder joint, the acromiodeltoid (Ad) and spinodeltoid (Sd) muscles. The Ad and Sd motor nuclei were both comprised of an average of 30–40 motoneurons distributed between spinal segments C5 and C7, in lateral regions of Rexed's lamina IX. Although there was considerable overlap among them, the center of the Ad motor nucleus was consistently located more rostral and medial than that for the Sd motor nucleus.

Vanilloid (capsaicin) receptors in the rat: distribution in the brain, regional differences in the spinal cord, axonal transport to the periphery, and depletion by systemic vanilloid treatment

Vanilloid (capsaicin) receptors were visualized by [3H]resiniferatoxin (RTX) autoradiography in the brain of newborn as well as adult (both control and colchicine-treated) rats. Specific labelling was seen in the brain stem only, in the nucleus of the solitary tract extending into the area postrema and the spinal sensory nucleus of the trigeminal nerve. Also, a strong signal was seen in the dorsal horn, dorsal root, trigeminal and nodose ganglia. Membranes obtained from the cervical, thoracic, and lumbar segments of the spinal cord showed similar affinities for RTX and likewise for capsaicin and capsazepine; maximal receptor density was similar in the cervical and thoracic segments (approximately 70 fmol/mg protein) but was twice as high in the lumbar segment. 24 h after ligation of the vagal or the sciatic nerves, a strong accumulation of specific RTX binding sites was observed mainly proximal to the ligature, implying intraaxonal receptor transport from the nodose and dorsal root ganglia, respectively, to the periphery. Systemic (s.c.) vanilloid treatment depleted specific [ 3H]RTX binding sites from the brain stem, the sensory (dorsal root as well as trigeminal) ganglia, and the spinal cord. RTX was approximately 200-fold more potent than capsaicin for eliminating vanilloid receptors from the spinal cord. The present results suggest a discrete expression of vanilloid receptors in the brain stem (sensory nuclei); although intrinsic vanilloid receptor-expressing neurons are thought to exist in the rat brain, they remain undetected by the present [ 3H]RTX autoradiography methodology.

Retrograde axonal transport of the alpha-subunit of the GTP-binding protein GZ in mouse sciatic nerve: a potential pathway for signal transduction in neurons.

We have utilized antibodies against the alpha subunit of GZ in fluorescence immunohistochemistry to determine whether this GTP-binding protein can translocate along nerves by intra-axonal transport. After ligation of the mouse sciatic nerve we found an increase in GZ-like immunoreactivity on the proximal and distal side with time, suggesting that the alpha subunit undergoes orthograde axonal transport and also returns to the cell body by retrograde axonal transport in the sciatic nerve. Unlike the retrograde transport of Gi alpha, shown in a previous study to be present in most sciatic axons, GZ alpha only accumulated in a subpopulation of axons, suggesting that different G-proteins could convey information specific to neuronal subtypes. These results support our proposal that GZ may play a second messenger role in communicating information from the terminals back to cell bodies. Gi alpha and GZ alpha may be representative of relatively stable signalling molecules by which the signal from some neurotrophic molecules can be translocated from the neuron periphery to the cell body without the need for the retrograde transport of the neurotrophic factor itself.

Sabeluzole administration does not enhance fast axonal transport in normal adult rat sciatic nerve.

Sabeluzole (R58735, Janssen Research Foundation) increased rates of axonal transport in short term tissue culture experiments and in rats with streptozotocin-induced diabetes. The drug was tested for its subacute (3 days) net effect on axonally transported substances in motor, sensory, and adrenergic axons of normal adult rats. Sabeluzol was given once daily for 3 days, 1 or 10 mg/kg/day intraperitoneally. Immunofluorescence was used to identify transported material. Three or 6 hr after crushing the sciatic nerves, to interrupt anterograde and retrograde intraaxonal transport, cytofluorimetric scanning was used to quantitate accumulated immunoreactive material. Compared with vehicle treated control rats, no clear differences in the net amounts of accumulated material, or in rates of accumulation, were detected in any axonal type. Since the short-term crush procedure interrupts ongoing axonal transport, the accumulation pattern reflects the transport characteristics in the crushed axons. The absence of clear increases in transport of several substances in this study indicates that sabeluzole did not enhance net axonal transport above control levels in peripheral axons of normal adult rats. Possible reasons for the discrepancy with earlier observations on the effect of sabeluzole on fast axonal transport is discussed.

Study of Sympathetic Innervation of Cranial Bones by Axonal Transport of Horseradish Peroxidase in the Rat: Preliminary Findings

This study performed by intraneuronal tracing directly demonstrates the presence of sympathetic postganglionic fibers in the cranial vault of the rat. Superior cervical sympathetic ganglia were injected with horseradish peroxidase (HRP), and after a 48-hour period, to permit anterograde axonal transport, the animals were sacrificed after in situ perfusion. An area of the calvaria that included portions of the frontal and parietal bones was fixed, decalcified and sectioned. HRP-containing axons were localized in the developing frontal and parietal bones of the calvaria. Adrenergic innervation was not demonstrated in sutural tissue (superior sagittal, coronal or metopic) by this technique. To our knowledge, this study is the first to trace sympathetic nerve fibers in the rat calvaria by intra-axonal transport of HRP.

An anatomical study of the origin of sympathetic and sensory innervation of the elbow and knee joint in the monkey

The origin of sympathetic and sensory innervation of the elbow and knee joint was studied by the intra-axonal transport method. After injection of WGA-HRP into the elbow and knee joint unilaterally in adult monkeys (macaca fascicularis), labelled cells were observed ipsilaterally in both the sympathetic and dorsal root ganglia. Following injection in the elbow joint the neurons were concentrated in the stellate and third to fourth thoracic (T 3-T 4) sympathetic ganglia, and in the fourth cervical to second thoracic spinal sensory ganglia. The knee joint injection resulted in labelling of sympathetic and spinal sensory ganglia, preferentially at the lumbo-sacral junction — the third lumbar to third sacral (L 3-S 3) ganglia. The functional implications are discussed in relation to pain mechanisms of joints and to an earlier hypothesis that the nervous system might be involved in the pathogenesis of chronic joint inflammation.