E3 ubiquitin ligases LNX1 and LNX2 localize at neuronal gap junctions formed by connexin36 in rodent brain and molecularly interact with connexin36.

Electrical synapses in the mammalian central nervous system (CNS) are increasingly recognized as highly complex structures for mediation of neuronal communication, both with respect to their capacity for dynamic short- and long-term modification in efficacy of synaptic transmission and their multimolecular regulatory and structural components. These two characteristics are inextricably linked, such that understanding of mechanisms that contribute to electrical synaptic plasticity requires knowledge of the molecular composition of electrical synapses and the functions of proteins associated with these synapses. Here, we provide evidence that the key component of gap junctions that form the majority of electrical synapses in the mammalian CNS, namely connexin36 (Cx36), directly interacts with the related E3 ubiquitin ligase proteins Ligand of NUMB protein X1 (LNX1) and Ligand of NUMB protein X2 (LNX2). This is based on immunofluorescence colocalization of LNX1 and LNX2 with Cx36-containing gap junctions in adult mouse brain versus lack of such coassociation in LNX null mice, coimmunoprecipitation of LNX proteins with Cx36, and pull-down of Cx36 with the second PDZ domain of LNX1 and LNX2. Furthermore, cotransfection of cultured cells with Cx36 and E3 ubiquitin ligase-competent LNX1 and LNX2 isoforms led to loss of Cx36-containing gap junctions between cells, whereas these junctions persisted following transfection with isoforms of these proteins that lack ligase activity. Our results suggest that a LNX protein mediates ubiquitination of Cx36 at neuronal gap junctions, with consequent Cx36 internalization, and may thereby contribute to intracellular mechanisms that govern the recently identified modifiability of synaptic transmission at electrical synapses.

Connexin36 localization along axon initial segments in the mammalian CNS.

Electrical synapses formed by gap junctions occur at a variety of neuronal subcellular sites in the mammalian central nervous system (CNS), including at somatic, dendritic and axon terminal compartments. Numerous electrophysiological studies using mice and rats, as well as computer modelling approaches, have predicted the additional occurrence of electrical synapses between axons near their emergence from neuronal somata. Here, we used immunofluorescence methods to search for localization of the neuronal gap junction-forming protein connexin36 (Cx36) along axon initial segments (AISs) labelled for the AIS marker ankyrinG. Immunofluorescent Cx36-puncta were found to be associated with AISs in several CNS regions of mice, including the spinal cord, inferior olive and cerebral cortex. Localization of Cx36-puncta at AISs was confirmed by confocal single scan and 3D imaging, immunofluorescence intensity profiling and high resolution structured illumination microscopy (SIM). AISs measuring up to 30 µm in length displayed typically a single Cx36-punctum and the incidence of these long AISs displaying Cx36-puncta ranged from 3% to 7% in the inferior olive and in various layers of the cerebral cortex. In the inferior olive, the gap junction associated protein zonula occludens-1 (ZO-1) was found to be co-localized with Cx36-puncta on AISs, indicating that these puncta have some of the molecular constituents of gap junctions. Our results add to the neuronal subcellular locations at which Cx36 is deployed, and raise possibilities for its involvement in novel functions in the AIS compartment.

Characterization of connexin31.1-deficient mice reveals impaired placental development.

The gap junction gene Connexin31.1 has been reported to be expressed predominantly in the epidermis of murine skin. To study the function of this gene, we generated mice in which the coding DNA of the Connexin31.1 gene was replaced by lacZ reporter coding DNA. Using beta-galactosidase staining, we have shown that lacZ/Connexin31.1 was expressed in the spinous and granular layers of the epidermis, in cells of olfactory epithelium and in the vomeronasal organ. During embryogenesis, Connexin31.1 was co-expressed with another isoform, Connexin31, in the post-implantation trophoblast cell lineage and, later in gestation, in placental glycogen cells. Although homozygous Connexin31.1-deficient mice were fertile and showed no morphological or functional defects in adult organs expressing this gene, 30% of the offspring expected according to Mendelian inheritance were lost between embryonic days 11.5 and 14.5 and surviving embryos were significantly reduced in weight near the end of pregnancy. Placentas of Connexin31.1-deficient embryos were reduced in weight and showed altered morphology of the spongiotrophoblast and labyrinth layer. The spongiotrophoblast formed a compact barrier at the decidual border that might restrict the maternal blood supply. We conclude that Connexin31.1 is critical for normal placental development but appears to be functionally compensated by other connexin isoforms in the embryo proper and adult mouse.

Evaluation of novel technique for studying ureteral function in vivo.

BACKGROUND AND PURPOSE: Knowledge of the ureteral response to instrumentation is limited. Ureterodynamic parameters such as intraureteral pressure, conduction velocity, direction of peristalsis, and electromyography have been measured using a variety of methods; however, these techniques are impractical for routine clinical use. The aim of this study was to evaluate a new commercial ureteral pressure transducer catheter, which records peristaltic frequency, conduction velocity, and intraureteral pressure. This device was assessed in an animal model and in patients who had undergone ureteroscopy. MATERIALS AND METHODS: An ambulatory urodynamic monitoring system was adapted to record the output from two pressure transducers mounted on a 4F ureteral catheter, which was inserted into the left ureters of six anesthetized pigs to record peristalsis. In six patients who had undergone ureteroscopy with or without stone removal, the recording catheter was inserted at the end of the procedure, and recovery of peristalsis was monitored for as long as 24 hours. RESULTS: The un-instrumented pig ureter showed spontaneous peristalsis immediately on catheter insertion, whereas the instrumented human ureter displayed a variable response that appeared to be related to previous physical or pharmacologic effects. CONCLUSIONS: Peristaltic frequency, pressure, and conduction velocity can be measured with the ureteral catheter described in both the experimental and clinical settings. Within the first 24 hours after ureteroscopy, peristaltic recovery is variable. Such information may enable both elucidation of the underlying mechanisms and improvement in the treatment of a variety of upper urinary-tract disorders.

An in vivo investigation of ulnar nerve sliding during upper limb movements.

BACKGROUND: Peripheral nerves straighten and stretch in order to accommodate increases in bed length during joint movements. The ulnar nerve is predicted to show large bed length changes, particularly on elbow flexion. The present study examines sliding of the ulnar nerve during limb movements, to determine how far these changes are accommodated by straightening and stretch. METHODS: Ultrasound imaging was used to measure longitudinal nerve sliding in the forearm and upper arm during 40 degrees wrist extension, 90 degrees elbow flexion and 50 degrees shoulder abduction. Nerve trunk folding in the upper arm was measured from still ultrasound images taken in a series of limb positions from 40 degrees shoulder abduction, elbow extended and wrist neutral to full elbow flexion, 90 degrees shoulder abduction and wrist extension, a position designed to stretch the ulnar nerve. FINDINGS: Wrist extension led to clear nerve sliding in the forearm with movements of up to 4 mm. However, shoulder abduction and elbow flexion caused remarkably little nerve movement. Images of the ulnar nerve showed considerable curvature with 40 degrees shoulder abduction and elbow extension but a much straighter path with the elbow flexed. INTERPRETATION: The ulnar nerve appears unloaded and follows a wavy path in most functional upper limb positions. During elbow and shoulder movements, changes in bed length appear to be accommodated largely by straightening of the nerve path, with only modest stretch of the nerve itself when the elbow flexes. The ulnar nerve is thus well adapted for the large changes in bed length that occur during limb movements.

In vivo study of nerve movement and mechanosensitivity of the median nerve in whiplash and non-specific arm pain patients.

Chronic pain following whiplash injury and non-specific arm pain (NSAP, previously termed diffuse repetitive strain injury) present clinicians with problems of diagnosis and management. In both patient groups there are clinical signs of altered nerve movement and increased nerve trunk mechanosensitivity. Previous studies of NSAP patients have identified altered median nerve movement at the wrist. The present study uses high frequency ultrasound imaging to examine changes to median nerve movement and clinical examination to assess altered mechanosensitivity of the median nerve. Longitudinal median nerve movement was measured in the forearm during maximal inspiration in nine post-whiplash patients with chronic neck and arm pain and eight controls subjects. Eight NSAP patients and seven controls were also studied. Transverse median nerve movement at the proximal carpal tunnel during 30 degrees wrist extension to 30 degrees flexion was also measured. A clinical examination of nerve trunk allodynia was performed in all subjects. Longitudinal nerve movement in the forearm was reduced by 71% in the post-whiplash patients and by 68% in NSAP patients compared to controls. In the whiplash patients the pattern of transverse median nerve movement at the proximal carpal tunnel was significantly different to controls (patient mean=2.57+/-0.80 mm (SEM) in a radial direction; control mean=0.39+/-0.52 mm in an ulnar direction). Signs of neural mechanosensitivity (i.e. painful responses to median nerve trunk and brachial plexus pressure and stretch) were apparent in both patients groups. Change in nerve tension and neural mechanosensitivity may contribute to symptoms in whiplash and NSAP patients.

Pressure and stretch mechanosensitivity of peripheral nerve fibres following local inflammation of the nerve trunk.

Patients with non-specific limb pain often show signs of nerve mechanosensitivity, i.e. local tenderness over nerve trunks and pain in response to limb movements that cause nerve stretch. In such patients a nerve lesion is not apparent, and it has been suggested that local neural inflammation may be a key factor. The present study examines the extent to which nerve fibres in regions of local inflammation respond to small stretches, and whether functional changes occur throughout the primary afferent neurone. A local neuritis was induced in adult rats by wrapping oxidised cellulose saturated in complete Freund's adjuvant (CFA) around the peroneal or sciatic nerves. A small cut was made in the perineurium of some of the peroneal lesioned animals. A- and C-fibre recordings were made 2-10 days post-surgery from filaments dissected proximal to the lesion. Local mechanosensitivity was assessed using a glass probe and by small stretches. Responses to stretch and local pressure were recorded in 7% of C- and 8% of A-fibres from the peroneal nerve following CFA treatment with the sheath opened. A smaller proportion of stretch sensitive fibres were seen in sciatic and peroneal nerves after CFA treatment alone (2% of C- and 3% of A-fibres), but such fibres were not seen in control preparations. The most responsive fibres fired to 3% stretch, which is within the range of nerve stretch seen during normal limb movements. Less than 1% of stretch sensitive fibres had peripheral fields, indicating that most had probably degenerated distally.

C-fibre Function During the 6 Weeks Following Brief Application of Capsaicin to a Cutaneous Nerve in the Rat.

The functional properties of afferent fibres, especially C-fibres, have been studied over the 46 days following application of capsaicinoids to a segment of the saphenous nerve in the rat. After 1 - 3 days, approximately half of the C-afferents were found to be non-conducting at or immediately distal to the treatment site. By 6 - 9 days there was some recovery of conduction at the treatment site, but few of the conducting fibres were excitable from the skin. From 12 days onwards, C-fibre conduction in the segment of nerve both proximal and distal to the treatment site was reduced and of the conducting fibres relatively few had cutaneous receptive fields. In the sample of C-fibres with cutaneous receptive fields the proportion of polymodal nociceptors was reduced markedly compared with control values. Analysis of the numbers of surviving units shows a large effect on C-polymodal nociceptors, but no significant change in the numbers of any other type of afferent unit. At 2 weeks, when C-fibre numbers were reduced to about one third, there was a virtual abolition of antidromic vasodilatation and an 85% fall in skin substance P levels. The synthetic capsaicinoid, NE-21610, had similar actions to capsaicin and was more potent in depleting skin substance P levels. These results confirm that capsaicinoids produce a selective local lesion on nociceptive C-fibres in rat cutaneous nerves. In addition, the transient and partial nature of functional recovery indicates that capsaicin may also cause a longer-term toxic action that inhibits significant regeneration.

Long-Term Reduction in the Number of C-Fibre Nociceptors Following Capsaicin Treatment of a Cutaneous Nerve in Adult Rats.

The structure and function of C- and A-fibres have been studied 3 - 12 months following a single 30 min exposure of the saphenous nerve of adult rats to 1% capsaicin. Examination of nerve cross-sections showed that the number of C-fibres was reduced by 36%, but A-fibres were unaffected. The remaining C-fibres included many that were unusually small in size. The changes were not restricted to the treatment site but were found over at least a further 10 mm proximal and distal to it. In treated nerves, the C-fibre component of the compound action potential was reduced in size relative to the Aalphabeta component. Single unit studies revealed that an unusually high proportion of C-fibres had no cutaneous receptive field (54%, compared with 28% in controls). There was no such change for A-fibres. The conduction velocity range for C-fibre units in treated nerves was almost normal, but this sample contained an unusually low proportion of polymodal nociceptors. Allowing for the fall in total numbers and the reduction in units with cutaneous receptive fields, it was calculated that overall the numbers of C-polymodal nociceptor units had fallen by 74%. No significant changes in number had occurred in the other classes of C-afferents. The reduction in the numbers of nociceptive C-fibres is likely to be the direct cause of the reduction in pain responses and in neurogenic inflammation that several groups have reported following treatment of adult rats with capsaicin. Reduction in the numbers of C-fibre is also likely to be the reason for reduced neuropeptide levels seen in tissues innervated by treated nerves.

Connexin29 and connexin32 at oligodendrocyte and astrocyte gap junctions and in myelin of the mouse central nervous system.

The cellular localization, relation to other glial connexins (Cx30, Cx32, and Cx43), and developmental expression of Cx29 were investigated in the mouse central nervous system (CNS) with an anti-Cx29 antibody. Cx29 was enriched in subcellular fractions of myelin, and immunofluorescence for Cx29 was localized to oligodendrocytes and myelinated fibers throughout the brain and spinal cord. Oligodendrocyte somata displayed minute Cx29-immunopositive puncta around their periphery and intracellularly. In developing brain, Cx29 levels increased during the first few postnatal weeks and were highest in the adult brain. Immunofluorescence labeling for Cx29 in oligodendrocyte somata was intense at young ages and was dramatically shifted in localization primarily to myelinated fibers in mature CNS. Labeling for Cx32 also was localized to oligodendrocyte somata and myelin and absent in Cx32 knockout mice. Cx29 and Cx32 were minimally colocalized on oligodendrocytes somata and partly colocalized along myelinated fibers. At gap junctions on oligodendrocyte somata, Cx43/Cx32 and Cx30/Cx32 were strongly associated, but there was minimal association of Cx29 and Cx43. Cx32 was very sparsely associated with astrocytic connexins along myelinated fibers. With Cx26, Cx30, and Cx43 expressed in astrocytes and Cx29, Cx32, and Cx47 expressed in oligodendrocytes, the number of connexins localized to gap junctions of glial cells is increased to six. The results suggested that Cx29 in mature CNS contributes minimally to gap junctional intercellular communication in oligodendrocyte cell bodies but rather is targeted to myelin, where it, with Cx32, may contribute to connexin-mediated communication between adjacent layers of uncompacted myelin.

Capsaicin: actions on nociceptive C-fibres and therapeutic potential.

Recent work on the excitatory action of capsaicin on somatic and visceral afferent neurones shows that depolarization is selective for C-fibre polymodal nociceptor afferents and involves opening a non-selective cation channel. Exposure to significantly suprathreshold amounts of capsaicin causes permanent degeneration of C-fibre afferents in adult rats. Functional changes in rats (hypalgesia, diminished neurogenic inflammation) are likely to be a direct consequence of the loss of C-fibre nociceptors, and so are the reductions in neuropeptide levels that follow adult capsaicin treatment. Clinical trials of topical capsaicin treatment for post-herpetic neuralgia have yielded promising results. The selective nature of the action of capsaicin in reducing just C-nociceptor activity may make it particularly useful for treating pain states triggered by C-fibre input.

A comparison of four tests for assessing the pain sensitivity of different subjects and test areas.

Four different testing methods were used to estimate the sensitivity of body surfaces to pain-causing stimuli for a group of 24 healthy male subjects. These methods were (a) determination of the heat pain threshold, (b) determination of the pain threshold for pinching of a skin fold, (c) discrimination of a sharp prick from a blunt contact, and (d) estimation of the pain caused by application of an ice-cold cylinder. Three different body regions were tested: the abdomen, the anterior surface of the neck and the lateral surface of the thigh. Of the three body regions, the thigh area required the strongest stimulation for pain in all 4 tests. The neck was the most sensitive for cold pain, and the abdomen had the lowest threshold for heat pain. There was a barely statistically significant tendency for subjects relatively sensitive on one test to be also relatively sensitive on other tests. Correlation coefficients between any two tests on the same subject were always less than 0.6. Possible reasons for the relative lack of agreement among the results of the different pain tests are discussed.

Sensory and autonomic function in the hands of patients with non-specific arm pain (NSAP) and asymptomatic office workers.

Chronic pain in the upper limb associated with repetitive movements of the arm and hand is often seen in patients in the absence of specific pathology such as epicondylitis, carpal tunnel syndrome and tenosynovitis. This condition has been given many names and will be referred to here as non-specific arm pain (NSAP). Previous work has shown elevated vibration thresholds and reduced flare suggesting a neuropathic cause for this condition. In order to examine this further the present study has assessed functions that involve both large sensory nerve fibres (vibration) and small dorsal root fibres (flare) and sympathetic fibres (vasoconstriction). NSAP patients and also a group of office workers who intensively used display screen equipment but who did not have NSAP were also studied along with an age-matched control group. In the median innervated area of the hand we measured flare responses to iontophoresis of histamine (a sensory C-fibre effect), and sympathetic vasoconstrictor responses to ice stimulation over C7. To tie in with previous studies, vibration threshold over areas of the hand innervated by the median, ulnar and radial nerves was also evaluated. All measures were carried out over both hands in each subject. Significant differences from controls were found on all three measures for the NSAP patient group and on two measures for the office workers. Flare area was reduced by 33% in the patients and by 30% in the office workers. Reflex vasoconstriction was reduced by 20% in the patient group but was not altered in office worker group. Over the median innervated area on the hand, vibration threshold was elevated by 47% in the patients and by 21% in the office workers. These results confirm previous findings showing reduced function associated with both small and large sensory fibres in the NSAP patients and additionally, for the first time, demonstrate a functional change related to sympathetic fibres. Office workers demonstrate a similar but smaller trend for reduced nerve function associated with both small and large sensory fibre function, but had no change in the sympathetic reflex.

Shoulder posture and median nerve sliding.

BACKGROUND: Patients with upper limb pain often have a slumped sitting position and poor shoulder posture. Pain could be due to poor posture causing mechanical changes (stretch; local pressure) that in turn affect the function of major limb nerves (e.g. median nerve). This study examines (1) whether the individual components of slumped sitting (forward head position, trunk flexion and shoulder protraction) cause median nerve stretch and (2) whether shoulder protraction restricts normal nerve movements. METHODS: Longitudinal nerve movement was measured using frame-by-frame cross-correlation analysis from high frequency ultrasound images during individual components of slumped sitting. The effects of protraction on nerve movement through the shoulder region were investigated by examining nerve movement in the arm in response to contralateral neck side flexion. RESULTS: Neither moving the head forward or trunk flexion caused significant movement of the median nerve. In contrast, 4.3 mm of movement, adding 0.7% strain, occurred in the forearm during shoulder protraction. A delay in movement at the start of protraction and straightening of the nerve trunk provided evidence of unloading with the shoulder flexed and elbow extended and the scapulothoracic joint in neutral. There was a 60% reduction in nerve movement in the arm during contralateral neck side flexion when the shoulder was protracted compared to scapulothoracic neutral. CONCLUSION: Slumped sitting is unlikely to increase nerve strain sufficient to cause changes to nerve function. However, shoulder protraction may place the median nerve at risk of injury, since nerve movement is reduced through the shoulder region when the shoulder is protracted and other joints are moved. Both altered nerve dynamics in response to moving other joints and local changes to blood supply may adversely affect nerve function and increase the risk of developing upper quadrant pain.

Quantitative in vivo studies of median nerve sliding in response to wrist, elbow, shoulder and neck movements.

OBJECTIVE: To examine median nerve sliding in response to upper limb movements in vivo. To determine whether the median nerve can be unloaded. DESIGN: Exploratory study in healthy subjects.Background. Impaired sliding may lead to neuropathic symptoms. In vivo results for neural dynamics in normal subjects are essential to understand changes in upper limb disorders. METHODS: Ultrasound imaging of the median nerve during 40 degrees wrist extension, 80 degrees shoulder abduction, 90 degrees elbow extension, and 35 degrees contralateral neck side flexion. Frame by frame cross-correlation of image sequences to measure nerve sliding and strain. RESULTS: Nerve excursion in the forearm and upper arm ranged from 0.3 mm for neck side flexion to 10.4 mm for elbow extension. Additional strain in the forearm for wrist extension was 1.1% (SEM, 0.2%), for shoulder abduction 1.0% (SEM, 0.2%), and for neck side flexion 0.1% (SEM, 0.1%). With the limb flexed, sliding was delayed and sometimes the nerve or the nerve fascicles had a wavy appearance. CONCLUSION: The median nerve is unloaded when the shoulder is adducted or elbow flexed. When the arm is extended (90 degrees shoulder abduction, 60 degrees wrist extension, and elbow straight) the total additional strain in the forearm will be 2.5-3.0%. Even in this position the strain is likely to be below levels that impair blood flow or conduction. Therefore, the median nerve appears well designed to cope with changes in bed length caused by limb movements. RELEVANCE: These results will provide baseline data that can be used to examine entrapment neuropathies.

Molecular and functional asymmetry at a vertebrate electrical synapse.

Electrical synapses are abundant in the vertebrate brain, but their functional and molecular complexities are still poorly understood. We report here that electrical synapses between auditory afferents and goldfish Mauthner cells are constructed by apposition of hemichannels formed by two homologs of mammalian connexin 36 (Cx36) and that, while Cx35 is restricted to presynaptic hemiplaques, Cx34.7 is restricted to postsynaptic hemiplaques, forming heterotypic junctions. This molecular asymmetry is associated with rectification of electrical transmission that may act to promote cooperativity between auditory afferents. Our data suggest that, in similarity to pre- and postsynaptic sites at chemical synapses, one side in electrical synapses should not necessarily be considered the mirror image of the other. While asymmetry based on the presence of two Cx36 homologs is restricted to teleost fish, it might also be based on differences in posttranslational modifications of individual connexins or in the complement of gap junction-associated proteins.

Longitudinal sliding of the median nerve in patients with non-specific arm pain.

In patients with non-specific arm pain (NSAP; also known as repetitive strain injury), there are clinical signs of altered median nerve sliding. It is possible that a restriction along the nerve course will lead to abnormal increases in local strain during limb movements, possibly contributing to symptoms. The present study uses ultrasound imaging to examine median nerve sliding through the proximal and distal nerve segments in 18 NSAP patients. Longitudinal nerve sliding was measured during metacarpophalangeal, wrist and elbow movements. During elbow movements, the angle of elbow extension at which the nerve begins to move was determined, since this was expected to decrease with a restriction through the shoulder. The results from this study were compared with previously reported data. Nerve movements ranged from 1.26 to 4.73 mm in patients compared with 1.43-5.57 mm in controls. There was no significant difference in nerve sliding (p>0.05) or in the angle of elbow extension at which the nerve began to move (mean=53.4 degrees in patients, 52.0 degrees in controls; p>0.05). In summary, restriction of median nerve sliding is unlikely to play a major role in NSAP. Therefore, painful responses during limb movements which tension the nerve are unlikely to result from abnormal increases in nerve strain.