Role of lipocalin 2 and its complex with matrix metalloproteinase-9 in oral cancer.

OBJECTIVES: Recent evidence demonstrated that lipocalin (LCN)2 is induced in many types of human cancer, while the detection of its complex with matrix metalloproteinase (MMP)-9 is correlated with the cancer disease status. We attempted to evaluate plasma expressions of LCN2, MMP-9, and their complex (LCN2/MMP-9) during the diagnostic work-up of patients with oral squamous cell carcinoma (OSCC) and investigated their correlations with disease progression. METHODS: In total, 195 patients with OSCC and 81 healthy controls were recruited. Expression levels of LCN2, MMP-9, and LCN2/MMP-9 were determined with immunoenzymatic assays. RESULTS: Patients with OSCC exhibited significantly higher levels of LCN2, MMP-9, and LCN2/MMP-9 compared with healthy controls (LCN2: P < 0.001; MMP-9: P < 0.001; LCN2/MMP-9: P < 0.01). Plasma levels of LCN2, MMP-9, and LCN2/MMP-9 in patients with OSCC were significantly correlated with each other and were associated with more-advanced clinical stages (P < 0.05) and/or a larger tumor size (P < 0.05), but were not associated with positive lymph-node metastasis or distal metastasis. CONCLUSION: Our results suggest that plasma levels of LCN2 and the LCN2/MMP-9 complex may be useful in non-invasively monitoring OSCC progression, while supporting their potential role as biomarkers of oral cancer disease status.

Regeneration of the active zone at the frog neuromuscular junction.

The active zone is a unique specialization of the presynaptic membrane and is believed to be the site of transmitter release. The formation of the active zone and the relationship of this process to transmitter release were studied at reinnervated neuromuscular junctions in the frog. At different times after a nerve crush, the cutaneous pectoris muscles were examined with intracellular recording recording and freeze-fracture electron microscopy. The P face of a normal active zone typically consists of two double rows of particles lined up in a continuous segment located opposite a junctional fold. In the initial stage of reinnervation, clusters of large intramembrane particles surrounding membrane elevations appeared on the P face of nerve terminals. Like normal active zones, these clusters were aligned with junctional folds. Vesicle openings, which indicate transmitter release, were seen at these primitive active zones, even though intramembrane particles were not yet organized into the normal pattern of two double rows. The length of active zones at this stage was only approximately 15% of normal. During the secondary stage, every junction was reinnervated and most active zones had begun to organize into the normal pattern with normal orientation. Unlike normal, there were often two or more discontinuous short segments of active zone aligned with the same junctional fold. The total length of active zone per junctional fold increased to one-third of normal, mainly because of the greater number of segments. In the third stage, the number of active zone segments per junctional fold showed almost no change when compared with the secondary stage. However, individual segments elongated and increased the total length of all active zone segments per junctional fold to about two-thirds of the normal length. The dynamic process culminated in the final stage, during which elongating active zones appeared to join together and the number of active zone segments per junctional fold decreased to normal. Thus, in most regions, regeneration of the active zones was complete. These results suggest that the normal organization of two double rows is not necessary for the active zone to be functional. Furthermore, localization of regenerating active zones is related to junctional folds and/or their associated structures.

Impulse blockade in frog cardiac ganglion does not resemble partial denervation in changing synaptic organization.

Partial denervation of parasympathetic neurons in the frog heart by surgical section of one vagus nerve results in a marked reorganization of functional synaptic connections made by the remaining vagus nerve. These changes are not simply due to a lack of impulse activity per se in the sectioned nerve because blockage of impulses in one vagus with tetrodotoxin-impregnated cuffs did not cause similar changes in the innervation pattern of the ganglion. Furthermore, tetrodotoxin-blocked vagal fibers retain their ability to sprout and can form new synapses on denervated neurons.

Intracranial pressure fluctuation during hemodialysis in renal failure patients with intracranial hemorrhage.

Coagulopathy in renal failure patients often makes them vulnerable to intracranial hemorrhage. Emergency decompression to remove the hematoma and to stop bleeding is always indicated. After the surgery, hemodialysis (HD) should be arranged to maintain the BUN/Cr. level, and I/O balance. During HD, intracranial pressure in all of the patients in this study fluctuated. This phenomenon always resulted in neurological deterioration in acute or chronic renal failure. We present intracranial pressure (ICP) changes during HD in five acute or chronic renal failure patients with intracranial hemorrhage. They all underwent craniectomy or craniotomy with ICP monitors implantation. Different HD protocols were arranged for these patients and then we observed clinical results. ICP elevated during HD and resulted in severe brain swelling. This situation was one of the clinical presentations of dialysis disequilibrium syndrome (DDS). Four patients died because of this complication and one survived. ICP fluctuation seemed to be correlated with the fluid amount and frequency of HD. The prevalence and pathophysiology of DDS remain unclear. Renal failure patient with intracranial hemorrhage may be complicated with DDS when HD was performed. An attempt to reduce the fluid amount and to increase the frequency of HD might help these patients.

Effect of hyperbaric oxygen on patients with traumatic brain injury.

Hyperbaric oxygen therapy (HBOT) is the medical therapeutic use of oxygen at a higher atmospheric pressure. The United States Food and Drug Administration have approved several clinical applications for HBOT, but HBOT in traumatic brain injury (TBI) patients has still remained in controversial. The purpose of our study is to evaluate the benefit of HBOT on the prognosis of subacute TBI patients. We prospectively enrolled 44 patients with TBI from November 1, 2004 to October 31, 2005. The study group randomly included 22 patients who received HBOT after the patients' condition stabilization, and the other 22 corresponding condition patients were assigned into the matched control group who were not treated with HBOT. The clinical conditions of the patients were evaluated with the Glasgow Coma Scale (GCS) and Glasgow Outcome Scale (GOS) before and 3 to 6 months after HBOT. The GCS of the HBOT group was improved from 11.1 to 13.5 in average, and from 10.4 to 11.5 (p < 0.05) for control group. Among those patients with GOS = 4 before the HBOT, significant GOS improvement was observed in the HBOT group 6 months after HBOT. Based on this study, HBOT can provide some benefits for the subacute TBI patients with minimal adverse side effects.

Schwann cells express active agrin and enhance aggregation of acetylcholine receptors on muscle fibers.

To explore novel roles of glial cells in synaptic function and formation, we examined the expression of agrin in frog Schwann cells and tested their role in the aggregation of acetylcholine receptors (AChRs). Using reverse transcription-PCR, we found that Schwann cells along nerve fibers in tadpoles expressed only the inactive agrin isoform B0 but began to also express active agrin isoforms B11 and B19 at approximately metamorphosis. During nerve regeneration in the adult, the expression of these active agrin isoforms in Schwann cells was upregulated, including the appearance of the most potent isoform, B8. This upregulation was induced by regenerating axons but not by nerve injury per se. In muscle cultures, the presence of adult Schwann cells enhanced the number and the total area of AChR aggregates 2.2- and 4.5-fold, respectively, and this enhancement was eliminated by heparin treatment. Furthermore, adult Schwann cells in culture expressed active agrin isoforms and produced agrin protein. Using a novel technique to selectively ablate perisynaptic Schwann cells (PSCs) at the neuromuscular junction, we found that PSCs also expressed active agrin isoforms B11 and B19, and these active isoforms were upregulated, including the appearance of B8, during reinnervation. Observation in vivo showed that extrajunctional AChR aggregates were associated with PSC sprouts after nerve injury and subsequent reinnervation. These results suggest that, contrary to the prevailing view that only neurons express active agrin, glial cells also express active agrin and play a role in the aggregation of AChRs both in vitro and in vivo.

PTX-sensitive and -insensitive synaptic modulation at the frog neuromuscular junction.

Pharmacological manipulations were used to examine the role of G proteins in modulating synaptic transmission at the frog neuromuscular junction. Pertussis toxin (PTX, a G protein antagonist) increased end-plate potential (epp) amplitude but had no effect on the amplitude or frequency of miniature end-plate potentials. Mastoparan (a G protein agonist) decreased epp amplitude, while suramin (an antagonist) increased epp amplitude. The results suggest that PTX-sensitive G proteins tonically modulate synaptic transmission by reducing the amount of transmitter released in response to presynaptic action potentials. We also showed that endogenous ATP decreased transmitter release via P2 receptor in a PTX-insensitive manner. Thus, at least two distinct mechanisms regulate neuromuscular transmission; one is coupled to PTX-sensitive G proteins and the other is not.

Synaptic transmission between rat spinal cord explants and dissociated superior cervical ganglion neurons in tissue culture.

Physiological properties of the synapses formed between explants of spinal cord and dissociated autonomic ganglion neurons in tissue culture were studied using intracellular and extracellular stimulation and recording techniques (as well as iontophoresis) with a culture perfusion system allowing continuous microscopic observation during repeated changes of the bathing medium. The principal neurons of the superior cervical ganglion (SCGN) were dissociated from perinatal rats and the spinal cord explants were obtained from 15-day rat fetuses; these were allowed to mature for 3-10 weeks in co-culture. Recordings from over 1000 SCGN established that: (a) spontaneous small depolarizations and action potentials occurred in 20% of the SCGN studied, (b) the EPSPs observed in SCGN after spinal cord stimulation were sensitive to decreased Ca2+ and increased Mg2+, as well as to D-tubocurare, hexamethonium and mecamylamine, but not to atropine (at 10(-6) M concentration) or to the alpha-adrenergic blocking agents phentolamine or phenoxybenzamine; no potentiation of the EPSPs was seen with neostigmate or eserine, (c) acetylcholine directly applied to the SCGN was seen to mimic the responses seen after spinal cord stimulation; tetrodotoxin blocked both direct and iontophoretically fired action potentials, with only a suprathreshold acetylcholine potential remaining. These synapses were not sensitive to alpha-bungarotoxin. It is concluded that the synapses formed by spinal cord neurites on principal SCGN in tissue culture are nicotinic cholinergic, and that the evoked EPSPs recorded in this study are thus similar to the orthodromic fast EPSPs observed in vivo. No slow synaptic responses were observed and no demonstrable effects were noted that could be attributed to adrenergic transmission.

Synaptic transmission between rat superior cervical ganglion neurons in dissociated cell cultures.

The principal neurons of the rat superior cervical ganglion (SCGN) when established as dissociated cells in tissue culture form synapses among themselves. In the present study we have examined this synaptic interaction when these neurons are co-cultured with several other types of tissues. Dissociated SCGN were prepared from perinatal rats and studied, after 3-4 weeks maturation, with intracellular recording techniques. Synaptic interactions between sympathetic neurons were demonstrated when these cells were: (a) grown with explants from newborn rat thoracic spinal cord, (b) when the SCGN had survived for several weeks subsequent to removal of the spinal cord explants, and (c) when the SCGN were grown in the presence of an adrenergic target (interscapular brown fat cells). Unidirectional, reciprocal, recurrent and complex chemical synaptic networks, consisting of convergence and divergence, characterized connections between SCGN. All synaptic responses were cholinergic since they were reversibly blocked by hexamethonium or mecamylamine but were not sensitive to 10(-5) M phenoxybenzamine. Removal of the spinal cord explants did not significantly alter the proportion of chemical synaptic interactions between SCGN (more than 25%) from matched cultures. Anatomical observations established that in cultures with brown fat, innervating neurites appeared on the fat cells; these neurites frequently expanded to form varicosities that resembled the adrenergic terminals normally seen on brown fat in the animal. Synaptic profiles also occurred on the neurons in these cultures and some of these were shown to be cholinergic. The proportion of neuronal interactions in the combined SCGN + fat cultures was low, however, suggesting that co-culture with target tissue might influence the frequency of interconnections developed between SCGN in culture. Other factors, such as the presence of non-neuronal cells, degree of dissociation, cellular density, culture age and the survival of certain types of SCGN in culture are discussed as variables related to the formation of synapses between SCGN. Non-rectified electrical coupling between SCGN was also observed in 17 out of 679 pairs (2.5%) of neurons. Attenuation factor for electrically coupled action potentials ranged between 1 and 43.5.

Induction of synaptic extracellular matrix molecules at ectopic neuromuscular junctions.

The induction of synapse-specific molecules recognized by peanut agglutinin (PNA) was examined at ectopic neuromuscular junctions in adult frog muscles using light and electron microscopy. In normal frog muscles, PNA specifically recognizes the extracellular matrix at neuromuscular junctions but not at extrajunctional regions. This report shows binding of PNA at ectopic neuromuscular junctions which were initially extrasynaptic and hence unrecognized by PNA. Results suggest that synapse-specific extracellular matrix molecules can be induced de novo at new junctional sites.

A lectin, peanut agglutinin, as a probe for the extracellular matrix in living neuromuscular junctions.

The extracellular matrix plays important roles in the differentiation of synapses. To identify molecules concentrated specifically in the synaptic extracellular matrix, fluorescently-labelled lectins were applied to neuromuscular junctions. A lectin, peanut agglutinin (PNA), stains the neuromuscular region selectively and irreversibly (up to at least 3 weeks in situ), outlining the periphery of the nerve terminal arborization in the frog. Snake neuromuscular junctions also stain intensely with fluorescent PNA, while mouse diaphragm staining is faint. At the electron microscopic level, the reaction products of horseradish peroxidase-conjugated PNA are found primarily in the extracellular matrix flanking Schwann cells in the frog endplate regions. Fluorescently labelled PNA does not affect synaptic potentials and can serve as a simple stain for correlating functional studies of living neuromuscular junctions. Moreover, it can be combined with a presynaptic dye to observe nerve terminals and synaptic extracellular matrix in the same junctions in situ. This report reveals the existence of synapse-specific carbohydrates associated with Schwann cell extracellular matrix in the frog neuromuscular junction. The specific binding and its physiological compatibility make PNA a useful probe for further investigation of synaptic differentiation, plasticity and maintenance.

Electrophysiological and freeze-fracture studies of changes following denervation at frog neuromuscular junctions.

1. Changes which occur at frog neuromuscular junctions following denervation have been studied by combining intracellular recording and freeze-fracture electron microscopy. 2. Shortly after nerve section, both neuromuscular transmission and intramembrane structures of neuromuscular junctions remain normal. 3. Later, neuromuscular transmission fails, beginning with the disappearance of end-plate potentials (e.p.p.s) and followed by the disappearance of miniature end-plate potentials (m.e.p.p.s). The frequency of m.e.p.p.s which persist after cessation of e.p.p.s is not increased dramatically in K+-rich or hypertonic solutions. 4. Concomitant with the changes of transmission are changes in intramembrane structures. The first sign of these changes in disruption of active zones, which become disorganized, fragmented or vanish. Nerve terminals then disintegrate and eventually are engulfed by Schwann cells. 5. When neuromuscular transmission has failed completely, former sites of the neuromuscular junction are occupied by Schwann cells. These cells develop transverse ridges which lie opposed to junctional folds, just like active zones of nerve terminals. However, the ridges on Schwann cells do not contain organized rows of particles or clusters of any synaptic organelles, even at later stages when Schwann cell m.e.p.p.s commence. 6. It is suggested that the failure of e.p.p.s involves at least an impairment of the transmitter release mechanism at the nerve terminal, which is probably associated with the disruption of active zones. The cessation of m.e.p.p.s is thought to be caused by the engulfment of terminals by Schwann cells.

Formation of the active zone at developing neuromuscular junctions in larval and adult bullfrogs.

Development of the presynaptic active zone was studied at neuromuscular junctions with freeze-fracture electron microscopy in larval and adult bullfrogs. In rudimentary larval neuromuscular junctions, clusters of active zone particles were scattered over the P-face of the presynaptic membrane. Vesicle openings were observed at these terminals even though active zone particles lacked the mature pattern of two double rows. Gradually, active zone particles became organized into rows, but they were still randomly located and oriented. Once junctional folds were observed in replicas, developing active zones were located opposite to the folds, as in mature terminals. Multiple terminals occupying the same junctional gutters were also observed. At the end of metamorphosis, most active zones were still immature in appearance and had only grown to one third of their mature length. After metamorphosis, the number of active zone segments aligned at the same junctional fold increased. These discontinuous short active zones then elongated, joined together, and finally formed the mature active zones. Signs suggesting synapse elimination such as disorganization of active zones, absence of intramembrane particles in varicosities, and exposed muscle membranes with patches of acetylcholine receptor aggregates were observed. In some multiply innervated junctions, one terminal had mature active zones with vesicle openings, the other in the same gutter displayed disorganized active zones without vesicle openings, although both terminals showed similar sizes and distributions of background particles. This study suggests that developing active zones, as is the case for regenerating active zones in the adult, are functional before the mature organization is formed. The sequence of development of active zones is also similar to that of regeneration except for the random location and orientation of early active zones in tadpoles. The comparison between regeneration and development further indicates that the process of active zone formation is related to junctional folds and/or associated structures. It is also suggested that synapse elimination may involve degenerative changes in presynaptic membranes, although direct evidence remains to be provided.

Disruption of active zones in frog neuromuscular junctions following treatment with proteolytic enzymes.

Frog neuromuscular junction treated with proteolytic enzymes to remove the basal lamina were studied with freeze-fracture techniques in order to examine the influence of the basal lamina in the maintenance of active zone ultrastructure. The active zone is believed to be the site of transmitter release and has a unique membrane organization and location in the neuromuscular junction. After removal of the basal lamina by successive treatment of 0.01% collagenase and 0.1% protease for 1 h each, active zone disruption was observed. Some active zones became segmented, and some were also randomly located and oriented, but they still had normal double-row particle organization. Others contained only clusters of large intramembrane particles. These disorganized active zones were still functional as indicated by the presence of vesicle openings. Some enzyme-treated junctions were also exposed to the membrane cholesterol probe, filipin, to examine the expression of membrane lipid heterogeneity in disrupted active zones. As in normal active zones, filipin-sterol complexes were absent. The densities of background particles in the presynaptic membranes and of large particles thought to be acetylcholine receptors were not significantly altered by the enzyme treatment. Although a direct effect of the enzymes on active zone ultrastructure can not be totally excluded, the present work is consistent with a maintenance role of the basal lamina in active zone organization and location.

Correlations between active zone ultrastructure and synaptic function studied with freeze-fracture of physiologically identified neuromuscular junctions.

The active zone is a unique presynaptic membrane specialization that is believed to be the site of neurotransmitter release. To examine directly the relationship between active zone ultrastructure and synaptic efficacy, frog neuromuscular junctions were studied with a new technique combining electrophysiology, light microscopy, and freeze-fracture of identified single muscle fibers. This technique allows correlations to be made between quantal content (measured in low Ca2+ and high Mg2+ Ringer solution), endplate size, and active zone structure at the same neuromuscular junctions. By measuring physiological and morphological variables at the same junctions, the validity of structure-function correlations is significantly improved. Synaptic quantal content in 91 physiologically identified muscle fibers varied considerably and was only poorly correlated with endplate size, as shown in previous studies. To measure the total length of endplate branches, either a modified cholinesterase stain or rhodamine-labeled peanut agglutinin stain was used. When the same identified muscle fibers were freeze-fractured, active zones were exposed in 17 junctions. In a replica that contained a large part of one nerve terminal, there was no detectable gradient in active zone structure along the length of 3 different nerve terminal branches identifiable with both light and electron microscopy. The results from these 17 identified junctions indicate that quantal content per unit terminal length is positively correlated with the amount of active zone per unit terminal length. The estimated total active zone length and total number of active zone particles per junction are also positively correlated with the quantal content in these identified junctions. This study suggests that active zone size and spacing are better indicators of transmitter release than is endplate size and that the active zone may play an important role in regulating synaptic efficacy at the neuromuscular junction.

Novel modulatory effect of L-type calcium channels at newly formed neuromuscular junctions.

This study aimed to examine changes of presynaptic voltage-sensitive calcium channel (VSCC) subtypes during synapse formation and regeneration in relation to transmitter release at the neuromuscular junction (NMJ). Synaptic potentials were recorded from developing rat NMJs and from regenerating mouse and frog NMJs. As in normal adult NMJs, evoked transmitter release was reduced by an N-type VSCC blocker in the frog and by a P/Q-type VSCC blocker in the mammal at immature NMJs; however, various L-type VSCC blockers, both dihydropyridine and nondihydropyridine antagonists, increased evoked but not spontaneous release in a dose-dependent manner at newly formed NMJs. This presynaptic potentiation disappeared as NMJs matured. A rapid intracellular Ca2+ buffer, bis(O-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM, prevented the potentiation effect of nifedipine, but a slow Ca2+ buffer, EGTA-AM, did not. Thus, the potentiation effect of L-type blockers requires Ca2+ transients. Pretreatment with Ca2(+)-activated K+ channel blockers, iberiotoxin or charybdotoxin, did not prevent potentiation by nifedipine at regenerating frog NMJs. Thus, Ca(2+)-activated K+ channels were not likely involved in this potentiation. In contrast, no additional potentiation by nifedipine was seen in muscles pretreated with pertussis toxin (PTX), a G-protein blocker, which by itself enhances evoked transmitter release at regenerating frog NMJs. These results suggest the existence of multiple subtypes of VSCCs at newly formed motor nerve terminals. In addition to the normal N- or P/Q-type VSCCs that mediate transmitter release, L-type VSCCs may play a novel modulatory role in evoked transmitter release by activating a mechanism linked to PTX-sensitive G-proteins during synapse maturation.

Extension of synaptic extracellular matrix during nerve terminal sprouting in living frog neuromuscular junctions.

Remodeling of the synaptic extracellular matrix (ECM) and its dynamic relationship with nerve terminal plasticity have been demonstrated in normal frog neuromuscular junctions (NMJs) in vivo (Chen et al., 1991). Our previous work has led to a hypothesis that extension of synaptic ECM precedes nerve terminal growth during synaptic remodeling. To test this hypothesis, the present study examined the changes of synaptic ECM in frog NMJs that were primarily undergoing nerve terminal growth and sprouting. Frog sartorius muscles were double stained with a fluorescent nerve terminal dye (4-Di-2-Asp) and rhodamine-tagged peanut agglutinin (PNA), which recognizes synaptic ECM. The double-labeled NMJs were visualized in vivo with video-enhanced fluorescence microscopy. Nerve sprouting was then induced in the muscle by grafting segments of the contralateral sciatic nerve. The identified NMJs were restrained and reexamined 2-3 months later. Extensive sprouting was observed in 46% of 167 identified NMJs. At junctional regions that showed extension or formation of new branches, synaptic ECM was commonly seen to have the same shape and distribution as the nerve terminal. However, extension of synaptic ECM beyond the corresponding nerve terminals, often by tens of microns, was observed in 29% of these newly formed junctional regions. This lack of correlation might be transient, as growth of nerve terminals following extended, PNA-stained ECM was seen. Examination with histological staining not only confirmed a lack of nerve terminal at the extended synaptic ECM region but also indicated an absence of AChE and postsynaptic junctional folds. The absence of these postsynaptic specializations at the extended, PNA-stained ECM region makes it unlikely that this region was previously occupied by nerve terminals that had retracted. Thus, the present study provides further findings consistent with the hypothesis that synaptic ECM precedes nerve terminal outgrowth and that the extension of synaptic ECM may play a role in synaptic remodeling.

Absence of sterol-specific complexes at active zones of degenerating and regenerating frog neuromuscular junctions.

Freeze-fracture combined with filipin treatment has been used as a cytochemical probe for membrane cholesterol. As previously shown at the frog neuromuscular junction, distinctive sterol-specific complexes were formed on the presynaptic membrane after filipin treatment, except at active zones. The absence of sterol-specific complexes from active zones was confirmed using two other cytochemical agents--digitonin and saponin. We also studied the maintenance and differentiation of the presynaptic membrane heterogeneity revealed by membrane cholesterol probes at degenerating and regenerating neuromuscular junctions. During degeneration, active zones in frog nerve terminals were disorganized, but still lacked sterol-specific complexes. After engulfing the degenerating nerve terminals, Schwann cells occupied the synaptic gutters and displayed a uniform distribution of sterol-specific complexes. Schwann cell ridges opposite the postjunctional folds also had prominent sterol-specific complexes in regions formerly occupied by active zones. By 2 weeks after nerve crush, nerve terminals reinvaded the endplate region and active zones began to regenerate. While the intramembrane particles of the early regenerating active zones were not arranged in the normal double-rowed organization, filipin-sterol complexes were nevertheless excluded from these primitive active zones. Areas of nerve terminal membrane opposite to junctional folds but lacking active zones were covered with filipin-sterol complexes. These results show that the normal double-rowed organization is not required for the expression of the membrane heterogeneity associated with the active zone. In addition, the absence of sterol-specific complexes is closely associated with the active zone particles and not simply the membrane regions opposite to the postjunctional folds. The membrane heterogeneity does not seem to be directly linked with the functional state of the active zone since it is still associated with degenerating active zones after transmission failure has occurred.

Effects of denervation on the distribution of peanut agglutinin binding molecules in frog muscles.

The extracellular matrix has been shown to play an important role in the differentiation of neuromuscular junctions during reinnervation in frogs. Peanut agglutinin, a lectin, is known to specifically bind to some glycoconjugates in the extracellular matrix at the frog neuromuscular junction and myotendinous junction. In order to determine if innervation has any role in regulating the specific binding of peanut agglutinin at neuromuscular junctions and myotendinous junctions, the distribution of peanut agglutinin binding was examined in muscles chronically denervated for various periods. Short-term denervated muscles (less than or equal to 2 months) showed no changes in peanut binding agglutinin binding at neuromuscular junctions and no extrajunctional binding. In contrast, long-term denervation (greater than 2 months - 7.5 months) resulted in altered peanut agglutinin distribution and a substantial reduction or a total loss in its binding at denervated neuromuscular junctions; binding at myotendinous junctions was not affected. Results of electron microscopic studies suggest that the presence of Schwann cells at denervated endplates delays the loss of peanut agglutinin binding. Reinnervation restores normal peanut agglutinin binding at neuromuscular junctions following long-term denervation. This report demonstrates that although the distribution of peanut agglutinin binding molecules is unchanged short-term denervation, intact innervation is necessary for the long-term maintenance of these molecules at neuromuscular junctions.

Synaptic remodeling revealed by repeated in vivo observations and electron microscopy of identified frog neuromuscular junctions.

This work aimed to examine the mechanism of synaptic remodeling using repeated in vivo observations, followed by electron microscopy, of identified frog neuromuscular junctions (NMJs). Our previous light microscopic studies suggested that extension of synaptic extracellular matrix (ECM) precedes, and may play a role in, nerve terminal (NT) growth during synaptic remodeling. To test this hypothesis, sartorius muscles were double labeled with a fluorescent dye, 4-(4-diethylamino-styryl)-N-methylpyridinium iodide, for NTs and rhodamine-conjugated peanut agglutinin for synaptic ECM. The double-labeled NMJs were observed in vivo with video-enhanced fluorescence microscopy. Two to three months after nerve sprouting was induced by a nerve graft, the same NMJs were restained and reexamined. After the final in vivo observations, the same NMJs were examined with semiserial thin section electron microscopy. Light microscopic observation of NMJs that showed synaptic ECM longer than the NT was confirmed with electron microscopy. At junctional branches where synaptic ECM extended beyond the NT, a Schwann cell process longer than the NT was observed in one example, whereas a Schwann cell with the same length as the NT was seen in other examples. In both cases, junctional folds were absent at the extended ECM region. In contrast, junctional folds were observed at the region vacated by a retracted NT. These results suggest that extension of synaptic ECM and Schwann cell processes may lead, and play a role in, the NT growth during the remodeling of adult synaptic connections.