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.

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.

The role of perisynaptic Schwann cells in development of neuromuscular junctions in the frog (Xenopus laevis).

Fluorescence microscopy was used to study the behavior of perisynaptic Schwann cells (PSCs) in relation to motor nerve terminals and postsynaptic clusters of acetylcholine receptors, during the development of the neuromuscular junction (NMJ) in the frog Xenopus laevis. Pectoral (supracoracoideus) muscles were labeled with monoclonal antibody 2A12 for Schwann cells, the dye FM4-64 for nerve terminals (NTs), alpha-bungarotoxin for acetylcholine receptors (AChRs), and Hoechst 33258 for cellular nuclei, in animals from tadpole stage 57 to fully grown adults. When muscle fibers first appeared in stage 57, NMJs consisted of tightly apposed NTs and AChRs and were only partially covered with PSCs or their processes. Within a few stages, PSCs fully occupied and overgrew the NMJs, extending fine sprouts between a few micrometers and hundreds of micrometers beyond the borders of the junction. Sprouts of PSCs were most abundant during the time when secondary myogenesis, synaptogenesis, and synaptic growth occurred at their highest rates. PSCs were recruited to NMJs during synaptic growth, at rates between 1.3 PSCs/100 microm junctional length early on and 0.4 PSCs/100 microm later. Shortly after metamorphosis, PSC sprouts disappeared and NMJs acquired the adult appearance, in which PSCs, NTs, and AChRs were mostly congruent. The results suggest that, although PSCs may not be required for initial nerve-muscle contacts, PSCs sprouts lead synaptic growth and play a role in the extension and maturation of developing NMJs.

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.

Reciprocal interactions between perisynaptic Schwann cells and regenerating nerve terminals at the frog neuromuscular junction.

The perisynaptic Schwann cell (PSC) has gained recent attention with respect to its roles in synaptic function, remodeling, and regeneration at the vertebrate neuromuscular junction (NMJ). Here we test the hypothesis that, following nerve injury, processes extended by PSCs guide regenerating nerve terminals (NTs) in vivo, and that the extension of sprouts by PSCs is triggered by the arrival of regenerating NTs. Frog NMJs were double-stained with a fluorescent dye, FM4-64, for NTs, and fluorescein isothiocyanate (FITC)-tagged peanut agglutinin (PNA) for PSCs. Identified NMJs were imaged in vivo repeatedly for several months after nerve injury. PSCs sprouted profusely beginning 3-4 weeks after nerve transection and, as reinnervation progressed, regenerating NTs closely followed the preceding PSC sprouts, which could extend tens to hundreds of microns beyond the original synaptic site. The pattern of reinnervation was dictated by PSC sprouts, which could form novel routes joining neighboring junctions or develop into new myelinated axonal pathways. In contrast to mammals, profuse PSC sprouting in frog muscles was not seen in response to axotomy alone, and did not occur at chronically denervated NMJs. Instead, sprouting coincided with the arrival of regenerating NTs. Immunofluorescent staining revealed that in muscle undergoing reinnervation 4 weeks after axotomy, 91% of NMJs bore PSC sprouts, compared to only 6% of NMJs in muscle that was chronically denervated for 4 weeks. These results suggest that reciprocal interactions between regenerating NTs and PSCs govern the process of reinnervation at frog NMJs: regenerating NTs induce PSCs to sprout, and PSC sprouts, in turn, lead and guide the elaboration of NTs.

Perisynaptic Schwann cells at neuromuscular junctions revealed by a novel monoclonal antibody.

This study aimed to generate a probe for perisynaptic Schwann cells (PSCs) to investigate the emerging role of these synapse-associated glial cells in the formation and maintenance of the neuromuscular junction (NMJ). We have obtained a novel monoclonal antibody, 2A12, which labels the external surface of PSC membranes at the frog NMJ. The antibody reveals PSC fine processes or "fingers" that are interposed between nerve terminal and muscle membrane, interdigitating with bands of acetylcholine receptors. This antibody also labels PSCs at the avian neuromuscular junction and recognizes a 200 kDa protein in Torpedo electric organs. In frog muscles, axotomy induces sprouting of PSC processes beyond clusters of acetylcholine receptors and acetylcholinesterase at denervated junctional branches. PSC branches often extend across several muscle fibers. At some junctions, PSC sprouts join the tips of neighboring branches. The average length of PSC sprouts is approximately 156 microm at 3-week denervated NMJs. PSC sprouting is accompanied by a significant increase in the number of Schwann cell bodies per NMJ. Following nerve regeneration, nerve terminals reinnervate the junction along the PSC processes. In vivo observations of normal frog muscles also show PSC processes longer than nerve terminals at some junctional branches. The results suggest that nerve injury induces profuse PSC sprouting that may play a role in guiding nerve terminal regeneration at frog NMJs. In addition, antibody 2A12 reveals the fine morphology of PSCs in relation to other synaptic elements and is a useful probe in elucidating the function of these synapse-associated glial cells in vivo.

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.

A Schwann cell matrix component of neuromuscular junctions and peripheral nerves.

Molecules localized to the synapse are potential contributors to processes unique to this specialized region, such as synapse formation and maintenance and synaptic transmission. We used an immunohistochemical strategy to uncover such molecules by generating antibodies that selectively stain synaptic regions and then using the antibodies to analyse their antigens. In this study, we utilized a monoclonal antibody, mAb 6D7, to identify and characterize an antigen concentrated at frog neuromuscular junctions and in peripheral nerves. In adult muscle, immunoelectron microscopy indicates that the antigen is located in the extracellular matrix around perisynaptic Schwann cell at the neuromuscular junction and in association with myelinated and nonmyelinated axons in peripheral nerves. The maintenance of the mAb 6D7 epitope is innervation-dependent but is muscle-independent; it disappears from the synaptic region within 2 weeks after denervation, but persists after muscle damage when the nerve is left intact. mAb 6D7 immunolabelling is also detected at the neuromuscular junction in developing tadpoles. Biochemical analyses of nerve extracts indicate that mAb 6D7 recognizes a glycoprotein of 127 kDa with both N- and O-linked carbohydrate moieties. Taken together, the results suggest that the antigen recognized by mAb 6D7 may be a novel component of the synaptic extracellular matrix overlying the terminal Schwann cell. The innervation-sensitivity of the epitope at the neuromuscular junction suggests a function in the interactions between nerves and Schwann cells.

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.

Differential blockade of voltage-sensitive calcium channels at the mouse neuromuscular junction by novel omega-conopeptides and omega-agatoxin-IVA.

This investigation assessed the ability of a variety of calcium channel blocking peptides to block synaptic transmission in the isolated mouse phrenic nerve-hemidiaphragm. The synthetic version of the naturally occurring N-type voltage-sensitive calcium channel (VSCC) blocker omega-conopeptide MVIIA (SNX-111) had no effect on nerve-evoked muscle contractions. The non-N-, non-L-type VSCC blocker, omega-conopeptide MVIIC (SNX-230), blocked neuromuscular transmission completely, as did the selective P-type VSCC blocker, omega-Aga-IVA. Subsequent evaluation of other synthetic omega-conopeptides and analogs disclosed a significant positive correlation between the test compounds' affinities for high-affinity SNX-230 brain binding sites and their neuromuscular blocking potencies. Quantal analysis of transmitter release showed that SNX-230 abolished evoked endplate potentials completely, but had little effect on the amplitude and frequency of spontaneous miniature endplate potentials. Perineural focal recordings of presynaptic currents showed that SNX-230 did not block the neuronal action potential. These and other findings indicated that SNX-230 prevents transmitter release at the mouse neuromuscular junction by blocking calcium channels at presynaptic nerve endings. These calcium channels correspond pharmacologically to VSCCs associated with high-affinity binding sites in rat brain and are most probably either of the P- or Q-type.

A novel omega-conopeptide for the presynaptic localization of calcium channels at the mammalian neuromuscular junction.

Voltage-sensitive Ca2+ channels are essential to transmitter release at the chemical synapse. To demonstrate the localization of voltage-sensitive Ca2+ channels in relation to the site of transmitter release, mouse neuromuscular junctions were double labelled with alpha-bungarotoxin and a novel voltage-sensitive Ca2+ channel probe, SNX-260, a synthetic analog of omega-conopeptide MVIIC. Similar to omega-conopeptide MVIIC, biotinylated SNX-260 blocked nerve-stimulated transmitter release at the mouse neuromuscular junction. Fluorescently-tagged biotinylated SNX-260 labelled the nerve terminal which appeared thinner than and was outlined by acetylcholine receptor clusters as seen in en face view. This SNX-260 labelling was inhibited by preincubation with unconjugated SNX-260. Side-views of the neuromuscular junction indicated that the SNX-260 labelling was on the synaptic side facing the acetylcholine receptor rather than on the nonsynaptic side of the nerve terminal. This presynaptic binding was confirmed by the absence of SNX-260 labelling in denervated muscles following a nerve cut or disjunction after collagenase treatment. Confocal microscopy revealed spots of SNX-260 labelling that may correlate with active zones. The SNX-260 labelling pattern was not affected by preincubation with unconjugated SNX-111 (omega-conopeptide MVIIA), an N-type voltage-sensitive Ca2+ channel blocker. These findings suggest that SNX-260 is a novel probe for localizing non-N type voltage-sensitive Ca2+ channels and that these voltage-sensitive Ca2+ channels are localized near the transmitter release sites at the mammalian motor nerve terminal membrane. The results are consistent with the suggestion that non-N, probably P/Q type voltage-sensitive Ca2+ channels mediate evoked transmitter release at the mammalian neuromuscular junction.

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.

The remodeling of synaptic extracellular matrix and its dynamic relationship with nerve terminals at living frog neuromuscular junctions.

The question of whether the synaptic extracellular matrix undergoes remodeling and how this remodeling is related to nerve terminal plasticity was examined in living neuromuscular junctions of adult frogs. Sartorius muscles were double stained with a fluorescent nerve terminal dye 4-(4-diethylamino-styryl)-N-methylpyridinium iodide (4-Di-2-Asp) and rhodamine-tagged peanut agglutinin (PNA) which recognizes synaptic extracellular matrix. Both nerve terminals and synaptic extracellular matrix in 200 identified normal junctions were visualized in vivo two or three times over a period of 2.6-6 months. The majority of neuromuscular junctions (NMJs) showed remodeling of both nerve terminals and synaptic extracellular matrix. Only 2.5% showed no changes in either synaptic element. The most commonly seen remodeling involved correlated changes in both nerve terminals and synaptic extracellular matrix. In this large group, while some junctions (20%) showed overall proportionate changes in all branches, most junctions (68%) showed disproportionate extension and/or retraction of some but not all individual branches. Another group of NMJs (9.5%) showed mismatched changes in the nerve terminal and synaptic extracellular matrix. In this group, some NMJs showed a decrease in the nerve terminal length without a corresponding reduction in synaptic extracellular matrix length. In other junctions that displayed extension of branches, the PNA-stained matrix was longer than the distal tip of the nerve terminal. Morphometric analysis indicated an average increase of 15.6% in total nerve terminal length and 13.6% in total synaptic extracellular matrix length. Although almost all NMJs displayed remodeling in at least one branch, about 50% of the 2201 individual branches examined did not show changes. The average change was 8.9% growth in the length of individual nerve terminal branches and 8.3% growth in the length of individual branches of synaptic extracellular matrix. There was no significant difference in the morphometry between the repeatedly observed junctions and the previously unobserved control junctions. Furthermore, junctions in which the synaptic extracellular matrix was longer than the nerve terminal also were seen in control as well as in experimental muscles. Cases where the nerve terminals were longer than the synaptic extracellular matrix were never observed in newly arising junctional branches. The present study has shown extensive remodeling in not only the nerve terminal but also the synaptic extracellular matrix in adult living frog NMJs. Results suggest that nerve terminals retract before the synaptic extracellular matrix. A hypothesis that extension of synaptic extracellular matrix precedes nerve terminal growth during synaptic remodeling is proposed.

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.

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.

Induction of active zones at ectopic neuromuscular junctions in the frog.

To examine de novo differentiation of the active zone, ectopic neuromuscular junctions were studied in adult frog muscles. Ectopic junctions induced by excising the original endplate region and implanting the nerve to an endplate-free site were examined by light and electron microscopy 4 weeks-1 year after operations. The earliest time point at which ectopic junction formation was detected with freeze fracture was 6 weeks postoperation, when clusters of active zone particles were observed scattered across the nerve ending. Subsequently, short active zones (6-10 weeks postoperation, length mean = 0.36 +/- 0.24 microns) composed of the characteristic 2 double rows of particles are detected. Before junctional folds are observed with freeze fracture, many active zones are parallel to each other and to the long axis of the nerve. The average angle 6-10 weeks postoperation is 27 degrees +/- 23 degrees. Even during these early stages of formation, active zones are functional. As time passes, active zones attain a more typical, perpendicular orientation (12-18 weeks postoperation, mean = 62 degrees +/- 24 degrees) and also increase in length (mean = 0.69 +/- 0.45 microns). However, even after 1 year, the orientation (angle, mean = 70 degrees +/- 22 degrees) and the length (mean = 0.78 +/- 0.63 microns) of active zones at ectopic junctions are still not well correlated with active zones at normal junctions (normal active zone angle mean = 85 degrees +/- 5 degrees, length mean = 1.00 +/- 0.57 microns).(ABSTRACT TRUNCATED AT 250 WORDS)

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.