Activity-dependent synaptic capture of transiting peptidergic vesicles.

Synapses require resources synthesized in the neuronal soma, but there are no known mechanisms to overcome delays associated with the synthesis and axonal transport of new proteins generated in response to activity, or to direct resources specifically to active synapses. Here, in vivo imaging of the Drosophila melanogaster neuromuscular junction reveals a cell-biological strategy that addresses these constraints. Peptidergic vesicles continually transit through resting terminals, but retrograde peptidergic vesicle flux is accessed following activity to rapidly boost neuropeptide content in synaptic boutons. The presence of excess transiting vesicles implies that synaptic neuropeptide stores are limited by the capture of peptidergic vesicles at the terminal, rather than by synthesis in the soma or delivery via the axon. Furthermore, activity-dependent capture from a pool of transiting vesicles provides a nerve terminal-based mechanism for directing distally and slowly generated resources quickly to active synapses. Finally, retrograde transport in the nerve terminal is regulated by activity.

NCAM 180 acting via a conserved C-terminal domain and MLCK is essential for effective transmission with repetitive stimulation.

NCAM 180 isoform null neuromuscular junctions are unable to effectively mobilize and exocytose synaptic vesicles and thus exhibit periods of total transmission failure during high-frequency repetitive stimulation. We have identified a highly conserved C-terminal (KENESKA) domain on NCAM that is required to maintain effective transmission and demonstrate that it acts via a pathway involving MLCK and probably myosin light chain (MLC) and myosin II. By perfecting a method of introducing peptides into adult NMJs, we tested the hypothesized role of proteins in this pathway by competitive disruption of protein-protein interactions. The effects of KENESKA and other peptides on MLCK and MLC activation and on failures in both wild-type and NCAM 180 null junctions supported this pathway, and serine phosphorylation of KENESKA was critical. We propose that this pathway is required to replenish synaptic vesicles utilized during high levels of exocytosis by facilitating myosin-driven delivery of synaptic vesicles to active zones or their subsequent exocytosis.

Heterogeneity in synaptic transmission along a Drosophila larval motor axon.

At the Drosophila melanogaster larval neuromuscular junction (NMJ), a motor neuron releases glutamate from 30-100 boutons onto the muscle it innervates. How transmission strength is distributed among the boutons of the NMJ is unknown. To address this, we created synapcam, a version of the Ca2+ reporter Cameleon. Synapcam localizes to the postsynaptic terminal and selectively reports Ca2+ influx through glutamate receptors (GluRs) with single-impulse and single-bouton resolution. GluR-based Ca2+ signals were uniform within a given connection (that is, a given bouton/postsynaptic terminal pair) but differed considerably among connections of an NMJ. A steep gradient of transmission strength was observed along axonal branches, from weak proximal connections to strong distal ones. Presynaptic imaging showed a matching axonal gradient, with higher Ca2+ influx and exocytosis at distal boutons. The results suggest that transmission strength is mainly determined presynaptically at the level of individual boutons, possibly by one or more factors existing in a gradient.

Activity-dependent liberation of synaptic neuropeptide vesicles.

Despite the importance of neuropeptide release, which is evoked by long bouts of action potential activity and which regulates behavior, peptidergic vesicle movement has not been examined in living nerve terminals. Previous in vitro studies have found that secretory vesicle motion at many sites of release is constitutive: Ca(2+) does not affect the movement of small synaptic vesicles in nerve terminals or the movement of large dense core vesicles in growth cones and endocrine cells. However, in vivo imaging of a neuropeptide, atrial natriuretic factor, tagged with green fluorescent protein in larval Drosophila melanogaster neuromuscular junctions shows that peptidergic vesicle behavior in nerve terminals is sensitive to activity-induced Ca(2+) influx. Specifically, peptidergic vesicles are immobile in resting synaptic boutons but become mobile after seconds of stimulation. Vesicle movement is undirected, occurs without the use of axonal transport motors or F-actin, and aids in the depletion of undocked neuropeptide vesicles. Peptidergic vesicle mobilization and post-tetanic potentiation of neuropeptide release are sustained for minutes.

The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity.

Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16-membered macrocycle. The toxic potential and the mechanism of action of GYM-A, highly purified from contaminated clams, have been assessed. GYM-A in isolated mouse phrenic hemidiaphragm preparations produced a concentration- and time-dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM-A paralyzed frog and mouse isolated neuromuscular preparations. Patch-clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM-A in a reversible manner. GYM-A also blocked, in a voltage-independent manner, homomeric human alpha7 nAChR expressed in Xenopus oocytes. Competition-binding assays confirmed that GYM-A is a powerful ligand interacting with muscle-type nAChR, heteropentameric alpha3beta2, alpha4beta2, and chimeric alpha7-5HT(3) neuronal nAChRs. Our data show for the first time that GYM-A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM-A detection in shellfish samples.

Neuromuscular synaptic function in mice lacking major subsets of gangliosides.

Gangliosides are a family of sialylated glycosphingolipids enriched in the outer leaflet of neuronal membranes, in particular at synapses. Therefore, they have been hypothesized to play a functional role in synaptic transmission. We have measured in detail the electrophysiological parameters of synaptic transmission at the neuromuscular junction (NMJ) ex vivo of a GD3-synthase knockout mouse, expressing only the O- and a-series gangliosides, as well as of a GM2/GD2-synthase*GD3-synthase double-knockout (dKO) mouse, lacking all gangliosides except GM3. No major synaptic deficits were found in either null-mutant. However, some extra degree of rundown of acetylcholine release at high intensity use was present at the dKO NMJ and a temperature-specific increase in acetylcholine release at 35 degrees C was observed in GD3-synthase knockout NMJs, compared with wild-type. These results indicate that synaptic transmission at the NMJ is not crucially dependent on the particular presence of most ganglioside family members and remains largely intact in the sole presence of GM3 ganglioside. Rather, presynaptic gangliosides appear to play a modulating role in temperature- and use-dependent fine-tuning of transmitter output.

Simulation studies of ultrashort, high-intensity electric pulse induced action potential block in whole-animal nerves.

A theoretical study of possible neuromuscular incapacitation based on the application of high-intensity, ultrashort electric pulses is presented. The analysis is applied to a rat, but the approach is general and can be extended to any whole-animal and applies for any arbitrary pulse waveform. It is hypothesized that repeatable and reversible action potential blocks in nerves can be attained based on the electroporation mechanism. Our numerical studies are based on the Hodgkin-Huxley distributed circuit representation of nerves, and incorporate a nodal analysis for the time-dependent and volumetric perturbing potentials and internal electric fields in whole animals. The predictions are compared to actual 600-ns experimental reports on rats and shown to be in very good agreement. Effective strength-duration plots for neuromuscular incapacitation are also generated.

Rolling blackout is required for synaptic vesicle exocytosis.

Rolling blackout (RBO) is a putative transmembrane lipase required for phospholipase C-dependent phosphatidylinositol 4,5-bisphosphate-diacylglycerol signaling in Drosophila neurons. Conditional temperature-sensitive (TS) rbo mutants display complete, reversible paralysis within minutes, demonstrating that RBO is acutely required for movement. RBO protein is localized predominantly in presynaptic boutons at neuromuscular junction (NMJ) synapses and throughout central synaptic neuropil, and rbo TS mutants display a complete, reversible block of both central and peripheral synaptic transmission within minutes. This phenotype appears limited to adults, because larval NMJs do not manifest the acute blockade. Electron microscopy of adult rbo TS mutant boutons reveals an increase in total synaptic vesicle (SV) content, with a concomitant shrinkage of presynaptic bouton size and an accumulation of docked SVs at presynaptic active zones within minutes. Genetic tests reveal a synergistic interaction between rbo and syntaxin1A TS mutants, suggesting that RBO is required in the mechanism of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated SV exocytosis, or in a parallel pathway necessary for SV fusion. The rbo TS mutation does not detectably alter SNARE complex assembly, suggesting a downstream requirement in SV fusion. We conclude that RBO plays an essential role in neurotransmitter release, downstream of SV docking, likely mediating SV fusion.

Induction of neuregulin signaling in mouse schwann cells in vivo mimics responses to denervation.

Neuregulins play crucial roles in early development of Schwann cells (SCs), but their roles in the activities of SCs during denervation and reinnervation of muscle are less clear. In the present study, the Tet-On system has been used in transgenic mice to enable inducible expression of a mutant, constitutively active neuregulin receptor (ErbB2) in SCs. This induction simulates neuregulin signaling to these cells. Reporter transgenes were used to show a tightly regulated, SC-selective expression in muscle. Induction leads to a number of changes in SCs at neuromuscular junctions that mimic the response to muscle denervation/reinnervation. These include process extension, soma migration, and proliferation. SCs also come to express nestin, a protein characteristic of their reaction to muscle denervation. This activation of SCs results in the sprouting of nerve terminals, and these sprouts follow the extensions of the SCs. However, these sprouts and their associated SCs disappear after the removal of the inducer. Last, induction of the active receptor is sufficient to rescue SCs in neonatal muscle from denervation-induced apoptosis. These findings show that the responses of SCs in muscle to denervation can be explained by induction of an autocrine/paracrine neuregulin signaling cascade suggested by previous molecular studies.

Increased asynchronous release and aberrant calcium channel activation in amyloid precursor protein deficient neuromuscular synapses.

Despite the critical roles of the amyloid precursor protein (APP) in Alzheimer's disease pathogenesis, its physiological function remains poorly established. Our previous studies implicated a structural and functional activity of the APP family of proteins in the developing neuromuscular junction (NMJ). Here we performed comprehensive analyses of neurotransmission in mature neuromuscular synapse of APP deficient mice. We found that APP deletion led to reduced paired-pulse facilitation and increased depression of synaptic transmission with repetitive stimulation. Readily releasable pool size and total releasable vesicles were not affected, but probability of release was significantly increased. Strikingly, the amount of asynchronous release, a measure sensitive to presynaptic calcium concentration, was dramatically increased, and pharmacological studies revealed that it was attributed to aberrant activation of N- and L-type Ca(2+) channels. We propose that APP modulates synaptic transmission at the NMJ by ensuring proper Ca(2+) channel function.

GaAs laser therapy reestablishes the morphology of the NMJ and nAChRs after injury due to bupivacaine.

BACKGROUND: Local anesthetics are used to relieve pre- and postoperative pain, acting on both sodium channels and nicotinic acetylcholine receptors (nAChR) at the neuromuscular junction (NMJ). Bupivacaine acts as a non-competitive antagonist and has limitations, such as myotoxicity, neurotoxicity, and inflammation. Low-level laser therapy (LLLT) has anti-inflammatory, regenerative, and analgesic effects. The aim of the present study was to evaluate the effects of a gallium arsenide laser (GaAs) on the morphology of the NMJ and nAChRs after application of bupivacaine in the sternomastoid muscle. METHODS: Thirty-two adult male Wistar rats received injections of bupivacaine 0.5% (Bupi: right antimere) and 0.9% sodium chloride (Cl: left antimere). Next, the animals were divided into a Control group (C) and a Laser group (LLLT). The laser group received LLLT (GaAs 904nm, 50mW, 4,8J) in both antimeres for five consecutive days. After seven days, the animals were euthanized and the surface portion of the sternomastoid muscle was removed, frozen, and subjected to morphological and morphometric analyses of the NMJs (nonspecific esterase reaction), confocal laser scanning, and an ultrastructural analysis. The nAChRs were quantified by Western blotting. RESULTS: In the chloride group, the morphology and morphometry of the NMJs remained stable. The maximum diameters of the NMJs were lower in the Bupi (15.048±1.985) and LLLT/Bupi subgroups (15.456±1.983) compared to the Cl (18.502±2.058) and LLLT/Cl subgroups (19.356±2.522) (p<0.05). Ultrastructurally, LLLT reduced myonecrosis observed after application of bupivacaine, with recovery in the junctional folds and active zone. There was an increase in the perimeter of the LLLT/Bupi subgroup (150.33) compared to the Bupi subgroup (74.69) (p<0.01) observed by confocal microscopy. There was also an increase in the relative planar area of the NMJ after LBI (8.75) compared to CBupi (4.80) (p<0.01). An analysis of the protein expression of nAChRα1 showed no major differences in the groups studied. There was an increase in protein expression of the ε subunit after application of LLLT (13.055) compared to Bupi (0.251) (p<0.01). Taken together, the present experiments indicate that there was a positive association of the α and γ subunits (p<0.05). CONCLUSIONS: These results demonstrate that LLLT at the dose used in this study reduced structural alterations in the NMJ and molecular changes in nAChRs triggered by bupivacaine, providing important data supporting the use of LLLT in therapeutic protocols for injuries triggered by local anesthetics.

Regulation of motoneuron excitability via motor endplate acetylcholine receptor activation.

Motoneuron populations possess a range of intrinsic excitability that plays an important role in establishing how motor units are recruited. The fact that this range collapses after axotomy and does not recover completely until after reinnervation occurs suggests that muscle innervation is needed to maintain or regulate adult motoneuron excitability, but the nature and identity of underlying mechanisms remain poorly understood. Here, we report the results of experiments in which we studied the effects on rat motoneuron excitability produced by manipulations of neuromuscular transmission and compared these with the effects of peripheral nerve axotomy. Inhibition of acetylcholine release from motor terminals for 5-6 d with botulinum toxin produced relatively minor changes in motoneuron excitability compared with the effect of axotomy. In contrast, the blockade of acetylcholine receptors with alpha-bungarotoxin over the same time interval produced changes in motoneuron excitability that were statistically equivalent to axotomy. Muscle fiber recordings showed that low levels of acetylcholine release persisted at motor terminals after botulinum toxin, but endplate currents were completely blocked for at least several hours after daily intramuscular injections of alpha-bungarotoxin. We conclude that the complete but transient blockade of endplate currents underlies the robust axotomy-like effects of alpha-bungarotoxin on motoneuron excitability, and the low level of acetylcholine release that remains after injections of botulinum toxin inhibits axotomy-like changes in motoneurons. The results suggest the existence of a retrograde signaling mechanism located at the motor endplate that enables expression of adult motoneuron excitability and depends on acetylcholine receptor activation for its normal operation.

Stimulated-single fiber electromyography monitoring of anti-sense induced changes in experimental autoimmune myasthenia gravis.

The neuromuscular weakness associated with myasthenia gravis (MG) can be transiently relieved by pharmacological inhibitors of acetylcholinesterase (AChE). Here, we expand the anticholinesterase repertoire to include 2'-O-methyl-protected antisense oligonucleotides targeted to AChE mRNA (EN101). Using stimulated-single fiber electromyography, we show that EN101 treatment of rats with experimental autoimmune myasthenia gravis (EAMG), improved the mean consecutive difference (MCD) and blocking for 24h. This treatment was more efficient than pyridostigmine and was accompanied by marked improvement in stamina and clinical profile.

Evidences for calcium-dependent inactivation of calcium current at the frog motor nerve terminal.

Assessment of calcium-dependent inactivation of calcium current in nerve terminals is not feasible due to technical reasons. Perineural measurement of calcium-flow, however, might be utilized as indirect means to evaluate synaptic currents. Using perineural recording from frog neuromuscular junction, supra-threshold stimuli applied to motor nerve in paired-pulse manner with varying inter-pulse intervals (5-50 ms) are demonstrated in this study to cause paired-pulse depression (PPD) of Ca(2+)-current. PPD of Ca(2+)-flow was reduced at lower extracellular Ca(2+) concentrations, in BAPTA-AM and EGTA-AM treated preparations and after replacing extracellular Ca(2+) with Sr(2+). Using perineural measurement of calcium current as an indirect model to investigate synaptic ionic activity, our findings demonstrate that PPD may be attributed to calcium-dependent inactivation of Ca(2+)-current, which may serve as negative feedback in response to massive Ca(2+) entry to motor nerve terminals. A putative sensor of Ca(2+)-current is also proposed in this study.

Short day lengths affect perinatal development of the male reproductive system in the Siberian hamster, Phodopus sungorus.

The Siberian hamster, Phodopus sungorus, breeds seasonally. In the laboratory, seasonal breeding can be controlled by photoperiod, which affects the duration of nightly melatonin secretion. Winterlike, short day lengths induce gonadal regression in adult animals, and pups born and maintained in short days undergo pubertal gonadal development later than animals born into long days. However, to date there have been no reports of gestational photoperiod affecting fetal development of reproductive systems. The spinal nucleus of the bulbocavernosus (SNB) and its target muscles, the bulbocavernosus (BC) and levator ani (LA), compose a sexually dimorphic, androgen-sensitive neuromuscular system involved in male reproduction. The SNB neuromuscular system was studied in male Siberian hamsters maintained from conception in short-day (8 h light, 16 h dark; 8L:16D) versus long-day (16L:8D) conditions. On the day of birth, and at postnatal (PN) days 2 and 18, the BC/LA muscles of hamsters gestated and raised in the short photoperiod were significantly reduced relative to those of their long-day counterparts. Testes weights were not significantly different between groups until day 18. Thus, photoperiod exposure during gestation and after birth affects perinatal development of the SNB system in this species, and these effects can be seen as early as the day of birth. Because photoperiod did not significantly affect testes weights until PN18, these results suggest that either perinatal photoperiod affects fetal androgen production without affecting testes weight or it influences BC/LA development independently from androgen.

Effects of experimental radiation-induced hypomyelinating neuropathy on motor end-plates and neuromuscular transmission.

Morphological and physiological techniques were used to study the effects of radiation-induced hypomyelinating neuropathy on the innervation of skeletal muscle in the mouse. The right sciatic nerve was crushed focally and 3 days later the hind limb exposed to 20Gy X-rays. After reinnervation of original end-plate sites there was extensive formation of ultraterminal sprouts and of new end-plates characterized by small nerve terminals and rudimentary postsynaptic folds. In vitro examination of soleus nerve-muscle preparations showed reduced frequency of spontaneous miniature end-plate potentials and low quantal content of evoked potentials. The findings indicate that hypomyelinating neuropathy may cause reduction in quantal release of neurotransmitter.

Electromyogram-triggered neuromuscular stimulation and stroke motor recovery of arm/hand functions: a meta-analysis.

Debate persists about the effectiveness of poststroke behavioral interventions for progress toward motor recovery. The current meta-analysis assessed the effect of electromyogram (EMG)-triggered neuromuscular stimulation on arm and hand functions. Computer searches of PubMed and Cochran databases, as well as hand searches of reference lists identified seven EMG-triggered neuromuscular stimulation studies. Outcome measures focused on arm and hand motor capability functions. In addition, the quality of each study was rated on three guidelines: randomization, double blind, and dropouts. After adjusting data for consistency in the arm/hand outcome measures and to avoid bias, five active stimulation studies were included in the analysis. Rehabilitation treatment in each study focused on wrist extension. The total number of individuals in the treatment groups was 47 whereas the control groups had 39 subjects. The meta-analysis revealed a significant overall mean effect size (delta=0.82, S.D.=0.59). A homogeneity test indicated that the pooled standardized effect sizes estimated the same treatment effect. A fail-safe test for null effect findings revealed that 15 studies were required to reduce the large effect (0.82) to a small effect (0.20). These improved wrist extension motor capabilities findings support EMG-triggered neuromuscular stimulation as an effective poststroke protocol.

Reduction of crotoxin-induced neuromuscular blockade by gamma radiation.

A comparative study between crotoxin and gamma irradiated crotoxin was performed on the indirectly evoked twitches and tetani of sciatic nerve-extensor digitorum longus muscle of rats. Crotoxin (3 to 14 microg/ml) decreased the amplitude of twitches and induced a slight tetanic fade, and irradiated crotoxin did not significantly affect either twitch amplitude or tetanic tension. Since gamma radiation reduced the neurotoxicity of crotoxin it may be useful for the production of anticrotalic serum.

Effect of ionizing radiation on neuromuscular junctions in mouse tongues.

Radiation damage to the neuromuscular junctions (NMJs) in mouse tongues was studied using local x-irradiation of the tongues with the rest of the body shielded. Transmission electron microscopy (TEM) revealed no significant morphological changes in the fine structures and organelles of the NMJs given 4 Gy. A dose of 8 Gy produced degenevative morphological changes associated with oxon terminal sprouting as early as 2 and 7 days following irradiation. Subsequently, 1-11 weeks later, severe degenerative changes were observed. The number of mitochondria was significantly decreased with increased occurrence of degenerative membranal features. The number of synaptic footplates without terminals or with multiple small terminals within one groove increased gradually with time. Most of these pathological changes persisted for at least 3 months after irradiation. However, the myofibres, blood vessels and interstitial cells appeared to be unaffected throughout the period of follow-up. The present study substantiates our previous reports of ageing-like changes in the tongues' NMJs induced by their excessive exposure to free radicals.

Some photoresponses of isolated tissue preparations to ultraviolet light in the presence of photosensitizers.

Responses of isolated tissue preparations to ultraviolet (UV) light were studied with and without the presence of photosensitizers like eosin, fluorescein and sodium nitrite. Exposure to UV light in the presence of sodium nitrite induced consistent relaxation of rat duodenum. The photorelaxation was found to be related to the concentration of sodium nitrite. Adrenergic or cholinergic mechanisms do not seem to be involved. The isolated rat duodenum preparation exhibited quantitatively consistent photoresponse for 3 to 4 hr at its normal tone obviating the need for additional spasmogens as needed with other preparations. The preparation is a suitable test model for the study of photobiologic response evoked by UV light.