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.

Degeneration and recovery of the neuromuscular junction after application of extracorporeal shock wave therapy.

It is known that free nerve endings are degenerated after application of shock waves. We therefore hypothesized that the application of shock waves to muscle induces dysfunction of neuromuscular transmission at neuromuscular junctions. We investigated changes in neuromuscular transmission in response to shock wave application. Sprague-Dawley rats were used in this study. Two thousand shock waves at an energy flux density of 0.18 mJ/mm(2) were applied to their right calf muscles. Neuromuscular junctions of gastrocnemius muscles were evaluated using rhodamine-α-bungarotoxin on the day of treatment (n = 5). Amplitude and latency of compound muscle action potentials were measured on the day of treatment and 1, 2, 4, 6, and 8 weeks after treatment (n = 10, each group). Degenerated acetylcholine receptors existed in all treated muscles. Although the action potential amplitude on the treated side was significantly less than on the control side from the day of treatment (25.1 ± 7.8 vs. 34.5 ± 9.1, p = 0.012) to 6 weeks (27.9 ± 7.2 vs. 34.5 ± 7.2, p = 0.037), there was no significant difference at 8 weeks. There was no significant difference in transmission latency between the groups. The application of shock waves to muscle induced a transient dysfunction of nerve conduction at neuromuscular junctions.

The Drosophila gene RanBPM functions in the mushroom body to regulate larval behavior.

BACKGROUND: In vertebrates, Ran-Binding Protein in the Microtubule Organizing Center (RanBPM) appears to function as a scaffolding protein in a variety of signal transduction pathways. In Drosophila, RanBPM is implicated in the regulation of germ line stem cell (GSC) niche organization in the ovary. Here, we addressed the role of RanBPM in nervous system function in the context of Drosophila larval behavior. METHODOLOGY/PRINCIPAL FINDINGS: We report that in Drosophila, RanBPM is required for larval feeding, light-induced changes in locomotion, and viability. RanBPM is highly expressed in the Kenyon cells of the larval mushroom body (MB), a structure well studied for its role in associative learning in Drosophila and other insects. RanBPM mutants do not display major disruption in nervous system morphology besides reduced proliferation. Expression of the RanBPM gene in the Kenyon cells is sufficient to rescue all behavioral phenotypes. Through genetic epistasis experiments, we demonstrate that RanBPM participates with the Drosophila orthologue of the Fragile X Mental Retardation Protein (FMRP) in the development of neuromuscular junction (NMJ). CONCLUSIONS/SIGNIFICANCE: We demonstrate that the RanBPM gene functions in the MB neurons for larval behavior. Our results suggest a role for this gene in an FMRP-dependent process. Taken together our findings point to a novel role for the MB in larval behavior.

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.

Turning off the central contribution to contractions evoked by neuromuscular electrical stimulation.

Neuromuscular electrical stimulation can generate contractions through both peripheral and central mechanisms. The peripheral mechanism involves the direct activation of motor axons, while the central mechanism involves the activation of sensory axons that recruit spinal neurons through a reflex pathway. For use in functional electrical stimulation. One must have control over turning the central mechanism on and off. We investigated whether inhibition developed through antagonist muscle (tibialis anterior, TA) contractions elicited by electrical stimulation or by volition can turn off the central mechanism in triceps surae. Both electrical stimulation and voluntary contractions of TA reduced or eliminated plantar flexion torque produced by the central mechanism, indicating that inhibition induced via these contractions can effectively turn off the central contribution to force. These findings suggest that patterns of electrical stimulation may be able to generate periodic muscle contractions by turning the central contribution to muscular contractions on and off.

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.

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.

The Drosophila ARC homolog regulates behavioral responses to starvation.

The gene encoding dARC1, one of three Drosophila homologs of mammalian activity-regulated cytoskeleton-associated protein (ARC), is upregulated in both seizure and muscular hypercontraction mutants. In this study we generate a null mutant for dArc1 and show that this gene is not involved in synaptic plasticity at the larval neuromuscular junction or in formation or decay of short-term memory of courtship conditioning, but rather is a modifier of stress-induced behavior. dARC1 is expressed in a number of neurosecretory cells and mutants are starvation-resistant, exhibiting an increased time of survival in the absence of food. Starvation resistance is likely due to the fact that dArc1 mutants lack the normal hyperlocomotor response to starvation, which is almost universal in the animal kingdom. dARC1 acts in insulin-producing neurons of the pars intercerebralis to control this behavior, but does not appear to be a general regulator of insulin signaling. This suggests that there are multiple modes of communication between the pars and the ring gland that control starvation-induced behavioral responses.

Synapse formation and elimination: role of activity studied in different models of adult muscle reinnervation.

Synapse competition and elimination are a general developmental process both in central and in peripheral nervous systems that is strongly activity dependent. Some common features regulate synapse competition, and one of these is an application to development of the Hebb's postulate of learning: repeated coincident spike activity in competing presynaptic inputs on the same target cell inhibits competition, whereas noncoincident activity promotes weakening of some of the inputs and ultimately their elimination. Here we report experiments that indicate that the development of muscle innervation (initial polyneuronal innervation and subsequent synapse elimination) follows the Hebb's paradigm. We utilized two different models of muscle reinnervation in the adult rat: 1) we crushed nerves going to soleus or extensor digitorum longus muscles, to activate regeneration of the presynaptic component of the neuromuscular junctions (NMJ), or 2) we injected the soleus muscle with Marcaine (a myotoxic agent) to activate regeneration of the postsynaptic component, the muscle fiber. A condition of transient polyneuronal innervation occurs during NMJ regeneration in both cases, although the two models differ insofar as the relative strength of the competing inputs is concerned. During the period of competition (a few days or weeks, in Marcaine or crush experiments, respectively), we imposed a synchronous firing pattern on the competing inputs by stimulating motor axons distal to a chronic conduction block and demonstrated that this procedure strongly inhibits synapse elimination, with respect to control muscles in which regeneration occurs under natural impulse activity of motoneurons.

Shp2 is dispensable in the formation and maintenance of the neuromuscular junction.

SHP2, a protein tyrosine phosphatase with two SH2 domains, has been implicated in regulating acetylcholine receptor (AChR) gene expression and cluster formation in cultured muscle cells. To understand the role of SHP2 in neuromuscular junction (NMJ) formation in vivo, we generated mus cle-specific deficient mice by using a loxP/Cre strategy since Shp2 null mutation causes embryonic lethality. Shp2(floxed/floxed) mice were crossed with mice expressing the Cre gene under the control of the human skeletal alpha-actin (HSA) promoter. Expression of SHP2 was reduced or diminished specifically in skeletal muscles of the conditional knockout (CKO) mice. The mutant mice were viable and fertile, without apparent muscle defects. The mRNA of the AChR alpha subunit and AChR clusters in CKO mice were localized in a narrow central region surrounding the phrenic nerve primary branches, without apparent change in intensity. AChR clusters colocalized with markers of synaptic vesicles and Schwann cells, suggesting proper differentiation of presynaptic terminals and Schwann cells. In comparison with age-matched littermates, no apparent difference was observed in the size and length of AChR clusters in CKO mice. Both the frequency and amplitude of mEPPs in CKO mice were similar to those in controls, suggesting normal neurotransmission when SHP2 was deficient. These results suggest that Shp2 is not required for NMJ formation and/or maintenance.

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.

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.

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.

Muscarinic autoreceptors modulate transmitter release through protein kinase C and protein kinase A in the rat motor nerve terminal.

We have used intracellular recording to investigate the existence of a functional link between muscarinic presynaptic acetylcholine (ACh) autoreceptors, the intracellular serine-threonine kinases-mediated transduction pathways and transmitter release in the motor nerve terminals of adult rats. We found the following. (1) Transmitter release was reduced by the M1 muscarinic acetylcholine receptor (mAChR) blocker pirenzepine and enhanced by the M2 blocker methoctramine. The unselective mAChR blocker atropine increased ACh release, which suggests the unmasking of another parallel release-potentiating mechanism. There are therefore two opposite, though finely balanced, M1-M2 mAChR-operated mechanisms that tonically modulate transmitter release. (2) Both M1 and M2 mechanisms were altered when protein kinase C (PKC), protein kinase A (PKA) or the P/Q-type calcium channel were blocked. (3) Both PKC and PKA potentiated release when they were specifically stimulated [with phorbol 12-myristate 13-acetate (PMA) and Sp-8-Br cAMPs, respectively], and both needed the P/Q channel. (4) In normal conditions PKC seemed not to be directly involved in transmitter release (the PKC blocker calphostin C did not reduce release), whereas PKA was coupled to potentiate release (the PKA blocker H-89 reduced release). However, when an imbalance of the M1-M2 mAChRs function was experimentally produced with selective blockers, an inversion of the kinase function occurred and PKC could then stimulate transmitter release, whereas PKA was uncoupled. (5) The muscarinic function may be explained by the existence of an M1-mediated increased PKC activity-dependent potentiation of release and an M2-mediated PKA decreased activity-dependent release reduction. These findings show that there is a precise interrelation pattern of the mAChRs, PKC and PKA in the control of the neurotransmitter release.

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.

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.

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.

Developmental consequences of neuromuscular junctions with reduced presynaptic calcium channel function.

Evoked neurotransmitter release at the Drosophila neuromuscular junction (NMJ) is regulated by the amount of calcium influx at the presynaptic nerve terminal, as for most chemical synapses. Calcium entry occurs via voltage-gated calcium channels. The temperature-sensitive Drosophila mutant, cac(TS2), has a reduced amount of calcium entry during evoked stimulation. We have used this mutation to examine homeostatic regulatory mechanisms during development of the NMJ on muscle 6 within the developing larva. The amplitude of the excitatory postsynaptic potentials are reduced for both the Ib and Is motor neurons in 3rd instar larvae which have been raised at 33 degrees C from the 1st instar stage. Larvae raised at 25 degrees C and larvae pulsed at 33 degrees C from the late 2nd instar for various lengths of time show a reduced synaptic efficacy as a 3rd instar. The results indicate that the nerve terminal cannot fully compensate physiologically in the regulation of synaptic transmission during larval life for a reduced amount of evoked calcium entry. Morphological comparisons of Ib and Is terminals in relation to length and numbers of varicosities are significantly reduced in cac(TS2), which also suggests a lack in homeostatic ability. These findings are relevant since many deficits in synaptic transmission in various systems are compensated for either physiologically or structural over development, but not in this case for reduced calcium entry during evoked transmission.

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.