Motor Nerve Terminals Research Articles

Anti-ganglioside antibodies are removed from circulation in mice by neuronal endocytosis.

SEE VAN DOORN AND JACOBS DOI101093/BRAIN/AWW078 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE : In axonal forms of Guillain-Barré syndrome, anti-ganglioside antibodies bind gangliosides on nerve surfaces, thereby causing injury through complement activation and immune cell recruitment. Why some nerve regions are more vulnerable than others is unknown. One reason may be that neuronal membranes with high endocytic activity, including nerve terminals involved in neurotransmitter recycling, are able to endocytose anti-ganglioside antibodies from the cell surface so rapidly that antibody-mediated injury is attenuated. Herein we investigated whether endocytic clearance of anti-ganglioside antibodies by nerve terminals might also be of sufficient magnitude to deplete circulating antibody levels. Remarkably, systemically delivered anti-ganglioside antibody in mice was so avidly cleared from the circulation by endocytosis at ganglioside-expressing plasma membranes that it was rapidly rendered undetectable in serum. A major component of the clearance occurred at motor nerve terminals of neuromuscular junctions, from where anti-ganglioside antibody was retrogradely transported to the motor neuron cell body in the spinal cord, recycled to the plasma membrane, and secreted into the surrounding spinal cord. Uptake at the neuromuscular junction represents a major unexpected pathway by which pathogenic anti-ganglioside antibodies, and potentially other ganglioside binding proteins, are cleared from the systemic circulation and also covertly delivered to the central nervous system.

Individual sympathetic postganglionic neurons coinnervate myenteric ganglia and smooth muscle layers in the gastrointestinal tract of the rat.

A full description of the terminal architecture of sympathetic axons innervating the gastrointestinal (GI) tract has not been available. To label sympathetic fibers projecting to the gut muscle wall, dextran biotin was injected into the celiac and superior mesenteric ganglia (CSMG) of rats. Nine days postinjection, animals were euthanized and stomachs and small intestines were processed as whole mounts (submucosa and mucosa removed) to examine CSMG efferent terminals. Myenteric neurons were counterstained with Cuprolinic Blue; catecholaminergic axons were stained immunohistochemically for tyrosine hydroxylase. Essentially all dextran-labeled axons (135 of 136 sampled) were tyrosine hydroxylase-positive. Complete postganglionic arbors (n = 154) in the muscle wall were digitized and analyzed morphometrically. Individual sympathetic axons formed complex arbors of varicose neurites within myenteric ganglia/primary plexus and, concomitantly, long rectilinear arrays of neurites within circular muscle/secondary plexus or longitudinal muscle/tertiary plexus. Very few CSMG neurons projected exclusively (i.e., ∼100% of an arbor's varicose branches) to myenteric plexus (∼2%) or smooth muscle (∼14%). With less stringent inclusion criteria (i.e., ≥85% of an axon's varicose branches), larger minorities of neurons projected predominantly to either myenteric plexus (∼13%) or smooth muscle (∼27%). The majority (i.e., ∼60%) of all individual CSMG postganglionics formed mixed, heterotypic arbors that coinnervated extensively (>15% of their varicose branches per target) both myenteric ganglia and smooth muscle. The fact that ∼87% of all sympathetics projected either extensively or even predominantly to smooth muscle, while simultaneously contacting myenteric plexus, is consistent with the view that these neurons control GI muscle directly, if not exclusively. J. Comp. Neurol. 524:2577-2603, 2016. © 2016 Wiley Periodicals, Inc.

Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles.

Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity. Synaptic vesicles loaded with pHrodo-BoNT/A-Hc exhibited a significantly reduced ability to fuse with the plasma membrane in mouse hippocampal nerve terminals when compared with pHrodo-dextran-containing synaptic vesicles and pHrodo-labeled anti-GFP nanobodies bound to VAMP2-pHluorin or vGlut-pHluorin. Similar results were also obtained at the amphibian neuromuscular junction. These results reveal that BoNT/A is internalized in a subpopulation of synaptic vesicles that are not destined to recycle, highlighting the existence of significant molecular and functional heterogeneity between synaptic vesicles.

Presynaptic Proteins as Markers of the Neurotoxic Activity of BmjeTX-I and BmjeTX-II Toxins from Bothrops marajoensis (Marajó Lancehead) Snake Venom.

Neuromuscular preparations exposed to B. marajoensis venom show increases in the frequency of miniature end-plate potentials and twitch tension facilitation followed by presynaptic neuromuscular paralysis, without evidences of muscle damage. Considering that presynaptic toxins interfere into the machinery involved in neurotransmitter release (synaptophysin, synaptobrevin, and SNAP25 proteins), the main objective of this communication is to analyze, by immunofluorescence and western blotting, the expression of the synaptic proteins, synaptophysin, synaptobrevin, and SNAP25 and by myography, light, and transmission electron microscopy the pathology of motor nerve terminals and skeletal muscle fibres of chick biventer cervicis preparations (CBC) exposed in vitro to BmjeTX-I and BmjeTX-II toxins from B. marajoensis venom. CBC incubated with toxins showed irreversible twitch tension blockade and unaffected KCl- and ACh-evoked contractures, and the positive colabelling of acetylcholine receptors confirmed that their action was primarily at the motor nerve terminal. Hypercontraction and loose myofilaments and synaptic vesicle depletion and motor nerve damage indicated that the toxins displayed both myotoxic and neurotoxic effect. The blockade resulted from interference on synaptophysin, synaptobrevin, and SNAP25 proteins leading to the conclusion that BmjeTX-I and BmjeTX-II affected neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal.

Correlation between afferent rearrangements and behavioral deficits after local excitotoxic insult in the mammalian vestibule: a rat model of vertigo symptoms.

ABSTRACTDamage to inner ear afferent terminals is believed to result in many auditory and vestibular dysfunctions. The sequence of afferent injuries and repair, as well as their correlation with vertigo symptoms, remains poorly documented. In particular, information on the changes that take place at the primary vestibular endings during the first hours following a selective insult is lacking. In the present study, we combined histological analysis with behavioral assessments of vestibular function in a rat model of unilateral vestibular excitotoxic insult. Excitotoxicity resulted in an immediate but transient alteration of the balance function that was resolved within a week. Concomitantly, vestibular primary afferents underwent a sequence of structural changes followed by spontaneous repair. Within the first two hours after the insult, a first phase of pronounced vestibular dysfunction coincided with extensive swelling of afferent terminals. In the next 24 h, a second phase of significant but incomplete reduction of the vestibular dysfunction was accompanied by a resorption of swollen terminals and fiber retraction. Eventually, within 1 week, a third phase of complete balance restoration occurred. The slow and progressive withdrawal of the balance dysfunction correlated with full reconstitution of nerve terminals. Competitive re-innervation by afferent and efferent terminals that mimicked developmental synaptogenesis resulted in full re-afferentation of the sensory epithelia. By deciphering the sequence of structural alterations that occur in the vestibule during selective excitotoxic impairment, this study offers new understanding of how a vestibular insult develops in the vestibule and how it governs the heterogeneity of vertigo symptoms.

The effects of ER morphology on synaptic structure and function in Drosophila melanogaster.

Hereditary spastic paraplegia (HSP) is a set of genetic diseases caused by mutations in one of 72 genes that results in age-dependent corticospinal axon degeneration accompanied by spasticity and paralysis. Two genes implicated in HSPs encode proteins that regulate endoplasmic reticulum (ER) morphology. Atlastin 1 (ATL1, also known as SPG3A) encodes an ER membrane fusion GTPase and reticulon 2 (RTN2, also known as SPG12) helps shape ER tube formation. Here, we use a new fluorescent ER marker to show that the ER within wild-type Drosophila motor nerve terminals forms a network of tubules that is fragmented and made diffuse upon loss of the atlastin 1 ortholog atl. atl or Rtnl1 loss decreases evoked transmitter release and increases arborization. Similar to other HSP proteins, Atl inhibits bone morphogenetic protein (BMP) signaling, and loss of atl causes age-dependent locomotor deficits in adults. These results demonstrate a crucial role for ER in neuronal function, and identify mechanistic links between ER morphology, neuronal function, BMP signaling and adult behavior.

Homocysteine aggravates ROS-induced depression of transmitter release from motor nerve terminals: potential mechanism of peripheral impairment in motor neuron diseases associated with hyperhomocysteinemia.

Homocysteine (HCY) is a pro-inflammatory sulphur-containing redox active endogenous amino acid, which concentration increases in neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). A widely held view suggests that HCY could contribute to neurodegeneration via promotion of oxidative stress. However, the action of HCY on motor nerve terminals has not been investigated so far. We previously reported that oxidative stress inhibited synaptic transmission at the neuromuscular junction, targeting primarily the motor nerve terminals. In the current study, we investigated the effect of HCY on oxidative stress-induced impairment of transmitter release at the mouse diaphragm muscle. The mild oxidant H2O2 decreased the intensity of spontaneous quantum release from nerve terminals (measured as the frequency of miniature endplate potentials, MEPPs) without changes in the amplitude of MEPPs, indicating a presynaptic effect. Pre-treatment with HCY for 2 h only slightly affected both amplitude and frequency of MEPPs but increased the inhibitory potency of H2O2 almost two fold. As HCY can activate certain subtypes of glutamate N-methyl D-aspartate (NMDA) receptors we tested the role of NMDA receptors in the sensitizing action of HCY. Remarkably, the selective blocker of NMDA receptors, AP-5 completely removed the sensitizing effect of HCY on the H2O2-induced presynaptic depressant effect. Thus, at the mammalian neuromuscular junction HCY largely increases the inhibitory effect of oxidative stress on transmitter release, via NMDA receptors activation. This combined effect of HCY and local oxidative stress can specifically contribute to the damage of presynaptic terminals in neurodegenerative motoneuron diseases, including ALS.

Ubiquitin-Synaptobrevin Fusion Protein Causes Degeneration of Presynaptic Motor Terminals in Mice.

Degeneration of motor nerve terminals occurs in amyotrophic lateral sclerosis (ALS) patients as well as in mouse models of ALS, leading to progressive paralysis. What causes a motor nerve terminal to degenerate remains unknown. Here we report on transgenic mice expressing a ubiquitinated synaptic vesicle protein (Ub(G76V)-GFP-Syb2) that develop progressive degeneration of motor nerve terminals. These mice may serve as a model for further elucidating the underlying cellular and molecular mechanisms of presynaptic nerve terminal degeneration.

Synaptic-like vesicles and candidate transduction channels in mechanosensory terminals.

This article summarises progress to date over an exciting and very enjoyable first 15 years of collaboration with Bob Banks. Our collaboration began when I contacted him with (to me) an unexpected observation that a dye used to mark recycling synaptic vesicle membrane at efferent terminals also labelled muscle spindle afferent terminals. This observation led to the re-discovery of a system of small clear vesicles present in all vertebrate primary mechanosensory nerve terminals. These synaptic-like vesicles (SLVs) have been, and continue to be, the major focus of our work. This article describes our characterisation of the properties and functional significance of these SLVs, combining our complementary skills: Bob’s technical expertise and encyclopaedic knowledge of mechanosensation with my experience of synaptic vesicles and the development of the styryl pyridinium dyes, of which the most widely used is FM1-43. On the way we have found that SLVs seem to be part of a constitutive glutamate secretory system necessary to maintain the stretch-sensitivity of spindle endings. The glutamate activates a highly unusual glutamate receptor linked to phospholipase D activation, which we have termed the PLD-mGluR. It has a totally distinct pharmacology first described in the hippocampus nearly 20 years ago but, like the SLVs that were first described over 50 years ago, has since been little researched. Yet, our evidence and literature searches suggest this glutamate/SLV/PLD-mGluR system is a ubiquitous feature of mechanosensory endings and, at least for spindles, is essential for maintaining mechanosensory function. This article summarises how this system integrates with the classical model of mechanosensitive channels in spindles and other mechanosensory nerve terminals, including hair follicle afferents and baroreceptors controlling blood pressure. Finally, in this time when there is an imperative to show translational relevance, I describe how this fascinating system might actually be a useful therapeutic drug target for clinical conditions such as hypertension and muscle spasticity. This has been a fascinating 15-year journey in collaboration with Bob who, as well as having an astute scientific mind, is also a great enthusiast, motivator and friend. I hope this exciting and enjoyable journey will continue well into the future.

Effects of IgG anti-GM1 monoclonal antibodies on neuromuscular transmission and calcium channel binding in rat neuromuscular junctions.

Guillain-Barré syndrome is a type of acute inflammatory neuropathy that causes ataxia and is associated with the IgG anti-GM1 antibody. However, the pathogenic role of the IgG anti-GM1 antibody and calcium channels in neuromuscular junctions (NMJs) remains unclear. Thus, the aim of the present study was to investigate the effects of the IgG anti-GM1 monoclonal antibody (mAb) on spontaneous muscle action potentials (SMAPs), and the effects of calcium channel blockers, in a rat spinal cord-muscle co-culture system. In addition, the binding of IgG anti-GM1 mAb to calcium channels was investigated in the rat hemidiaphragm. The frequency of SMAPs in the innervated muscle cells was acutely inhibited by the IgG anti-GM1 mAb; however, this effect was blocked by the N-type calcium channel blocker, ω-conotoxin GVIA (30 nM). Furthermore, the P/Q-type calcium channel blocker, ω-agatoxin IVA (10 nM), was found to partially block the IgG anti-GM1 mAb-induced inhibitory effect in the spinal cord-muscle co-culture system. Immunohistochemical analysis of the rat hemidiaphragm indicated that IgG anti-GM1 mAb binding overlapped with anti-Cav2.2 (α1B) antibody binding in the nerve terminal. In addition, IgG anti-GM1 mAb binding partially overlapped with anti-Cav2.1 (α1A) antibody binding. Thus, the results demonstrated that the IgG anti-GM1 mAb binds to calcium channels in the nerve terminals of NMJs. Therefore, the inhibitory effect of IgG anti-GM1 mAb on SMAPs may involve N-type and P/Q-type calcium channels in motor nerve terminals at the NMJ.

Different dynamin blockers interfere with distinct phases of synaptic endocytosis during stimulation in motoneurones.

Neurotransmitter release requires a tight coupling between synaptic vesicle exocytosis and endocytosis with dynamin being a key protein in that process. We used imaging techniques to examine the time course of endocytosis at mouse motor nerve terminals expressing synaptopHluorin, a genetically encoded reporter of the synaptic vesicle cycle. We separated two sequential phases of endocytosis taking place during the stimulation train: early and late endocytosis. Freshly released synaptic vesicle proteins are preferentially retrieved during the early phase, which is very sensitive to dynasore, an inhibitor of dynamin GTPase activity. Synaptic vesicle proteins pre-existing at the plasma membrane before the stimulation are preferentially retrieved during the late phase, which is very sensitive to myristyl trimethyl ammonium bromide (MitMAB), an inhibitor of the dynamin-phospholipid interaction. Synaptic endocytosis is essential at nerve terminals to maintain neurotransmitter release by exocytosis. Here, at the neuromuscular junction of synaptopHluorin (spH) transgenic mice, we have used imaging to study exo- and endocytosis occurring simultaneously during nerve stimulation. We observed two endocytosis components, which occur sequentially during stimulation. The early component of endocytosis apparently internalizes spH molecules freshly exocytosed. This component was sensitive to dynasore, a blocker of dynamin 1 GTPase activity. In contrast, this early component was resistant to myristyl trimethyl ammonium bromide (MiTMAB), a competitive agent that blocks dynamin binding to phospholipid membranes. The late component of endocytosis is likely to internalize spH molecules that pre-exist at the plasma membrane before stimulation starts. This component was blocked by MiTMAB, perhaps by impairing the binding of dynamin or other key endocytic proteins to phospholipid membranes. Our study suggests the co-existence of two sequential synaptic endocytosis steps taking place during stimulation that are susceptible to pharmacological dissection: an initial step, preferentially sensitive to dynasore, that internalizes vesicular components immediately after they are released, and a MiTMAB-sensitive step that internalizes vesicular components pre-existing at the plasma membrane surface. In addition, we report that post-stimulus endocytosis also has several components with different sensitivities to dynasore and MiTMAB.

Therapeutic Effect of Dexamethasone for Noise-induced Hearing Loss: Systemic Versus Intratympanic Injection in Mice.

Dexamethasone is commonly used clinically to treat noise-induced hearing loss (NIHL) because the drug exerts multiple anti-inflammatory effects. In the present study, we investigated the post-noise therapeutic effects of dexamethasone given systemically or via intratympanic injection in the mouse. Twenty-four C57BL/6J mice were used. Eighteen experimental mice were exposed to 110 dB sound pressure level white noise and then divided into three groups: the noise, intraperitoneal dexamethasone injection (IP), and intratympanic dexamethasone injection (IT) groups. Dexamethasone (3 mg/kg/d) was injected intraperitoneally for five successive days in the IP group. Intratympanic injections were given on post-noise days 1 and 4 in the IT group. We compared hearing levels, the architecture of the organ of Corti (OC), and the microscopic appearance of the medial olivocochlear efferent terminals (MOC ETs) among the groups. Both the IP and IT groups exhibited hearing recovery as revealed by auditory brainstem responses (ABRs), but recovery was not apparent in distortion product otoacoustic emissions (DPOAEs). OC degeneration as revealed by light microscopy was most extensive in the noise group and least extensive in the IP group. Scanning electron microscopy showed that the OC ultrastructure was better preserved in the IP than the IT group. Confocal microscopy showed that the ETs were shrunken in all noise-exposed groups as compared to the control group, but more shrunken in the dexamethasone-treated groups. Transmission electron microscopy showed that the MOC ET-outer hair cell (OHC) synapses were damaged in all noise-exposed groups, but the extent of degeneration was less in the IT than in the noise group. Dexamethasone exerts reliable therapeutic effects when used to treat NIHL. It seems that the protective effects may differ by the routes of administration as the OCs were better preserved in the IP group and the ET-OHC synapses were more intact in the IT group.

Deterioration of the Medial Olivocochlear Efferent System Accelerates Age-Related Hearing Loss in Pax2-Isl1 Transgenic Mice.

The development, maturation, and maintenance of the inner ear are governed by temporal and spatial expression cascades of transcription factors that form a gene regulatory network. ISLET1 (ISL1) may be one of the major players in this cascade, and in order to study its role in the regulation of inner ear development, we produced a transgenic mouse overexpressing Isl1 under the Pax2 promoter. Pax2-regulated ISL1 overexpression increases the embryonic ISL1(+) domain and induces accelerated nerve fiber extension and branching in E12.5 embryos. Despite these gains in early development, the overexpression of ISL1 impairs the maintenance and function of hair cells of the organ of Corti. Mutant mice exhibit hyperactivity, circling behavior, and progressive age-related decline in hearing functions, which is reflected in reduced otoacoustic emissions (DPOAEs) followed by elevated hearing thresholds. The reduction of the amplitude of DPOAEs in transgenic mice was first detected at 1 month of age. By 6-9 months of age, DPOAEs completely disappeared, suggesting a functional inefficiency of outer hair cells (OHCs). The timing of DPOAE reduction coincides with the onset of the deterioration of cochlear efferent terminals. In contrast to these effects on efferents, we only found a moderate loss of OHCs and spiral ganglion neurons. For the first time, our results show that the genetic alteration of the medial olivocochlear (MOC) efferent system induces an early onset of age-related hearing loss. Thus, the neurodegeneration of the MOC system could be a contributing factor to the pathology of age-related hearing loss.

Control of autophagosome axonal retrograde flux by presynaptic activity unveiled using botulinum neurotoxin type a.

Botulinum neurotoxin type A (BoNT/A) is a highly potent neurotoxin that elicits flaccid paralysis by enzymatic cleavage of the exocytic machinery component SNAP25 in motor nerve terminals. However, recent evidence suggests that the neurotoxic activity of BoNT/A is not restricted to the periphery, but also reaches the CNS after retrograde axonal transport. Because BoNT/A is internalized in recycling synaptic vesicles, it is unclear which compartment facilitates this transport. Using live-cell confocal and single-molecule imaging of rat hippocampal neurons cultured in microfluidic devices, we show that the activity-dependent uptake of the binding domain of the BoNT/A heavy chain (BoNT/A-Hc) is followed by a delayed increase in retrograde axonal transport of BoNT/A-Hc carriers. Consistent with a role of presynaptic activity in initiating transport of the active toxin, activity-dependent uptake of BoNT/A in the terminal led to a significant increase in SNAP25 cleavage detected in the soma chamber compared with nonstimulated neurons. Surprisingly, most endocytosed BoNT/A-Hc was incorporated into LC3-positive autophagosomes generated in the nerve terminals, which then underwent retrograde transport to the cell soma, where they fused with lysosomes both in vitro and in vivo. Blocking autophagosome formation or acidification with wortmannin or bafilomycin A1, respectively, inhibited the activity-dependent retrograde trafficking of BoNT/A-Hc. Our data demonstrate that both the presynaptic formation of autophagosomes and the initiation of their retrograde trafficking are tightly regulated by presynaptic activity.

Pharmacologically distinct nicotinic acetylcholine receptors drive efferent-mediated excitation in calyx-bearing vestibular afferents.

Electrical stimulation of vestibular efferent neurons rapidly excites the resting discharge of calyx/dimorphic (CD) afferents. In turtle, this excitation arises when acetylcholine (ACh), released from efferent terminals, directly depolarizes calyceal endings by activating nicotinic ACh receptors (nAChRs). Although molecular biological data from the peripheral vestibular system implicate most of the known nAChR subunits, specific information about those contributing to efferent-mediated excitation of CD afferents is lacking. We sought to identify the nAChR subunits that underlie the rapid excitation of CD afferents and whether they differ from α9α10 nAChRs on type II hair cells that drive efferent-mediated inhibition in adjacent bouton afferents. We recorded from CD and bouton afferents innervating the turtle posterior crista during electrical stimulation of vestibular efferents while applying several subtype-selective nAChR agonists and antagonists. The α9α10 nAChR antagonists, α-bungarotoxin and α-conotoxin RgIA, blocked efferent-mediated inhibition in bouton afferents while leaving efferent-mediated excitation in CD units largely intact. Conversely, 5-iodo-A-85380, sazetidine-A, varenicline, α-conotoxin MII, and bPiDDB (N,N-dodecane-1,12-diyl-bis-3-picolinium dibromide) blocked efferent-mediated excitation in CD afferents without affecting efferent-mediated inhibition in bouton afferents. This pharmacological profile suggested that calyceal nAChRs contain α6 and β2, but not α9, nAChR subunits. Selective blockade of efferent-mediated excitation in CD afferents distinguished dimorphic from calyx afferents by revealing type II hair cell input. Dimorphic afferents differed in having higher mean discharge rates and a mean efferent-mediated excitation that was smaller in amplitude yet longer in duration. Molecular biological data demonstrated the expression of α9 in turtle hair cells and α4 and β2 in associated vestibular ganglia.

Internalization and retrograde axonal trafficking of tetanus toxin in motor neurons and trans-synaptic propagation at central synapses exceed those of its C-terminal-binding fragments.

The prominent tropism of tetanus toxin (TeTx) towards peripheral nerves with retrograde transport and transfer to central neurons render it an invaluable probe for exploring fundamental neuronal processes such as endocytosis, retrograde trafficking and trans-synaptic transport to central neurons. While the specificity of TeTx to nerve cells has been attributed to its binding domains (HC and HCC), molecular determinants of the long-range trafficking that ensure its central delivery and induction of spastic paralysis remain elusive. Here, we report that a protease-inactive TeTx mutant (TeTIM) fused to core streptavidin (CS) proved superior to CS-HC and CS-HCC fragments in antagonizing the internalization of the active toxin in cultured spinal cord neurons. Also, in comparison to CS-HC and CS-HCC, CS-TeTIM undergoes faster clearance from motor nerve terminals after peripheral injection, and is detected in a greater number of neurons in the spinal cord and brain stem ipsi-lateral to the administration site. Consistent with trans-synaptic transfer from motor neurons to inter-neurons, CS-TeTIM infiltrated non-cholinergic cells in the spinal cord; in contrast, the retrograde spread of CS-HC was largely restricted to neurons stained for choline acetyltransferase. Peripheral injection of CS-TeTIM conjugated to a lentivirus encoding mutated SNAP-25, resistant to cleavage by botulinum neurotoxin A, E and C1, rendered spontaneous excitatory postsynaptic currents in motor neurons resilient to challenge by type A toxin in vitro, whereas the same virus conjugated to CS-HC proved ineffective. These findings indicate that full-length inactive TeTx greatly exceeds HC and HCC in targeting and invading motor nerve terminals at the periphery and exploits more efficiently the retrograde transport and trans-synaptic transfer mechanisms of motor neurons to arrive at central neurons. Such qualities render TeTIM a more suitable research probe and neuron-targeting vehicle for retro-axonal delivery of viral vectors to the CNS.

The novel protein kinase C epsilon isoform at the adult neuromuscular synapse: location, regulation by synaptic activity-dependent muscle contraction through TrkB signaling and coupling to ACh release.

BackgroundProtein kinase C (PKC) regulates a variety of neural functions, including neurotransmitter release. Although various PKC isoforms can be expressed at the synaptic sites and specific cell distribution may contribute to their functional diversity, little is known about the isoform-specific functions of PKCs in neuromuscular synapse. The present study is designed to examine the location of the novel isoform nPKCε at the neuromuscular junction (NMJ), their synaptic activity-related expression changes, its regulation by muscle contraction, and their possible involvement in acetylcholine release.ResultsWe use immunohistochemistry and confocal microscopy to demonstrate that the novel isoform nPKCε is exclusively located in the motor nerve terminals of the adult rat NMJ. We also report that electrical stimulation of synaptic inputs to the skeletal muscle significantly increased the amount of nPKCε isoform as well as its phosphorylated form in the synaptic membrane, and muscle contraction is necessary for these nPKCε expression changes. The results also demonstrate that synaptic activity-induced muscle contraction promotes changes in presynaptic nPKCε through the brain-derived neurotrophic factor (BDNF)-mediated tyrosine kinase receptor B (TrkB) signaling. Moreover, nPKCε activity results in phosphorylation of the substrate MARCKS involved in actin cytoskeleton remodeling and related with neurotransmission. Finally, blocking nPKCε with a nPKCε-specific translocation inhibitor peptide (εV1-2) strongly reduces phorbol ester-induced ACh release potentiation, which further indicates that nPKCε is involved in neurotransmission.ConclusionsTogether, these results provide a mechanistic insight into how synaptic activity-induced muscle contraction could regulate the presynaptic action of the nPKCε isoform and suggest that muscle contraction is an important regulatory step in TrkB signaling at the NMJ.

TRPV1 Activation Modulates Acetylcholine Release at Myoneural Junctions

Transient receptor potential (TRP) proteins are non-selective cation channel proteins that are expressed throughout the body. Previous studies show that TRP Vanilloid 1 (TRPV1), capsaicin (CAP) receptor, is expressed in sensory neurons. Here we report that TRPV1 protein is expressed at the motor nerve terminals and that activation of TRPV1 by CAP suppresses acetylcholine release, in vitro. We analyzed twitch tension, spontaneous and nerve stimulus evoked acetylcholine release from phrenic nerve diaphragm nerve muscle preparations (NMP) isolated from isoflurane anesthetized adult wild type mice and that genetically lacked TRPV1 (TRPV1-/-). When acutely applied to isolated NMP, 1 µM CAP produced a concentration-dependent decline of twitch tension and a significant decline in the amplitude of stimulus evoked endplate currents (EPCs) and quantal content without any effect on the miniature endplate currents (mEPCs). The suppression of nerve stimulus evoked acetylcholine release by CAP was antagonized by capsazepine (CPZ; 10 µM), a TRPV1 antagonist. However, CAP did not suppress phrenic nerve stimulus evoked acetylcholine release in TRPV1−/− mice. Also, CAP treatment, in vitro, interfered with the localization of adapter protein 2 in cholinergic Neuro 2a cells. Wortmannin, (WMN; 10 µM; non-selective phosphoinositol kinase inhibitor), mimicked the effects of CAP by inhibiting the acetylcholine exocytosis. Our data suggest that TRPV1 proteins expressed at the motor nerve terminal are coupled to the exo-endocytic mechanisms to regulate neuromuscular functions.

Effects of IgM Anti-Galnac-GD1a Monoclonal Antibody on Neuromuscular Transmission and Calcium Channel Binding in the Rat Neuromuscular Junction

Guillain-Barre syndrome with antibodies against ganglioside N-acetylgalactosaminyl GD1a (GalNAc-GD1a) is characterized by a rapid onset of predominantly distal pure motor neuropathy. However, the pathogenic role of anti- GalNAc-GD1a antibody and calcium channels in neuromuscular junctions (NMJs) remains unclear. We investigated the effects of IgM anti-GalNAc-GD1a monoclonal antibody (IgM anti-GalNAc-GD1a mAb) on spontaneous muscle action potentials (SMAP) in a rat spinal cord–muscle co-culture system and the localization of IgM anti-GalNAc- GD1a mAb and calcium channel binding in the rat hemi-diaphragm. Immunohistochemistry of the rat hemidiaphragm showed that IgM anti-GalNAc-GD1a mAb binding overlapped with anti-neurofilament 200 antibody and α- bungarotoxin staining, demonstrating that IgM anti-GalNAc-GD1a mAb was localized at the motor nerve terminal. Moreover, IgM anti-GalNAc-GD1a mAb binding overlapped with anti-Cav2.1 antibody in the nerve terminal. We suggest that the inhibitory effect of IgM anti-GalNAc-GD1a mAb on SMAP is related to the GalNAc-GD1a epitope on P/Q-type calcium channels in motor nerve terminals at NMJs.

Loss of β2-laminin alters calcium sensitivity and voltage-gated calcium channel maturation of neurotransmission at the neuromuscular junction.

Neuromuscular junctions from β2-laminin-deficient mice exhibit lower levels of calcium sensitivity. Loss of β2-laminin leads to a failure in switching from N- to P/Q-type voltage-gated calcium channel (VGCC)-mediated transmitter release that normally occurs with neuromuscular junction maturation. The motor nerve terminals from β2-laminin-deficient mice fail to up-regulate the expression of P/Q-type VGCCs clusters and down-regulate N-type VGCCs clusters, as they mature. There is decreased co-localisation of presynaptic specialisations in β2-laminin-deficient neuromuscular junctions as a consequence of lesser P/Q-type VGCC expression. These findings support the idea that β2-laminin is critical in the organisation and maintenance of active zones at the neuromuscular junction via its interaction with P/Q-type VGCCs, which aid in stabilisation of the synapse. β2-laminin is a key mediator in the differentiation and formation of the skeletal neuromuscular junction. Loss of β2-laminin results in significant structural and functional aberrations such as decreased number of active zones and reduced spontaneous release of transmitter. In vitro β2-laminin has been shown to bind directly to the pore forming subunit of P/Q-type voltage-gated calcium channels (VGCCs). Neurotransmission is initially mediated by N-type VGCCs, but by postnatal day 18 switches to P/Q-type VGCC dominance. The present study investigated the changes in neurotransmission during the switch from N- to P/Q-type VGCC-mediated transmitter release at β2-laminin-deficient junctions. Analysis of the relationship between quantal content and extracellular calcium concentrations demonstrated a decrease in the calcium sensitivity, but no change in calcium dependence at β2-laminin-deficient junctions. Electrophysiological studies on VGCC sub-types involved in transmitter release indicate N-type VGCCs remain the primary mediator of transmitter release at matured β2-laminin-deficient junctions. Immunohistochemical analyses displayed irregularly shaped and immature β2-laminin-deficient neuromuscular junctions when compared to matured wild-type junctions. β2-laminin-deficient junctions also maintained the presence of N-type VGCC clustering within the presynaptic membrane, which supported the functional findings of the present study. We conclude that β2-laminin is a key regulator in development of the NMJ, with its loss resulting in reduced transmitter release due to decreased calcium sensitivity stemming from a failure to switch from N- to P/Q-type VGCC-mediated synaptic transmission.