Mammalian Neuromuscular Junction Research Articles

Neuronal LRP4 directs the development, maturation and cytoskeletal organization of Drosophila peripheral synapses.

Synaptic development requires multiple signaling pathways to ensure successful connections. Transmembrane receptors are optimally positioned to connect the synapse and the rest of the neuron, often acting as synaptic organizers to synchronize downstream events. One such organizer, the LDL receptor-related protein LRP4, is a cell surface receptor that has been most well-studied postsynaptically at mammalian neuromuscular junctions. Recent work, however, identified emerging roles, but how LRP4 acts as a presynaptic organizer and the downstream mechanisms of LRP4 are not well understood. Here, we show that LRP4 functions presynaptically at Drosophila neuromuscular synapses, acting in motoneurons to instruct pre- and postsynaptic development. Loss of presynaptic LRP4 results in multiple defects, impairing active zone organization, synapse growth, physiological function, microtubule organization, synaptic ultrastructure and synapse maturation. We further demonstrate that LRP4 promotes most aspects of presynaptic development via a downstream SR-protein kinase, SRPK79D. These data demonstrate a function for presynaptic LRP4 as a peripheral synaptic organizer, highlight a downstream mechanism conserved with its CNS function in Drosophila, and underscore previously unappreciated but important developmental roles for LRP4 in cytoskeletal organization, synapse maturation and active zone organization.

B-092 Limited Clinical Utility of N-type Antibody Testing in Lambert-Eaton Myasthenic Syndrome

Abstract Background Lambert-Eaton myasthenic syndrome (LEMS) is caused by IgG-mediated disruption of certain voltage-gated Ca2+-channel (VGCC) functions and diminished peripheral motor nerve-stimulated release of acetylcholine by presynaptic terminals at the neuromuscular junction and selected autonomic synapses. Both P/Q-type (Cav2.1) and N-type (Cav.2) channels are the targets of IgG-mediated autoimmunity in LEMS. Functional presynaptic VGCCs at the healthy mature mammalian neuromuscular junction are P/Q-type. About 60% of LEMS cases are paraneoplastic, of which a majority are associated with small-cell lung cancer (SCLC-LEMS). LEMS can also have a non-paraneoplastic etiology (idiopathic, non-tumor LEMS [NT-LEMS]). It is important to have reliable autoantibody biomarkers to aid the serological diagnosis of LEMS and to distinguish between SCLC-LEMS and NT-LEMS, because the treatment and prognosis differ. Previous studies have reported seropositivity for SOX1/AGNA-1-IgG in ∼ 64% of SCLC-LEMS cases, but in no NT-LEMS cases. When coexisting with P/Q-type VGCC IgG in LEMS patients, IgGs of both N-type VGCC and SOX1/AGNA-1 IgG specificities have been suggested as useful biomarkers for predicting SCLC. Here we compared the clinical utility of serological tests for P/Q-type and N-type VGCCs and SOX1/AGNA-1 IgGs as diagnostic aids for LEMS and for differentiating paraneoplastic from idiopathic LEMS. Methods This is a retrospective study of 102 patients with a clinical (electrophysiologically confirmed) diagnosis of LEMS who were tested for both P/Q-type and N-type VGCC IgGs by radioimmunoprecipitation assays performed at the Mayo Clinic Neuroimmunology Laboratory. 95 patients had comprehensive clinical screening for cancer; 66 had NT-LEMS, 21 had SCLC-LEMS and 8 had LEMS with non-SCLC cancers. Historical data were reviewed retrospectively. Of the NT-LEMS and SCLC-LEMS patients, 76 were tested for SOX1/AGNA-1 IgG. 122 healthy sex and age-matched controls were tested. Results Among the 102 LEMS patients, 92 (90%) were P/Q-VGCC IgG positive (P/Q+) and 26 (26%) were N-VGCC IgG positive (N+); 67 (66%) were P/Q+ only, 1 (0.9%) was N+ only, 25 (25%) were P/Q+N+, and 9 (9%) were seronegative for both. Three of the 122 healthy control subjects (2.5%) were N+ only, the rest were seronegative. The frequency of isolated N-type VGCC seropositivity did not differ significantly for LEMS patients and healthy controls (P = 0.69). All 21 SCLC-LEMS cases (100%) were P/Q-VGCC IgG positive, compared with 58 of the 66 NT-LEMS cases (88%). The rate of N-type VGCC IgG positivity in P/Q+ LEMS patients did not differ significantly for SCLC-LEMS (5 of 21) and NT-LEMS (13 of 58) diagnoses (24% vs 22%, P = 0.90). The frequency of SOX1/AGNA-1 IgG was 35% for the SCLC-LEMS cases (7 of 20), but none of the NT-LEMS cases (n = 56) was SOX1/AGNA-1 IgG positive. Conclusion N-type VGCC improved neither sensitivity for LEMS nor positive predictive value for cancer and has limited diagnostic utility in assessing patients with suspected LEMS. PQ-type VGCC IgG negativity does not exclude a diagnosis of LEMS. SOX1/AGNA-1 positivity has high specificity but low sensitivity for SCLC. Therefore, while antibody testing confirms a diagnosis of autoimmune LEMS, electrophysiology (to confirm neurological diagnosis) and body imaging (for cancer detection) remain important for the diagnostic evaluation.

NMJ-related diseases beyond the congenital myasthenic syndromes.

Neuromuscular junctions (NMJs) are a special type of chemical synapse that transmits electrical stimuli from motor neurons (MNs) to their innervating skeletal muscle to induce a motor response. They are an ideal model for the study of synapses, given their manageable size and easy accessibility. Alterations in their morphology or function lead to neuromuscular disorders, such as the congenital myasthenic syndromes, which are caused by mutations in proteins located in the NMJ. In this review, we highlight novel potential candidate genes that may cause or modify NMJs-related pathologies in humans by exploring the phenotypes of hundreds of mouse models available in the literature. We also underscore the fact that NMJs may differ between species, muscles or even sexes. Hence the importance of choosing a good model organism for the study of NMJ-related diseases: only taking into account the specific features of the mammalian NMJ, experimental results would be efficiently translated to the clinic.

Postsynaptic Calcium Extrusion at the Mouse Neuromuscular Junction Alkalinizes the Synaptic Cleft.

Neurotransmission is shaped by extracellular pH. Alkalization enhances pH-sensitive transmitter release and receptor activation, whereas acidification inhibits these processes and can activate acid-sensitive conductances in the synaptic cleft. Previous work has shown that the synaptic cleft can either acidify because of synaptic vesicular release and/or alkalize because of Ca2+ extrusion by the plasma membrane ATPase (PMCA). The direction of change differs across synapse types. At the mammalian neuromuscular junction (NMJ), the direction and magnitude of pH transients in the synaptic cleft during transmission remain ambiguous. We set out to elucidate the extracellular pH transients that occur at this cholinergic synapse under near-physiological conditions and identify their sources. We monitored pH-dependent changes in the synaptic cleft of the mouse levator auris longus using viral expression of the pseudoratiometric probe pHusion-Ex in the muscle. Using mice from both sexes, a significant and prolonged alkalization occurred when stimulating the connected nerve for 5 s at 50 Hz, which was dependent on postsynaptic intracellular Ca2+ release. Sustained stimulation for a longer duration (20 s at 50 Hz) caused additional prolonged net acidification at the cleft. To investigate the mechanism underlying cleft alkalization, we used muscle-expressed GCaMP3 to monitor the contribution of postsynaptic Ca2+ Activity-induced liberation of intracellular Ca2+ in muscle positively correlated with alkalization of the synaptic cleft, whereas inhibiting PMCA significantly decreased the extent of cleft alkalization. Thus, cholinergic synapses of the mouse NMJ typically alkalize because of cytosolic Ca2+ liberated in muscle during activity, unless under highly strenuous conditions where acidification predominates.SIGNIFICANCE STATEMENT Changes in synaptic cleft pH alter neurotransmission, acting on receptors and channels on both sides of the synapse. Synaptic acidification has been associated with a myriad of diseases in the central and peripheral nervous system. Here, we report that in near-physiological recording conditions the cholinergic neuromuscular junction shows use-dependent bidirectional changes in synaptic cleft pH-immediate alkalinization and a long-lasting acidification under prolonged stimulation. These results provide further insight into physiologically relevant changes at cholinergic synapses that have not been defined previously. Understanding and identifying synaptic pH transients during and after neuronal activity provides insight into short-term synaptic plasticity synapses and may identify therapeutic targets for diseases.

Simulations of active zone structure and function at mammalian NMJs predict that loss of calcium channels alone is not sufficient to replicate LEMS effects.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune-mediated neuromuscular disease thought to be caused by autoantibodies against P/Q-type voltage-gated calcium channels (VGCCs), which attack and reduce the number of VGCCs within transmitter release sites (active zones; AZs) at the neuromuscular junction (NMJ), resulting in neuromuscular weakness. However, patients with LEMS also have antibodies to other neuronal proteins, and about 15% of patients with LEMS are seronegative for antibodies against VGCCs. We hypothesized that a reduction in the number of P/Q-type VGCCs alone is not sufficient to explain LEMS effects on transmitter release. Here, we used a computational model to study a variety of LEMS-mediated effects on AZ organization and transmitter release constrained by electron microscopic, pharmacological, immunohistochemical, voltage imaging, and electrophysiological observations. We show that models of healthy AZs can be modified to predict the transmitter release and short-term facilitation characteristics of LEMS and that in addition to a decrease in the number of AZ VGCCs, disruption in the organization of AZ proteins, a reduction in AZ number, a reduction in the amount of synaptotagmin, and the compensatory expression of L-type channels outside the remaining AZs are important contributors to LEMS-mediated effects on transmitter release. Furthermore, our models predict that antibody-mediated removal of synaptotagmin in combination with disruption in AZ organization alone could mimic LEMS effects without the removal of VGCCs (a seronegative model). Overall, our results suggest that LEMS pathophysiology may be caused by a collection of pathological alterations to AZs at the NMJ, rather than by a simple loss of VGCCs.NEW & NOTEWORTHY We used a computational model of the active zone (AZ) in the mammalian neuromuscular junction to investigate Lambert-Eaton myasthenic syndrome (LEMS) pathophysiology. This model suggests that disruptions in presynaptic active zone organization and protein content (particularly synaptotagmin), beyond the simple removal of presynaptic calcium channels, play an important role in LEMS pathophysiology.

Muscarinic Receptors in Developmental Axonal Competition at the Neuromuscular Junction

In recent years, we have studied by immunohistochemistry, intracellular recording, and western blotting the role of the muscarinic acetylcholine receptors (mAChRs; M1, M2, and M4 subtypes) in the mammalian neuromuscular junction (NMJ) during development and in the adult. Here, we evaluate our published data to emphasize the mAChRs’ relevance in developmental synaptic elimination and their crosstalk with other metabotropic receptors, downstream kinases, and voltage-gated calcium channels (VGCCs). The presence of mAChRs in the presynaptic membrane of motor nerve terminals allows an autocrine mechanism in which the secreted acetylcholine influences the cell itself in feedback. mAChR subtypes are coupled to different downstream pathways, so their feedback can move in a broad range between positive and negative. Moreover, mAChRs allow direct activity-dependent interaction through ACh release between the multiple competing axons during development. Additional regulation from pre- and postsynaptic sites (including neurotrophic retrograde control), the agonistic and antagonistic contributions of adenosine receptors (AR; A1 and A2A), and the tropomyosin-related kinase B receptor (TrkB) cooperate with mAChRs in the axonal competitive interactions which lead to supernumerary synapse elimination that achieves the optimized monoinnervation of musculoskeletal cells. The metabotropic receptor-driven balance between downstream PKA and PKC activities, coupled to developmentally regulated VGCC, explains much of how nerve terminals with different activities finally progress to their withdrawal or strengthening.

Structure and Function of the Mammalian Neuromuscular Junction.

The mammalian neuromuscular junction (NMJ) comprises a presynaptic terminal, a postsynaptic receptor region on the muscle fiber (endplate), and the perisynaptic (terminal) Schwann cell. As with any synapse, the purpose of the NMJ is to transmit signals from the nervous system to muscle fibers. This neural control of muscle fibers is organized as motor units, which display distinct structural and functional phenotypes including differences in pre- and postsynaptic elements of NMJs. Motor units vary considerably in the frequency of their activation (both motor neuron discharge rate and duration/duty cycle), force generation, and susceptibility to fatigue. For earlier and more frequently recruited motor units, the structure and function of the activated NMJs must have high fidelity to ensure consistent activation and continued contractile response to sustain vital motor behaviors (e.g., breathing and postural balance). Similarly, for higher force less frequent behaviors (e.g., coughing and jumping), the structure and function of recruited NMJs must ensure short-term reliable activation but not activation sustained for a prolonged period in which fatigue may occur. The NMJ is highly plastic, changing structurally and functionally throughout the life span from embryonic development to old age. The NMJ also changes under pathological conditions including acute and chronic disease. Such neuroplasticity often varies across motor unit types. © 2022 American Physiological Society. Compr Physiol 12:1-36, 2022.

ACh Transfers: Homeostatic Plasticity of Cholinergic Synapses.

The field of homeostatic plasticity continues to advance rapidly, highlighting the importance of stabilizing neuronal activity within functional limits in the context of numerous fundamental processes such as development, learning, and memory. Most homeostatic plasticity studies have been focused on glutamatergic synapses, while the rules that govern homeostatic regulation of other synapse types are less understood. While cholinergic synapses have emerged as a critical component in the etiology of mammalian neurodegenerative disease mechanisms, relatively few studies have been conducted on the homeostatic plasticity of such synapses, particularly in the mammalian nervous system. An exploration of homeostatic mechanisms at the cholinergic synapse may illuminate potential therapeutic targets for disease management and treatment. We will review cholinergic homeostatic plasticity in the mammalian neuromuscular junction, the autonomic nervous system, central synapses, and in relation to pathological conditions including Alzheimer disease and DYT1 dystonia. This work provides a historical context for the field of cholinergic homeostatic regulation by examining common themes, unique features, and outstanding questions associated with these distinct cholinergic synapse types and aims to inform future research in the field.

Homeostatic Plasticity of the Mammalian Neuromuscular Junction.

The mammalian neuromuscular junction (NMJ) is an ideal preparation to study synaptic plasticity. Its simplicity- one input, one postsynaptic target- allows experimental manipulations and mechanistic analyses that are impossible at more complex synapses. Homeostatic synaptic plasticity attempts to maintain normal function in the face of perturbations in activity. At the NMJ, 3 aspects of activity are sensed to trigger 3 distinct mechanisms that contribute to homeostatic plasticity: Block of presynaptic action potentials triggers increased quantal size secondary to increased release of acetylcholine from vesicles. Simultaneous block of pre- and postsynaptic action potentials triggers an increase in the probability of vesicle release. Block of acetylcholine binding to acetylcholine receptors during spontaneous fusion of single vesicles triggers an increase in the number of releasable vesicles as well as increased motoneuron excitability. Understanding how the NMJ responds to perturbations of synaptic activity informs our understanding of its response to diverse neuromuscular diseases.

Dendrimer-2PMPA Delays Muscle Function Loss and Denervation in a Murine Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease where muscle weakness and neuromuscular junction (NMJ) denervation precede motor neuron cell death. Although acetylcholine is the canonical neurotransmitter at the mammalian NMJ synapse, glutamate has recently been identified as a critical neurotransmitter for NMJ development and maintenance. One source of glutamate is through the catabolism of N-acetyl-aspartyl-glutamate (NAAG), which is found in mM concentrations in mammalian motoneurons, where it is released upon stimulation and hydrolyzed to glutamate by the glial enzyme glutamate carboxypeptidase II (GCPII). Using the SOD1G93A model of ALS, we found an almost fourfold elevation of GCPII enzymatic activity in SOD1G93A versus WT muscle and a robust increase in GCPII expression which was specifically associated with activated macrophages infiltrating the muscle. 2-(Phosphonomethyl)pentanedioic acid (2PMPA) is a potent GCPII inhibitor which robustly blocks glutamate release from NAAG but is highly polar with limited tissue penetration. To improve this, we covalently attached 2PMPA to a hydroxyl polyamidoamine (PAMAM-G4-OH) dendrimer delivery system (D-2PMPA) which is known to target activated macrophages in affected tissues. Systemic D-2PMPA therapy (20 mg/kg 2PMPA equivalent; IP 2 × /week) was found to localize in muscle macrophages in SOD1G93A mice and completely normalize the enhanced GCPII activity. Although no changes in body weight or survival were observed, D-2PMPA significantly improved grip strength and inhibited the loss of NMJ innervation in the gastrocnemius muscles. Our finding that inhibiting elevated GCPII activity in SOD1G93A muscle can prolong muscle function and delay NMJ denervation may have early therapeutic implications for ALS patients.

Homeostatic plasticity induced by increased acetylcholine release at the mouse neuromuscular junction

At the neuromuscular junction (NMJ), changes to the size of the postsynaptic potential induce homeostatic compensation. At the Drosophila NMJ, increased glutamate release causes a compensatory decrease in quantal content, but it is unknown if this mechanism operates at the cholinergic mammalian NMJ. We addressed this question by recording endplate potentials (EPP) and muscle contraction in 3-month and 24-month ChAT-ChR2-EYFP mice that overexpress vesicular acetylcholine transporter and release more acetylcholine per vesicle. At 3 months, the quantal content of EPPs from ChAT-ChR2-EYFP mice were not different from WT controls, however tetanic depression was greater, and quantal size during high-frequency stimulation and the size of the readily releasable pool (RRP) were decreased. At 24 months of age, quantal content was reduced in ChAT-ChR2-EYFP mice, which normalized synaptic depression despite smaller RRP. The effect of pancuronium on indirect evoked muscle twitch was not different between groups. These results indicate that an increase in the amount of acetylcholine per vesicle induces two distinct age-dependent homeostatic mechanisms compensating excessive acetylcholine release.

Constructing and Tuning Excitatory Cholinergic Synapses: The Multifaceted Functions of Nicotinic Acetylcholine Receptors in Drosophila Neural Development and Physiology.

Nicotinic acetylcholine receptors (nAchRs) are widely distributed within the nervous system across most animal species. Besides their well-established roles in mammalian neuromuscular junctions, studies using invertebrate models have also proven fruitful in revealing the function of nAchRs in the central nervous system. During the earlier years, both in vitro and animal studies had helped clarify the basic molecular features of the members of the Drosophila nAchR gene family and illustrated their utility as targets for insecticides. Later, increasingly sophisticated techniques have illuminated how nAchRs mediate excitatory neurotransmission in the Drosophila brain and play an integral part in neural development and synaptic plasticity, as well as cognitive processes such as learning and memory. This review is intended to provide an updated survey of Drosophila nAchR subunits, focusing on their molecular diversity and unique contributions to physiology and plasticity of the fly neural circuitry. We will also highlight promising new avenues for nAchR research that will likely contribute to better understanding of central cholinergic neurotransmission in both Drosophila and other organisms.

Conservation and Innovation: Versatile Roles for LRP4 in Nervous System Development.

As the nervous system develops, connections between neurons must form to enable efficient communication. This complex process of synaptic development requires the coordination of a series of intricate mechanisms between partner neurons to ensure pre- and postsynaptic differentiation. Many of these mechanisms employ transsynaptic signaling via essential secreted factors and cell surface receptors to promote each step of synaptic development. One such cell surface receptor, LRP4, has emerged as a synaptic organizer, playing a critical role in conveying extracellular signals to initiate diverse intracellular events during development. To date, LRP4 is largely known for its role in development of the mammalian neuromuscular junction, where it functions as a receptor for the synaptogenic signal Agrin to regulate synapse development. Recently however, LRP4 has emerged as a synapse organizer in the brain, where new functions for the protein continue to arise, adding further complexity to its already versatile roles. Additional findings indicate that LRP4 plays a role in disorders of the nervous system, including myasthenia gravis, amyotrophic lateral sclerosis, and Alzheimer’s disease, demonstrating the need for further study to understand disease etiology. This review will highlight our current knowledge of how LRP4 functions in the nervous system, focusing on the diverse developmental roles and different modes this essential cell surface protein uses to ensure the formation of robust synaptic connections.

The Metabolic Stability of the Nicotinic Acetylcholine Receptor at the Neuromuscular Junction.

The clustering and maintenance of nicotinic acetylcholine receptors (AChRs) at high density in the postsynaptic membrane is a hallmark of the mammalian neuromuscular junction (NMJ). The regulation of receptor density/turnover rate at synapses is one of the main thrusts of neurobiology because it plays an important role in synaptic development and synaptic plasticity. The state-of-the-art imaging revealed that AChRs are highly dynamic despite the overall structural stability of the NMJ over the lifetime of the animal. This review highlights the work on the metabolic stability of AChRs at developing and mature NMJs and discusses the role of synaptic activity and the regulatory signaling pathways involved in the dynamics of AChRs.

A high-affinity, partial antagonist effect of 3,4-diaminopyridine mediates action potential broadening and enhancement of transmitter release at NMJs

3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach ∼1 μM in serum) are the Food and Drug Administration (FDA)-approved treatment for neuromuscular weakness caused by Lambert–Eaton myasthenic syndrome. Canonically, 3,4-DAP is thought to block voltage-gated potassium (Kv) channels, resulting in prolongation of the presynaptic action potential (AP). However, recent reports have shown that low millimolar concentrations of 3,4-DAP have an off-target agonist effect on the Cav1 subtype (“L-type”) of voltage-gated calcium (Cav) channels and have speculated that this agonist effect might contribute to 3,4-DAP effects on transmitter release at the NMJ. To address 3,4-DAP’s mechanism(s) of action, we first used the patch-clamp electrophysiology to characterize the concentration-dependent block of 3,4-DAP on the predominant presynaptic Kv channel subtypes found at the mammalian NMJ (Kv3.3 and Kv3.4). We identified a previously unreported high-affinity (1–10 μM) partial antagonist effect of 3,4-DAP in addition to the well-known low-affinity (0.1–1 mM) antagonist activity. We also showed that 1.5-μM DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-μM 3,4-DAP broadened the AP waveform in a dose-dependent manner, independent of Cav1 calcium channels. Finally, we demonstrated that 1.5- or 100-μM 3,4-DAP augmented transmitter release in a dose-dependent manner and this effect was also independent of Cav1 channels. From these results, we conclude that low micromolar concentrations of 3,4-DAP act solely on Kv channels to mediate AP broadening and enhance transmitter release at the NMJ.

Lack of Desmin in Mice Causes Structural and Functional Disorders of Neuromuscular Junctions.

Desmin, the major intermediate filament (IF) protein in muscle cells, interlinks neighboring myofibrils and connects the whole myofibrillar apparatus to myonuclei, mitochondria, and the sarcolemma. However, desmin is also known to be enriched at postsynaptic membranes of neuromuscular junctions (NMJs). The pivotal role of the desmin IF cytoskeletal network is underscored by the fact that over 120 mutations of the human DES gene cause hereditary and sporadic myopathies and cardiomyopathies. A subgroup of human desminopathies comprises autosomal recessive cases resulting in the complete abolition of desmin protein. In these patients, who display a more severe phenotype than the autosomal dominant cases, it has been reported that some individuals also suffer from a myasthenic syndrome in addition to the classical occurrence of myopathy and cardiomyopathy. Since further studies on the NMJ pathology are hampered by the lack of available human striated muscle biopsy specimens, we exploited homozygous desmin knock-out mice which closely mirror the striated muscle pathology of human patients lacking desmin protein. Here, we report on the impact of the lack of desmin on the structure and function of NMJs and the transcription of genes coding for postsynaptic proteins. Desmin knock-out mice display a fragmentation of NMJs in soleus, but not in the extensor digitorum longus muscle. Moreover, soleus muscle fibers show larger NMJs. Further, transcription levels of acetylcholine receptor (AChR) genes are increased in muscles from desmin knock-out mice, especially of the AChRγ subunit, which is known as a marker of muscle fiber regeneration. Electrophysiological recordings depicted a pathological decrement of nerve-dependent endplate potentials and an increased rise time of the nerve-independent miniature endplate potentials. The latter appears related to the fragmentation of NMJs in desmin knockout mice. Our study highlights the essential role of desmin for the structural and functional integrity of mammalian NMJs.

Tumor necrosis factor alpha mediates neuromuscular synapse elimination

During the development of mammalian neuromuscular junction (NMJ), the original supernumerary axon inputs are gradually eliminated, finally leaving each muscle fiber innervated by a single axon terminal. However, the molecular cues that mediate the elimination of redundant axon inputs remain unclear. Here we show that tumor necrosis factor-α (TNFα) expressed in postsynaptic muscle cells plays an important role in presynaptic axonal elimination at the NMJ. We found that intramuscular injection of TNFα into the levator auris longus (LAL) muscles caused disassociation of presynaptic nerve terminals from the postsynaptic acetylcholine receptor (AChR) clusters. By contrast, genetic ablation of TNFα globally or specifically in skeletal muscle cells, but not in motoneurons or Schwann cells, delayed the synaptic elimination. Moreover, ablation of TNFα in muscle cells attenuated the tendency of activity-dependent competition in a motoneuron–muscle coculture system. These results suggest a role of postsynaptic TNFα in the elimination of redundant synaptic inputs.

Muscle specific kinase protects dystrophic mdx mouse muscles from eccentric contraction-induced loss of force-producing capacity.

Adeno-associated viral vector was used to elevate the expression of muscle specific kinase (MuSK) and rapsyn (a cytoplasmic MuSK effector protein) in the tibialis anterior muscle of wild-type and dystrophic (mdx) mice. In mdx mice, enhanced expression of either MuSK or rapsyn ameliorated the acute loss of muscle force associated with strain injury. Increases in sarcolemmal immunolabelling for utrophin and β-dystroglycan suggest a mechanism for the protective effect of MuSK in mdx muscles. MuSK also caused subtle changes to the structure and function of the neuromuscular junction, suggesting novel roles for MuSK in muscle physiology and pathophysiology. Muscle specific kinase (MuSK) has a well-defined role in stabilizing the developing mammalian neuromuscular junction, but MuSK might also be protective in some neuromuscular diseases. In the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy, limb muscles are especially fragile. We injected the tibialis anterior muscle of 8-week-old mdx and wild-type (C57BL10) mice with adeno-associated viral vectors encoding either MuSK or rapsyn (a cytoplasmic MuSK effector protein) fused to green fluorescent protein (MuSK-GFP and rapsyn-GFP, respectively). Contralateral muscles injected with empty vector served as controls. One month later mice were anaesthetized with isoflurane and isometric force-producing capacity was recorded from the distal tendon. MuSK-GFP caused an unexpected decay in nerve-evoked tetanic force, both in wild-type and mdx muscles, without affecting contraction elicited by direct electrical stimulation of the muscle. Muscle fragility was probed by challenging muscles with a strain injury protocol consisting of a series of four strain-producing eccentric contractions in vivo. When applied to muscles of mdx mice, eccentric contraction produced an acute 27% reduction in directly evoked muscle force output, affirming the susceptibility of mdx muscles to strain injury. mdx muscles overexpressing MuSK-GFP or rapsyn-GFP exhibited significantly milder force deficits after the eccentric contraction challenge (15% and 14%, respectively). The protective effect of MuSK-GFP in muscles of mdx mice was associated with increased immunolabelling for utrophin and β-dystroglycan in the sarcolemma. Elevating the expression of MuSK or rapsyn revealed several distinct synaptic and extrasynaptic effects, suggesting novel roles for MuSK signalling in muscle physiology and pathophysiology.

Introducing a mammalian nerve-muscle preparation ideal for physiology and microscopy, the transverse auricular muscle in the ear of the mouse

A new mammalian neuromuscular preparation is introduced for physiology and microscopy of all sorts: the intrinsic muscle of the mouse ear. The great utility of this preparation is demonstrated by illustrating how it has permitted us to develop a wholly new technique for staining muscle T-tubules, the critical conductive-elements in muscle. This involves sequential immersion in dilute solutions of osmium and ferrocyanide, then tannic acid, and then uranyl acetate, all of which totally blackens the T-tubules but leaves the muscle pale, thereby revealing that the T-tubules in mouse ear-muscles become severely distorted in several pathological conditions. These include certain mouse-models of muscular dystrophy (specifically, dysferlin-mutations), certain mutations of muscle cytoskeletal proteins (specifically, beta-tubulin mutations), and also in denervation-fibrillation, as observed in mouse ears maintained with in vitro tissue-culture conditions. These observations permit us to generate the hypothesis that T-tubules are the “Achilles' heel” in several adult-onset muscular dystrophies, due to their unique susceptibility to damage via muscle lattice-dislocations. These new observations strongly encourage further in-depth studies of ear-muscle architecture, in the many available mouse-models of various devastating human muscle-diseases. Finally, we demonstrate that the delicate and defined physical characteristics of this 'new' mammalian muscle are ideal for ultrastructural study, and thereby facilitate the imaging of synaptic vesicle membrane recycling in mammalian neuromuscular junctions, a topic that is critical to myasthenia gravis and related diseases, but which has, until now, completely eluded electron microscopic analysis. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Inhibition of Kv2.1 Potassium Channels by MiDCA1, A Pre-Synaptically Active PLA2-Type Toxin from Micrurus dumerilii carinicauda Coral Snake Venom.

MiDCA1, a phospholipase A2 (PLA2) neurotoxin isolated from Micrurus dumerilii carinicauda coral snake venom, inhibited a major component of voltage-activated potassium (Kv) currents (41 ± 3% inhibition with 1 μM toxin) in mouse cultured dorsal root ganglion (DRG) neurons. In addition, the selective Kv2.1 channel blocker guangxitoxin (GxTx-1E) and MiDCA1 competitively inhibited the outward potassium current in DRG neurons. MiDCA1 (1 µM) reversibly inhibited the Kv2.1 current by 55 ± 8.9% in a Xenopus oocyte heterologous system. The toxin showed selectivity for Kv2.1 channels over all the other Kv channels tested in this study. We propose that Kv2.1 channel blockade by MiDCA1 underlies the toxin’s action on acetylcholine release at mammalian neuromuscular junctions.