Sympathetic innervation controls homeostasis of neuromuscular junctions in health and disease.

The distribution and function of sympathetic innervation in skeletal muscle have largely remained elusive. Here we demonstrate that sympathetic neurons make close contact with neuromuscular junctions and form a network in skeletal muscle that may functionally couple different targets including blood vessels, motor neurons, and muscle fibers. Direct stimulation of sympathetic neurons led to activation of muscle postsynaptic ß2-adrenoreceptor (ADRB2), cAMP production, and import of the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PPARGC1A) into myonuclei. Electrophysiological and morphological deficits of neuromuscular junctions upon sympathectomy and in myasthenic mice were rescued by sympathicomimetic treatment. In conclusion, this study identifies the neuromuscular junction as a target of the sympathetic nervous system and shows that sympathetic input is crucial for synapse maintenance and function.

Formation of cholinergic synapse-like specializations at developing murine muscle spindles.

Muscle spindles are complex stretch-sensitive mechanoreceptors. They consist of specialized skeletal muscle fibers, called intrafusal fibers, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ-motoneurons. We show that AChRs are concentrated at the γ-motoneuron endplate as well as in the equatorial region where they colocalize with the sensory nerve ending. In addition to the AChRs, the contact site between sensory nerve ending and intrafusal muscle fiber contains a high concentration of choline acetyltransferase, vesicular acetylcholine transporter and the AChR-associated protein rapsyn. Moreover, bassoon, a component of the presynaptic cytomatrix involved in synaptic vesicle exocytosis, is present in γ-motoneuron endplates but also in the sensory nerve terminal. Finally, we demonstrate that during postnatal development of the γ-motoneuron endplate, the AChR subunit stoichiometry changes from the γ-subunit-containing fetal AChRs to the ε-subunit-containing adult AChRs, similar and approximately in parallel to the postnatal subunit maturation at the neuromuscular junction. In contrast, despite the onset of ε-subunit expression during postnatal development the γ-subunit remains detectable in the equatorial region by subunit-specific antibodies as well as by analysis of muscle spindles from mice with genetically-labeled AChR γ-subunits. These results demonstrate an unusual maturation of the AChR subunit composition at the annulospiral endings and suggest that in addition to the recently described glutamatergic secretory system, the sensory nerve terminals are also specialized for cholinergic synaptic transmission, synaptic vesicle storage and exocytosis.

A new mouse model for the slow-channel congenital myasthenic syndrome induced by the AChR εL221F mutation.

We have generated a new mouse model for congenital myasthenic syndromes by inserting the missense mutation L221F into the ε subunit of the acetylcholine receptor by homologous recombination. This mutation has been identified in man to cause a mild form of slow-channel congenital myasthenic syndrome with variable penetrance. In our mouse model we observe as in human patients prolonged endplate currents. The summation of endplate potentials may account for a depolarization block at increasing stimulus frequencies, moderate reduced muscle strength and tetanic fade. Calcium and intracellular vesicle accumulation as well as junctional fold loss and organelle degeneration underlying a typical endplate myopathy, were identified. Moreover, a remodeling of neuromuscular junctions occurs in a muscle-dependent pattern expressing variable phenotypic effects. Altogether, this mouse model provides new insight into the pathophysiology of congenital myasthenia and serves as a new tool for deciphering signaling pathways induced by excitotoxicity at peripheral synapses.

Time lapse in vivo visualization of developmental stabilization of synaptic receptors at neuromuscular junctions.

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.

Voluntary exercise induces anxiety-like behavior in adult C57BL/6J mice correlating with hippocampal neurogenesis.

Several studies investigated the effect of physical exercise on emotional behaviors in rodents; resulting findings however remain controversial. Despite the accepted notion that voluntary exercise alters behavior in the same manners as antidepressant drugs, several studies reported opposite or no effects at all. In an attempt to evaluate the effect of physical exercise on emotional behaviors and brain plasticity, we individually housed C57BL/6J male mice in cages equipped with a running wheel. Three weeks after continuous voluntary running we assessed their anxiety- and depression-like behaviors. Tests included openfield, dark-light-box, elevated O-maze, learned helplessness, and forced swim test. We measured corticosterone metabolite levels in feces collected over a 24-h period and brain-derived neurotrophic factor (BDNF) in several brain regions. Furthermore, cell proliferation and adult hippocampal neurogenesis were assessed using Ki67 and Doublecortin. Voluntary wheel running induced increased anxiety in the openfield, elevated O-maze, and dark-light-box and higher levels of excreted corticosterone metabolites. We did not observe any antidepressant effect of running despite a significant increase of hippocampal neurogenesis and BDNF. These data are thus far the first to indicate that the effect of physical exercise in mice may be ambiguous. On one hand, the running-induced increase of neurogenesis and BDNF seems to be irrelevant in tests for depression-like behavior, at least in the present model where running activity exceeded previous reports. On the other hand, exercising mice display a more anxious phenotype and are exposed to higher levels of stress hormones such as corticosterone. Intriguingly, numbers of differentiating neurons correlate significantly with anxiety parameters in the openfield and dark-light-box. We therefore conclude that adult hippocampal neurogenesis is a crucial player in the genesis of anxiety.

A mouse model for congenital myasthenic syndrome due to MuSK mutations reveals defects in structure and function of neuromuscular junctions.

In the muscle-specific tyrosine kinase receptor gene MUSK, a heteroallelic missense and a null mutation were identified in a patient suffering from a congenital myasthenic syndrome (CMS). We generated one mouse line carrying the homozygous missense mutation V789M in musk (musk(V789M/V789M) mice) and a second hemizygous line, resembling the patient genotype, with the V789M mutation on one allele and an allele lacking the kinase domain (musk(V789M/-) mice). We report here that musk(V789M/V789M) mice present no obvious abnormal phenotype regarding weight, muscle function and viability. In contrast, adult musk(V789M/-) mice suffer from severe muscle weakness, exhibit shrinkage of pelvic and scapular regions and hunchback. Musk(V789M/-) diaphragm develops less force upon direct or nerve-induced stimulation. A profound tetanic fade is observed following nerve-evoked muscle contraction, and fatigue resistance is severely impaired upon a train of tetanic nerve stimulations. Electrophysiological measurements indicate that fatigable muscle weakness is due to impaired neurotransmission as observed in a patient suffering from a CMS. The diaphragm of adult musk(V789M/-) mice exhibits pronounced changes in endplate architecture, distribution and innervation pattern. Thus, the missense mutation V789M in MuSK acts as a hypomorphic mutation and leads to insufficiency in MuSK function in musk(V789M/-) mutants. These mutant mice represent valuable models for elucidating the roles of MuSK for synapse formation, maturation and maintenance as well as for studying the pathophysiology of a CMS due to MuSK mutations.

Differential muscle-driven synaptic remodeling in the neuromuscular junction after denervation.

We used knock-in mice that express green fluorescent protein (GFP)-labeled embryonic-type acetylcholine receptors to investigate postsynaptic responses to denervation of fast-twitch and slow-twitch muscle fibers, and to visualize the integration of newly synthesized GFP-labeled embryonic-type receptors into adult synapses. The embryonic-type receptors are transiently expressed and incorporated into the denervated endplates. They replaced synaptic adult-type receptors in a directed fashion, starting from the endplate's periphery and proceeding to its central regions. The progress of embryonic-type receptor expression with respect to transcriptional control is a transient, short-term activation mechanism. The less pronounced increase in the expression levels of the GFP-labeled receptors revealed a differential shift in the integration and degradation processes that constitute the dynamic equilibrium of the synaptic receptor pool. Therefore, we were able to model the changes in the total receptor load of the neuromuscular endplate following denervation as a function of the abundance of available receptors and the initial receptor load of the endplate.

AChR channel conversion and AChR-adjusted neuronal survival during embryonic development.

We generated knock-in mice that express GFP-labeled embryonic-type acetylcholine receptors (AChR) to follow postsynaptic differentiation and innervation during embryonic development and to visualize the postnatally occurring channel conversion from embryonic- to adult-type AChR. The dynamics of AChRgamma/AChRepsilon conversion at the neuromuscular junction indicates that muscle-specific programs of receptor subunit gene transcription control AChR replacement. While conversion proceeds from peripheral to central regions for individual endplates, it does not occur simultaneously for all endplates. Although GFP-labeled receptors were expressed at reduced levels, the localization of postsynaptic sites and synapse formation was not noticeably altered. However, these alterations correlated with a striking reduction of motoneuron programmed cell death, transient increase of neurite growth and axon branching. This animal model refines the view on reciprocal neuromuscular signaling and suggests that motoneuron survival and axon branching are directly regulated by AChR function to enable optimal innervation and timing of neurally evoked muscle contraction.

Motor Endplate-Anatomical, Functional, and Molecular Concepts in the Historical Perspective.

By mediating voluntary muscle movement, vertebrate neuromuscular junctions (NMJ) play an extraordinarily important role in physiology. While the significance of the nerve-muscle connectivity was already conceived almost 2000 years back, the precise cell and molecular biology of the NMJ have been revealed in a series of fascinating research activities that started around 180 years ago and that continues. In all this time, NMJ research has led to fundamentally new concepts of cell biology, and has triggered groundbreaking advancements in technologies. This review tries to sketch major lines of thought and concepts on NMJ in their historical perspective, in particular with respect to anatomy, function, and molecular components. Furthermore, along these lines, it emphasizes the mutual benefit between science and technology, where one drives the other. Finally, we speculate on potential major future directions for studies on NMJ in these fields.

Changes in acetylcholine receptor function induce shifts in muscle fiber type composition.

AChRepsilon(-/-) mice lack epsilon-subunits of the acetylcholine receptor and thus fail to express adult-type receptors. The expression of fetal-type receptors throughout postnatal life alters postsynaptic signal transduction and causes a fast-to-slow fiber type transition, both in slow-twitch soleus muscle and in fast-twitch extensor digitorum longus muscle. In comparison to wild-type muscle, the proportion of type 1 slow fibers is significantly increased (6%), whereas the proportion of fast fibers is reduced (in soleus, type 2A by 12%, and in extensor digitorum longus, type 2B/2D by 10%). The increased levels of troponin I(slow) transcripts clearly support a fast-to-slow fiber type transition. Shifts of protein and transcript levels are not restricted to 'myogenic' genes but also affect 'synaptogenic' genes. Clear increases are observed for acetylcholine receptor alpha-subunits and the postsynaptically located utrophin. Although the fast-to-slow fiber type transition appears to occur in a coordinated manner in both muscle types, muscle-specific differences are retained. Most prominently, the differential expression level of the synaptic regulator MuSK is significantly lower in extensor digitorum muscle than in soleus muscle. The results show a new quality in muscle plasticity, in that changes in the functional properties of endplate receptors modulate the contractile properties of skeletal muscles. Muscle thus represents a self-matching system that adjusts contractile properties and synaptic function to variable functional demands.

Peptide-toxin tools for probing the expression and function of fetal and adult subtypes of the nicotinic acetylcholine receptor.

Although the neuromuscular nicotinic acetylcholine receptor (nAChR) is one of the most intensively studied ion channels in the nervous system, the differential roles of fetal and adult subtypes of the nAChR under normal and pathological conditions are still incompletely defined. Until recently, no pharmacological tools distinguished between fetal and adult subtypes. Waglerin toxins (from snake venom) and alphaA(S)-conotoxins (from cone-snail venom) have provided such tools. Because these peptides were characterized by different research groups using different methods, we have: 1) more extensively tested their subtype selectivity, and 2) begun to explore how these peptides may be used in concert to elucidate expression patterns and functions of fetal and adult nAChRs. In heterologous expression systems and native tissues, Waglerin-1 and an alphaA(S)-conotoxin analog, alphaA-OIVA[K15N], are high-affinity, highly selective inhibitors of the adult and fetal muscle nAChRs, respectively. We have used the peptides and their fluorescent derivatives to explore the expression and function of the fetal and adult nAChR subtypes. While fluorescent derivatives of these peptides indicated a gradual transition from fetal to adult muscle nAChRs in mice during the first 2 weeks postnatal, we unexpectedly observed a steeper transition in functional expression in the mouse diaphragm muscle using electrophysiology. As a toolkit of pharmacological agents with complementary specificity, alphaA-OIVA[K15N] and Waglerin-1 should have further utility in determining the roles of fetal and adult nAChR subtypes in development, in mature tissues, and under pathological conditions.

Fiber types of the intrinsic whisker muscle and whisking behavior.

Some rodent species show rhythmic bouts of vibrissal protractions and retractions, referred to as whisking, that are among the fastest movements performed by mammals. To better understand the muscular basis of whisking, we compared (1) whisker movements of two whisking species (mouse, rat) and a non-whisking species (guinea pig), (2) the muscle fiber composition of intrinsic whisker muscles of whisking and a non-whisking species, and (3) the muscle fiber composition of intrinsic whisker muscles and of selected skeletal muscles. Using high-speed videography, we found that mice, rats, and guinea pigs can generate fast and large-amplitude whisker movements. Guinea pigs do not show bouts of fast, strictly rhythmic whisker movements, and the average speed of their whisker movements is much lower than in mice and rats. Analysis of mRNA expression of myosin heavy chain isoforms, myofibrillar ATPase staining, and antibody labeling indicate that in all three species intrinsic whisker muscles are composed predominantly of type 2B muscle fibers. Intrinsic whisker muscles of mice consisted of type 2B (> or =90%) and type 2D fibers. In rats we observed, in addition to type 2B/2D fibers, approximately 10% of slow type 1 fibers, and in guinea pigs we observed approximately 3% of slow type 1 fibers and 20% of type 2A fibers. Type 2B fibers have high levels of anaerobic glycolytic enzymes providing a rapid source of ATP and high maximum velocity of contraction but are less fatigue resistant than other muscle fiber types. The high percentage of type 2B fibers distinguishes the intrinsic whisker musculature from skeletal muscles and may have evolved for fast scanning of the sensory environment.

High-efficiency transfection of individual neurons using modified electrophysiology techniques.

Transfection of cells by electroporation is a widely used and efficient method. Recently, it has been shown that single neurons in brain slice cultures can be transfected using micropipettes loaded with plasmid DNA expression constructs. However, the transfection efficiencies were very low. Routine employment of single-cell electroporation (SCE) for transfection of neurons requires high and reliable efficiency together with good cell survival. Here, we describe the modification of electrophysiology techniques for SCE leading to very simple and efficient (up to 80%) transfection of neurons in organotypic rat hippocampus and mouse cortex slice cultures. Electroporation-mediated transfection was visualized in real-time by two-photon microscopy at the cellular level using fluorescently labeled oligonucleotides and plasmid DNA. Small oligonucleotides enter the cell immediately during pulse application while large plasmids remain localized for more than 10 min at the cell membrane before they enter the cell by an, as yet, unknown process. SCE does not affect the electrophysiology of transgene-expressing cells. Expression of several neuronal green fluorescent protein-tagged proteins demonstrates that the method can be employed to analyze subcellular trafficking and targeting in single living neurons.

The neuromuscular junction: selective remodeling of synaptic regulators at the nerve/muscle interface.

The peripheral synapses between motoneurons and skeletal muscle fibers, the neuromuscular junctions, are ideal to investigate the general principles of synaptogenesis that depend on the interaction of activity-dependent and activity-independent signals. Much has been learned from gene "knock out" mouse models that helped to identify major synaptic regulators. The "knock out" approach, however, may not distinguish between changes arising from the disruption of molecular signaling pathways and changes caused by the absence of synaptic transmission. To circumvent these problems, postsynaptic activity was modulated in mouse models by specifically targeting endplate receptors or the activity of synaptic regulators such as MuSK. Both regulators have multiple functions and acetylcholine receptors are not just signal transducers but regulate the localization and architecture of endplates. The results show that detailed analysis of mouse models will help to understand the complexity in mechanisms that regulate synaptic remodeling.

Novel mouse model reveals distinct activity-dependent and -independent contributions to synapse development.

The balanced action of both pre- and postsynaptic organizers regulates the formation of neuromuscular junctions (NMJ). The precise mechanisms that control the regional specialization of acetylcholine receptor (AChR) aggregation, guide ingrowing axons and contribute to correct synaptic patterning are unknown. Synaptic activity is of central importance and to understand synaptogenesis, it is necessary to distinguish between activity-dependent and activity-independent processes. By engineering a mutated fetal AChR subunit, we used homologous recombination to develop a mouse line that expresses AChR with massively reduced open probability during embryonic development. Through histological and immunochemical methods as well as electrophysiological techniques, we observed that endplate anatomy and distribution are severely aberrant and innervation patterns are completely disrupted. Nonetheless, in the absence of activity AChRs form postsynaptic specializations attracting motor axons and permitting generation of multiple nerve/muscle contacts on individual fibers. This process is not restricted to a specialized central zone of the diaphragm and proceeds throughout embryonic development. Phenotypes can be attributed to separate activity-dependent and -independent pathways. The correct patterning of synaptic connections, prevention of multiple contacts and control of nerve growth require AChR-mediated activity. In contrast, myotube survival and acetylcholine-mediated dispersal of AChRs are maintained even in the absence of AChR-mediated activity. Because mouse models in which acetylcholine is entirely absent do not display similar effects, we conclude that acetylcholine binding to the AChR initiates activity-dependent and activity-independent pathways whereby the AChR modulates formation of the NMJ.

Role of Myosin Va in the plasticity of the vertebrate neuromuscular junction in vivo.

BACKGROUND: Myosin Va is a motor protein involved in vesicular transport and its absence leads to movement disorders in humans (Griscelli and Elejalde syndromes) and rodents (e.g. dilute lethal phenotype in mice). We examined the role of myosin Va in the postsynaptic plasticity of the vertebrate neuromuscular junction (NMJ). METHODOLOGY/PRINCIPAL FINDINGS: Dilute lethal mice showed a good correlation between the propensity for seizures, and fragmentation and size reduction of NMJs. In an aneural C2C12 myoblast cell culture, expression of a dominant-negative fragment of myosin Va led to the accumulation of punctate structures containing the NMJ marker protein, rapsyn-GFP, in perinuclear clusters. In mouse hindlimb muscle, endogenous myosin Va co-precipitated with surface-exposed or internalised acetylcholine receptors and was markedly enriched in close proximity to the NMJ upon immunofluorescence. In vivo microscopy of exogenous full length myosin Va as well as a cargo-binding fragment of myosin Va showed localisation to the NMJ in wildtype mouse muscles. Furthermore, local interference with myosin Va function in live wildtype mouse muscles led to fragmentation and size reduction of NMJs, exclusion of rapsyn-GFP from NMJs, reduced persistence of acetylcholine receptors in NMJs and an increased amount of punctate structures bearing internalised NMJ proteins. CONCLUSIONS/SIGNIFICANCE: In summary, our data show a crucial role of myosin Va for the plasticity of live vertebrate neuromuscular junctions and suggest its involvement in the recycling of internalised acetylcholine receptors back to the postsynaptic membrane.

Development of the neuromuscular junction.

The differentiation of the neuromuscular junction is a multistep process requiring coordinated interactions between nerve terminals and muscle. Although innervation is not needed for muscle production, the formation of nerve-muscle contacts, intramuscular nerve branching, and neuronal survival require reciprocal signals from nerve and muscle to regulate the formation of synapses. Following the production of muscle fibers, clusters of acetylcholine receptors (AChRs) are concentrated in the central regions of the myofibers via a process termed "prepatterning". The postsynaptic protein MuSK is essential for this process activating possibly its own expression, in addition to the expression of AChR. AChR complexes (aggregated and stabilized by rapsyn) are thus prepatterned independently of neuronal signals in developing myofibers. ACh released by branching motor nerves causes AChR-induced postsynaptic potentials and positively regulates the localization and stabilization of developing synaptic contacts. These "active" contact sites may prevent AChRs clustering in non-contacted regions and counteract the establishment of additional contacts. ACh-induced signals also cause the dispersion of non-synaptic AChR clusters and possibly the removal of excess AChR. A further neuronal factor, agrin, stabilizes the accumulation of AChR at synaptic sites. Agrin released from the branching motor nerve may form a structural link specifically to the ACh-activated endplates, thereby enhancing MuSK kinase activity and AChR accumulation and preventing dispersion of postsynaptic specializations. The successful stabilization of prepatterned AChR clusters by agrin and the generation of singly innervated myofibers appear to require AChR-mediated postsynaptic potentials indicating that the differentiation of the nerve terminal proceeds only after postsynaptic specializations have formed.

Synapse disassembly and formation of new synapses in postnatal muscle upon conditional inactivation of MuSK.

The muscle-specific-kinase MuSK is required for the formation of acetylcholine receptor clusters during embryonic development, but its physiological role in adult muscle is not known. We used the loxP/Cre system in mice to conditionally inactivate MuSK whereby expression of Cre recombinase increases during postnatal development. The MuSK-inactivated mice develop myasthenic symptoms and die prematurely due to severe muscle weakness. The postnatal inactivation of MuSK causes loss of acetylcholine receptors and disassembly of the postsynaptic organization and innervating axons retract but start to grow and branch extensively. Due to the mosaic expression of Cre recombinase, MuSK is not globally inactivated and new synapses are formed aberrantly patterned across the diaphragm. Our findings demonstrate that MuSK kinase activity is required throughout postnatal development to hold up MuSK and AChR levels at endplates. Thus, MuSK and AChR together maintain the functional and structural integrity of the postsynaptic architecture and prevent axon growth.

Mutation of single murine acetylcholine receptor subunits reveals differential contribution of P121 to acetylcholine binding and channel opening.

The nicotinic acetylcholine receptor (AChR) is a heteropentameric, ligand-gated ion channel at the neuromuscular junction, where it is responsible for signal transduction between the motorneuron and the muscle. Point mutations in the subunits of the receptor change the channel's electrophysiological properties and underlie inherited forms of muscle weakness, the congenital myasthenic syndromes. One point mutation (P121L) has been identified in the epsilon-subunit of patients suffering from the fast-channel congenital myasthenic syndrome, which is evoked by reduced AChR openings. We introduced the P121L mutation into all murine AChR subunits and performed electrophysiological studies in Xenopus laevis oocytes. The P121L mutation in the epsilon-subunit of the adult mouse AChR affected ligand binding and channel gating in a manner similar to that described for human AChR. At equivalent positions in the alpha- and beta-subunits, the mutation caused only minor electrophysiological changes. Mutation of the delta-subunit had similar, but less pronounced functional consequences compared to epsilonP121L, reflecting the asymmetry of the acetylcholine binding sites and the dominant effect of the alpha-epsilon site on channel opening.

Acetylcholine receptor channel subtype directs the innervation pattern of skeletal muscle.

Acetylcholine receptors (AChRs) mediate synaptic transmission at the neuromuscular junction, and structural and functional analysis has assigned distinct functions to the fetal (alpha2beta(gamma)delta) and adult types of AChR (alpha2beta(epsilon)delta). Mice lacking the epsilon-subunit gene die prematurely, showing that the adult type is essential for maintenance of neuromuscular synapses in adult muscle. It has been suggested that the fetally and neonatally expressed AChRs are crucial for muscle differentiation and for the formation of the neuromuscular synapses. Here, we show that substitution of the fetal-type AChR with an adult-type AChR preserves myoblast fusion, muscle and end-plate differentiation, whereas it substantially alters the innervation pattern of muscle by the motor nerve. Mutant mice form functional neuromuscular synapses outside the central, narrow end-plate band region in the diaphragm, with synapses scattered over a wider muscle territory. We suggest that one function of the fetal type of AChR is to ensure an orderly innervation pattern of skeletal muscle.