Forked end: a novel transmembrane protein involved in neuromuscular specificity in drosophila identified by gain‐of‐function screening

The Drosophila neuromuscular connectivity provides an excellent model system for studies on target recognition and selective synapse formation. To identify molecules involved in neuromuscular recognition, we conducted gain‐of‐function screening for genes whose forced expression in all muscles alters the target specificity. We report here the identification of a novel transmembrane protein, Forked end (FEND), encoded by the fend gene, by the said screening. When the FEND expression was induced in all muscles, motoneurons that normally innervate muscle 12 formed ectopic synapses on a neighboring muscle 13. The target specificity of these motoneurons was also altered in the loss‐of‐function mutant of fend. During embryonic development, fend mRNA was detected in a subset of cells in the central nervous system and in the periphery. These results suggest that FEND is a novel axon guidance molecule involved in neuromuscular specificity. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 205–214, 2002     

Functional dissection of drosophila capricious: its novel roles in neuronal pathfinding and selective synapse formation

Drosophila Capricious (CAPS) is a transmembrane protein with leucine-rich repeat (LRR) motifs, expressed on small subsets of neurons and muscles, including muscle 12 and the motoneurons that innervate it (muscle 12 MNs). Panmuscle ectopic expression of CAPS alters the target specificity of muscle 12 MNs, indicating that CAPS can function in muscles as a target recognition molecule. In this study, we first examined the effect of ectopic panneural expression of CAPS on the motoneuronal circuit. We found that panneural expression of CAPS alters the pathfinding of muscle 12 MNs. The defect appeared to be caused by changes in the steering behavior of muscle 12 MNs at a specific choice point along their pathway to the target muscle. These results revealed a novel function of CAPS in axon pathfinding. We then performed deletion analyses of CAPS. We expressed CAPS lacking the intracellular domain in all neurons or in all muscles, and studied their ability to induce the pathfinding and targeting phenotypes. We found that the function of muscularly expressed CAPS in target recognition is intracellular domain dependent, whereas the function of neurally expressed CAPS in pathfinding is not, suggesting that CAPS may function in neurons and muscles in a different manner. The requirement of the intracellular domain for the function of muscularly expressed CAPS suggests the presence of a signaling event within muscle cells that is essential for selective synapse formation.     

Control of muscle fibre and motoneuron diversification

Recent studies have elucidated both the mechanism of early formation of diverse muscle fibre types and the matching of diverse populations of motoneurons to their appropriate muscle targets. Highlights include the demonstration that distinct signals are necessary for the formation of several distinct myoblast populations in the vertebrate somite, the identification of motoneuron subtypes, studies of how motoneurons target appropriate muscles, and rapid progress on the Drosophila neuromuscular system. We propose a model in which four classes of decision control the patterning of both motoneurons and muscles.     

The spinal nucleus of the bulbocavernosus: firsts in androgen-dependent neural sex differences

Cell number in the spinal nucleus of the bulbocavernosus (SNB) of rats was the first neural sex difference shown to differentiate under the control of androgens, acting via classical intracellular androgen receptors. SNB motoneurons reside in the lumbar spinal cord and innervate striated muscles involved in copulation, including the bulbocavernosus (BC) and levator ani (LA). SNB cells are much larger and more numerous in males than in females, and the BC/LA target muscles are reduced or absent in females. The relative simplicity of this neuromuscular system has allowed for considerable progress in pinpointing sites of hormone action, and identifying the cellular bases for androgenic effects. It is now clear that androgens act at virtually every level of the SNB system, in development and throughout adult life. In this review we focus on effects of androgens on developmental cell death of SNB motoneurons and BC/LA muscles; the establishment and maintenance of SNB motoneuron soma size and dendritic length; BC/LA muscle morphology and physiology; and behaviors controlled by the SNB system. We also describe new data on neurotherapeutic effects of androgens on SNB motoneurons after injury in adulthood.     

Testosterone metabolites differentially maintain adult morphology in a sexually dimorphic neuromuscular system

The lumbar spinal cord of rats contains the sexually dimorphic, steroid‐sensitive spinal nucleus of the bulbocavernosus (SNB). Androgens are necessary for the development of the SNB neuromuscular system, and in adulthood, continue to influence the morphology and function of the motoneurons and their target musculature. However, estrogens are also involved in the development of the SNB system, and are capable of maintaining function in adulthood. In this experiment, we assessed the ability of testosterone metabolites, estrogens and nonaromatizable androgens, to maintain neuromuscular morphology in adulthood. Motoneuron and muscle morphology was assessed in adult normal males, sham‐castrated males, castrated males treated with testosterone, dihydrotestosterone, estradiol, or left untreated, and gonadally intact males treated with the 5α‐reductase inhibitor finasteride or the aromatase inhibitor fadrozole. After 6 weeks of treatment, SNB motoneurons were retrogradely labeled with cholera toxin‐HRP and reconstructed in three dimensions. Castration resulted in reductions in SNB target muscle size, soma size, and dendritic morphology. Testosterone treatment after castration maintained SNB soma size, dendritic morphology, and elevated target muscle size; dihydrotestosterone treatment also maintained SNB dendritic length, but was less effective than testosterone in maintaining both SNB soma size and target muscle weight. Treatment of intact males with finasteride or fadrozole did not alter the morphology of SNB motoneurons or their target muscles. In contrast, estradiol treatment was completely ineffective in preventing castration‐induced atrophy of the SNB neuromuscular system. Together, these results suggest that the maintenance of adult motoneuron or muscle morphology is strictly mediated by androgens. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 70: 206–221, 2010.     

Nerve-muscle interactions regulate motor terminal growth and myoblast distribution during muscle development

Interactions between motoneurons and muscles influence many aspects of neuromuscular development in all animals. These interactions can be readily investigated during adult muscle development in holometabolous insects. In this study, the development of the dorsolongitudinal flight muscle (DLM) and its innervation is investigated in the moth, Manduca sexta, to address the specificity of neuromuscular interactions. The DLM develops from an anlage containing both regressed larval template fibers and imaginal myoblasts. In the adult, each fiber bundle (DLM1-5) is innervated by a single motoneuron (MN1-MN5), with the dorsal-most fiber bundle (DLM5) innervated by a mesothoracic motoneuron (MN5). The DLM failed to develop following complete denervation because myoblasts failed to accumulate in the DLM anlage. After lesioning MN1-4, MN5 retained its specificity for the DLM5 region of the anlage and failed to rescue DLM1-4. Thus specific innervation of the DLM fiber bundles does not depend on interactions among motoneurons. Myoblast accumulation, but not myonuclear proliferation, increased around the MN5 terminals, producing a hypertrophied adult DLM5. Therefore, motoneurons compete for uncommitted myoblasts. MN5 terminals subsequently grew more rapidly over the hypertrophied DLM5 anlage, indicating that motoneuron terminal expansion is regulated by the size of the target muscle anlage.     

Synaptic defects in the spinal and neuromuscular circuitry in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a major genetic cause of death in childhood characterized by marked muscle weakness. To investigate mechanisms underlying motor impairment in SMA, we examined the spinal and neuromuscular circuitry governing hindlimb ambulatory behavior in SMA model mice (SMNΔ7). In the neuromuscular circuitry, we found that nearly all neuromuscular junctions (NMJs) in hindlimb muscles of SMNΔ7 mice remained fully innervated at the disease end stage and were capable of eliciting muscle contraction, despite a modest reduction in quantal content. In the spinal circuitry, we observed a ～28% loss of synapses onto spinal motoneurons in the lateral column of lumbar segments 3-5, and a significant reduction in proprioceptive sensory neurons, which may contribute to the 50% reduction in vesicular glutamate transporter 1(VGLUT1)-positive synapses onto SMNΔ7 motoneurons. In addition, there was an increase in the association of activated microglia with SMNΔ7 motoneurons. Together, our results present a novel concept that synaptic defects occur at multiple levels of the spinal and neuromuscular circuitry in SMNΔ7 mice, and that proprioceptive spinal synapses could be a potential target for SMA therapy.     

Competitive mechanisms underlying synapse elimination in the lumbrical muscle of the rat

The process of neuromuscular synapse elimination has been studied in the fourth deep lumbrical (4DL) muscle of the rat, a preparation which offers technical advantages for some types of experimental work. Studies have been performed both during development and in adult denervated muscles undergoing reinnervation. Results indicate that synapse elimination is dependent upon competition between motoneurons. Cellular mechanisms underlying this competition have also been explored. Both neuromuscular activity and muscle fiber type recognition appear to play a role, but positional cues appear unimportant in this small muscle.     

Connectin: a homophilic cell adhesion molecule expressed on a subset of muscles and the motoneurons that innervate them in Drosophila.

Each abdominal hemisegment in the Drosophila embryo contains a stereotyped array of 30 muscles, each specifically innervated by one or a few motoneurons. We screened 11,000 enhancer trap lines, isolated several expressing beta-galactosidase in small subsets of muscle fibers prior to innervation, and identified two of these as inserts in connectin and Toll, members of the leucine-rich repeat gene family. Connectin contains a signal sequence, ten leucine-rich repeats, and a putative phosphatidylinositol membrane linkage; in S2 cells, connectin can mediate homophilic cell adhesion. Connectin is expressed on the surface of eight muscles, the motoneurons that innervate them, and several glial cells along the pathways leading to them. During synapse formation, the protein localizes to synaptic sites; afterward, it largely disappears. Thus, connectin is a novel cell adhesion molecule whose expression suggests a role in target recognition.     

Chronic application of curare does not increase the level of motoneuron survival-promoting activity in limb muscle extracts during the naturally occurring motoneuron cell death period

Naturally occurring motoneuron cell death during development is a well-described phenomenon and the existence of a survival factor provided by target muscles has been postulated. Blockade of activity by chronic application of a neuromuscular junction blocker rescues almost all motoneurons from cell death. The present study was conducted in order to examine the possibility that the motoneuron survival-promoting activity in muscles increases following activity blockade. Cell culture was used to assess the degree of motoneuron survival-promoting activity present in muscle extracts. Embryonic chick motoneurons were labeled by injecting the water-insoluble fluorescent dye, DiI (Molecular Probes, Inc.) into the spinal nerves both before and during the cell death period. The labeled cells extending long neurites were counted after 2 days of culture as viable motoneurons in low-density heterogeneous cell cultures. The culture medium, Ham F12/DMEM (1:1 mixture) supplemented with 10% horse serum, 5% chick serum, and 5% fetal calf serum, was employed as a basic culture medium for assessing motoneuron survival factor, since it supported the survival of a significantly higher number of motoneurons derived from embryos before cell death than those during the cell death period, thus representing the motoneuron's requirement for survival factor in vivo. The number of surviving motoneurons clearly increased in proportion to the amount of muscle extract added to the culture medium. In comparison with control chick embryos, the dose-response relation between the number of surviving motoneurons and the amount of muscle extract added did not change when embryos were used after chronic application of curare. These results therefore indicate that survival factor derived from target muscle is crucial to the in vitro motoneurons during the cell death period, but do not support the idea that inactive muscle contains a higher amount of the survival factor.     

Peptidergic motoneurons in the buccal ganglia of Aplysia californica Immunocytochemical,morphological, and physiological characterizations

Summary We used physiological recordings, intracellular dye injections and immunocytochemistry to further identify and characterize neurons in the buccal ganglia of Aplysia calif ornica expressing Small Cardioactive Peptide-like immunoreactivity (SCP-LI). Neurons were identified based upon soma size and position, input from premotor cells B4 and B5, axonal projections, muscle innervation patterns, and neuromuscular synaptic properties. SCP-LI was observed in several large ventral neurons including B6, B7, B9, B10, and B11, groups of s1 and s2 cluster cells, at least one cell located at a branch point of buccal nerve n2, and the previously characterized neurons B1, B2 and B15.B6, B7, B9, B10 and B11 are motoneurons to intrinsic muscles of the buccal mass, each displaying a unique innervation pattern and neuromuscular plasticity. Combined, these motoneurons innervate all major intrinsic buccal muscles (I1/I3, I2, I4, I5, I6). Correspondingly, SCP-LI processes were observed on all of these muscles. Innervation of multiple nonhomologous buccal muscles by individual motoneurons having extremely plastic neuromuscular synapses, represents a unique form of neuromuscular organization which is prevalent in this system. Our results show numerous SCPergic buccal motoneurons with widespread ganglionic processes and buccal muscle innervation, and support extensive use of SCPs in the control of feeding musculature.Abbreviations SCP-LI small cardioactive peptide-like immunoreactivity - PSC postsynaptic current - EPSP excitatory postsynaptic potential - IPSP inhibitory postsynaptic potential - FI facilitation index - TMR time to maximal response     

Cellular mechanisms governing synaptic development in Drosophila melanogaster

The neuromuscular connections of Drosophila are ideally suited for studying synaptic function and development. Hypotheses about cell recognition can be tested in a simple array of pre-and postsynaptic elements. Drosophila muscle fibers are multiply innervated by individually identifiable motoneurons. The neurons express several synaptic cotransmitters, including glutamate, proctolin, and octopamine, and are specialized by their synaptic morphology, neurotransmitters, and connectivity. During larval development the initial motoneuron endings grow extensively over the surface of the muscle fibers, and differentiate synaptic boutons of characteristic morphology. While considerable growth occurs postembryonically, the initial wiring of motoneurons to muscle fibers is accomplished during mid-to-late embryogenesis (stages 15–17). Efferent growth cones sample multiple muscle fibers with rapidly moving filopodia. Upon reaching their target muscle fibers, the growth cones rapidly differentiate into synaptic contacts whose morphology prefigures that of the larval junction. Mismatch experiments show that growth cones recognize specific muscle fibers, and can do so when the surrounding musculature is radically altered. However, when denied their normal targets, motoneurons can establish functional synapses on alternate muscle fibers. Blocking synaptic activity with either injected toxins or ion channel mutants does not derange synaptogenesis, but may influence the number of motor ending processes. The molecular mechanisms governing cellular recognition during synaptogenesis remain to be identified. However, several cell surface glycoproteins known to mediate cellular adhesion events in vitro are expressed by the developing synapses. Furthermore, enhancer detector lines have identified genes with expression restricted to small subsets of muscle fibers and /or motoneurons during the period of synaptogenesis. These observations suggest that in Drosophila a mechanism of target chemoaffinity may be involved in the genesis of stereotypic synaptic wiring. © 1993 John Wiley & Sons, Inc.     

Independent development of sensory and motor innervation patterns in embryonic chick hindlimbs

Previous studies suggest that sensory axon outgrowth is guided by motoneurons, which are specified to innervate particular target muscles. Here we present evidence that questions this conclusion. We have used a new approach to assess the pathfinding abilities of bona fide sensory neurons, first by eliminating motoneurons after neural crest cells have coalesced into dorsal root ganglia (DRG) and second by challenging sensory neurons to innervate muscles in a novel environment created by shifting a limb bud rostrally. The resulting sensory innervation patterns mapped with the lipophilic dyes DiI and DiA showed that sensory axons projected robustly to muscles in the absence of motoneurons, if motoneurons were eliminated after DRG formation. Moreover, sensory neurons projected appropriately to their usual target muscles under these conditions. In contrast, following limb shifts, muscle sensory innervation was often derived from inappropriate segments. In this novel environment, sensory neurons tended to make more "mistakes" than motoneurons. Whereas motoneurons tended to innervate their embryologically correct muscles, sensory innervation was more widespread and was generally from more rostral segments than normal. Similar results were obtained when motoneurons were eliminated in embryos with limb shifts. These findings show that sensory neurons are capable of navigating through their usual terrain without guidance from motor axons. However, unlike motor axons, sensory axons do not appear to actively seek out appropriate target muscles when confronted with a novel terrain. These findings suggest that sensory neuron identity with regard to pathway and target choice may be unspecified or quite plastic at the time of initial axon outgrowth.     

The resilience of the size principle in the organization of motor unit properties in normal and reinnervated adult skeletal muscles

Henneman's size principle relates the input and output properties of motoneurons and their muscle fibers to size and is the basis for size-ordered activation or recruitment of motor units during movement. After nerve injury and surgical repair, the relationship between motoneuron size and the number and size of the muscle fibers that the motoneuron reinnervates is initially lost but returns with time, irrespective of whether the muscles are self- or cross-reinnervated by the regenerated axons. Although the return of the size relationships was initially attributed to the recovery of the cross-sectional area of the reinnervated muscle fibers and their force per fiber, direct enumeration of the innervation ratio and the number of muscle fibers per motoneuron demonstrated that a size-dependent branching of axons accounts for the size relationships in normal muscle, as suggested by Henneman and his colleagues. This same size-dependent branching accounts for the rematching of motoneuron size and muscle unit size in reinnervated muscles. Experiments were carried out to determine whether the daily amount of neuromuscular activation of motor units accounts for the size-dependent organization and reorganization of motor unit properties. The normal size-dependent matching of motoneurons and their muscle units with respect to the numbers of muscle fibers per motoneuron was unaltered by synchronous activation of all of the motor units with the same daily activity. Hence, the restored size relationships and rematching of motoneuron and muscle unit properties after nerve injuries and muscle reinnervation sustain the normal gradation of muscle force during movement by size-ordered recruitment of motor units and the process of rate coding of action potentials. Dynamic modulation of size of muscle fibers and their contractile speed and endurance by neuromuscular activity allows for neuromuscular adaptation in the context of the sustained organization of the neuromuscular system according to the size principle.     

Plasticity in the cockroach neuromuscular system

The retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase extracellularly injected into a leg muscle was used to identify the regenerating cockroach motor neurons that have grown an axonal branch into that muscle. At least 66% of the animals with crushed nerve roots eventually reform the original innervation pattern of this muscle with no mistakes. In spite of this apparent specificity the cockroach neuromuscular system can express plasticity as evidenced by the correction of mistakes made at early stages of regeneration. These mistakes are corrected through elimination during the time interval between 40 and 60 days after nerve crush. In addition, when the distal segments of the leg are removed, thus depriving some motor neurons of their normal target muscles, many of them form stable inappropriate axonal branches in denervated as well as fully innervated muscles. These observations are discussed in terms of possible mechanisms responsible for the specificity of the cellular interactions and in terms of their relevance to understanding the development of vertebrate neuromuscular systems.     

Glutamatergic motoneurons in the stomatogastric ganglion of the mantis shrimp Squilla oratoria

1. Transmitters of motoneurons in the stomatogastric ganglion (STG) of Squilla were identified by analyzing the excitatory neuromuscular properties of muscles in the posterior cardiac plate (pcp) and pyloric regions. 2. Bath and iontophoretic applications of glutamate produce depolarizations in these muscles. The pharmacological experiments and desensitization of the junctional receptors elucidate the glutamatergic nature of the excitatory junctional potentials (EJPs) evoked in the constrictor and dilator muscles. The reversal potentials for the excitatory junctional current (EJC) and for the glutamate-induced current are almost the same. 3. Some types of dilator muscle show sensitivity to both glutamate and acetylcholine (ACh) exogenously applied. The pharmacological evidence and desensitization of the junctional receptors indicate the glutamatergic nature of neuromuscular junctions in these dually sensitive muscles. The reversal potentials for the EJC and for the ACh-induced current are not identical. 4. Glutamate is a candidate as an excitatory neuro-transmitter at the neuromuscular junctions which the STG motoneurons named PCP, PY, PD, LA and VC make with the identified muscles. Kainic and quisqualic acids which act on glutamate receptors are potent excitants of these muscles. Extrajunctional receptors to ACh are present in two types of the muscle innervated by LA and VC. 5. Neurotransmitters used by the STG motoneurons of stomatopods are compared to those of decapods.     

Maintenance of the spinal nucleus of the bulbocavernosus neuromuscular system is not influenced by physiological levels of glucocorticoids

The spinal nucleus of the bulbocavernosus (SNB) neuromuscular system mediates sexual reflexes, and is highly sexually dimorphic in rats. While maintenance of this system in adulthood is mainly dependent on androgens, there is also evidence to suggest that glucocorticoids may have a catabolic effect. We conducted a series of studies to fully examine the influence of basal glucocorticoids on the size of the SNB motoneurons and the associated bulbocavernosus (BC) and levator ani (LA) muscles. Specifically, we examined whether the muscles and motoneurons of the SNB neuromuscular system are affected by: (1) blockade of endogenous glucocorticoids via delivery of the antagonist RU-486 at doses ranging from low to high, (2) removal of endogenous glucocorticoids via adrenalectomy, or (3) restoration of physiological corticosterone levels via implants following adrenalectomy. In each study, we found that muscle and motoneuron size were unaffected by glucocorticoid manipulation. In contrast to previous results with supraphysiological levels of glucocorticoids, our results indicate that basal, nonstress levels of glucocorticoids do not influence the size of the BC/LA muscles or their associated SNB motoneurons.     

Abnormal development of the neuromuscular junction in Nedd4-deficient mice

Nedd4 (neural precursor cell expressed developmentally down-regulated gene 4) is an E3 ubiquitin ligase highly conserved from yeast to humans. The expression of Nedd4 is developmentally down-regulated in the mammalian nervous system, but the role of Nedd4 in mammalian neural development remains poorly understood. Here we show that a null mutation of Nedd4 in mice leads to perinatal lethality: mutant mice were stillborn and many of them died in utero before birth (between E15.5-E18.5). In Nedd4 mutant embryos, skeletal muscle fiber sizes and motoneuron numbers are significantly reduced. Surviving motoneurons project axons to their target muscles on schedule, but motor nerves defasciculate upon reaching the muscle surface, suggesting that Nedd4 plays a critical role in fine-tuning the interaction between the nerve and the muscle. Electrophysiological analyses of the neuromuscular junction (NMJ) demonstrate an increased spontaneous miniature endplate potential (mEPP) frequency in Nedd4 mutants. However, the mutant neuromuscular synapses are less responsive to membrane depolarization, compared to the wildtypes. Ultrastructural analyses further reveal that the pre-synaptic nerve terminal branches at the NMJs of Nedd4 mutants are increased in number, but decreased in diameter compared to the wildtypes. These ultrastructural changes are consistent with functional alternation of the NMJs in Nedd4 mutants. Unexpectedly, Nedd4 is not expressed in motoneurons, but is highly expressed in skeletal muscles and Schwann cells. Together, these results demonstrate that Nedd4 is involved in regulating the formation and function of the NMJs through non-cell autonomous mechanisms.