Acetylcholine Receptor Binding Properties at the Rat Neuromuscular Junction During Aging

Specific binding characteristics of acetylcholine receptors at the diaphragm neuromuscular junction of rats aged 10 (mature adult) and 28 (aged) months were assayed by measuring 125I-alpha-bungarotoxin binding. Maximal binding to intact tissue samples was greater in the older rats; this could be attributed to an age-related increase in terminal branching. The toxin concentration at which half-maximal binding occurred increased in the older rats. Binding kinetics were assayed in finely minced tissue samples, and the association rate constant was observed to decrease in the 28-month animals. Retardation of the initial rate of toxin binding by d-tubocurarine (dTC) in minced tissue was described by a two-component nonlinear Hofstee plot; IC50 values (7.1-7.2 microM and 39.0-46.5 nM) were about the same for both age groups, but there was a significant shift toward the low-affinity values in the aged rats. Rhodamine-conjugated alpha-bungarotoxin was used to visualize receptor localization. There were no major changes in receptor distribution, and nerve terminals were consistently associated with receptors and vice versa. The data indicate a shift toward lower binding affinity during aging, which may involve changes either in one of the two toxin-binding sites on individual receptors, in dTC blocking of the channel moiety, or in receptor types.     

Patterning and organization of motor neuron dendrites in the Drosophila larva

Precise patterns of motor neuron connectivity depend on the proper establishment and positioning of the dendritic arbor. However, how different motor neurons orient their dendrites to selectively establish synaptic connectivity is not well understood. The Drosophila neuromuscular system provides a simple model to investigate the underlying organizational principles by which distinct subclasses of motor neurons orient their dendrites within the central neuropil. Here we used genetic mosaic techniques to characterize the diverse dendritic morphologies of individual motor neurons from five main nerve branches (ISN, ISNb, ISNd, SNa, and SNc) in the Drosophila larva. We found that motor neurons from different nerve branches project their dendrites to largely stereotyped mediolateral domains in the dorsal region of the neuropil providing full coverage of the receptive territory. Furthermore, dendrites from different motor neurons overlap extensively, regardless of subclass, suggesting that repulsive dendrite-dendrite interactions between motor neurons do not influence the mediolateral positioning of dendritic fields. The anatomical data in this study provide important information regarding how different subclasses of motor neurons organize their dendrites and establishes a foundation for the investigation of the mechanisms that control synaptic connectivity in the Drosophila motor circuit.     

Acetylcholinesterase function is dispensable for sensory neurite growth but is critical for neuromuscular synapse stability

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine. In addition, AChE is thought to play several 'non-classical' roles that do not require catalytic function. Most prominent among these is facilitation of neurite growth. Here, we report that the zebrafish zieharmonika (zim) locus encodes AChE. We show that one mutant zim allele is caused by a pre-mature stop codon, resulting in a truncated protein that lacks both the catalytic site and the carboxy-terminal neuritogenic domain. To explore the 'non-classical' role of AChE, we examined embryos mutant for this allele. In contrast to previous results using a catalytic-inactive allele, our analysis demonstrates that AChE is dispensable for muscle fiber development and Rohon-Beard sensory neuron growth and survival. Moreover, we show that in the absence of AChE, acetylcholine receptor clusters at neuromuscular junctions initially assemble, but that these clusters are not maintained. Taken together, our results demonstrate that AChE is dispensable for its proposed non-classical roles in muscle fiber formation and sensory neuron development, but is crucial for regulating the stability of neuromuscular synapses.     

Neuromuscular synapses mediate motor axon branching and motoneuron survival during the embryonic period of programmed cell death

The embryonic period of motoneuron programmed cell death (PCD) is marked by transient motor axon branching, but the role of neuromuscular synapses in regulating motoneuron number and axonal branching is not known. Here, we test whether neuromuscular synapses are required for the quantitative association between reduced skeletal muscle contraction, increased motor neurite branching, and increased motoneuron survival. We achieved this by comparing agrin and rapsyn mutant mice that lack acetylcholine receptor (AChR) clusters. There were significant reductions in nerve-evoked skeletal muscle contraction, increases in intramuscular axonal branching, and increases in spinal motoneuron survival in agrin and rapsyn mutant mice compared with their wild-type littermates at embryonic day 18.5 (E18.5). The maximum nerve-evoked skeletal muscle contraction was reduced a further 17% in agrin mutants than in rapsyn mutants. This correlated to an increase in motor axon branch extension and number that was 38% more in agrin mutants than in rapsyn mutants. This suggests that specializations of the neuromuscular synapse that ensure efficient synaptic transmission and muscle contraction are also vital mediators of motor axon branching. However, these increases in motor axon branching did not correlate with increases in motoneuron survival when comparing agrin and rapsyn mutants. Thus, agrin-induced synaptic specializations are required for skeletal muscle to effectively control motoneuron numbers during embryonic development.     

Peripheral nervous system defects in a mouse model for peroxisomal biogenesis disorders

Peroxisome biogenesis disorders (PBD) are autosomal recessive disorders in humans characterized by skeletal, eye and brain abnormalities. Despite the fact that neurological deficits, including peripheral nervous system (PNS) defects, can be observed at birth in some PBD patients including those with PEX10 mutations, the embryological basis of the PNS defects is unclear. Using a forward genetic screen, we identified a mouse model for Pex10 deficiency that exhibits neurological abnormalities during fetal development. Homozygous Pex10 mutant mouse embryos display biochemical abnormalities related to a PBD deficiency. During late embryogenesis, Pex10 homozygous mutant mice experience progressive loss of movement and at birth they become cyanotic and die shortly thereafter. Homozygous Pex10 mutant fetuses display decreased integrity of axons and synapses, over-extension of axons in the diaphragm and decreased Schwann cell numbers. Our neuropathological, molecular and electrophysiological studies provide new insights into the embryological basis of the PNS deficits in a PBD model. Our findings identify PEX10 function, and likely other PEX proteins, as an essential component of the spinal locomotor circuit.     

Acetylcholine Synthesis in the Schwann Cell and Axon in the Giant Nerve Fiber of the Squid

Abstract: Acetyltransferase enzymatic activity was detected and measured in homogenates obtained from intact nerve fibers and their separate cellular components, in the tropical squid Sepioteuthis sepioidea. The levels of acetylcholine synthesis were determined in pooled samples of whole stellar nerve, intact giant nerve fiber, extruded axoplasm, axoplasm-free giant nerve fiber sheaths, and small nerve fibers. The values found per mg of protein for the axoplasm-free sheaths are about 3–9 times those of the extruded axoplasm, and comparable to those found for the intact giant nerve fiber. These experimental findings settle the question of whether the Schwann cells of the giant nerve fiber of S. sepioidea , under physiological conditions, contain acetyltransferase activity and are able to synthesize acetylcholine.     

Multiple Molecular Forms of Acetylcholine Receptors in Cultured Skeletal Muscle Cells: Subcellular Localization and Characterization

Abstract: Skeletal muscle cells of newborn rats, cultured in the absence of neuronal influence, were found to contain two types of cell surface acetylcholine receptors as demonstrated by isoelectric focusing. The isoelectric points of the two types of receptors were indistinguishable from those of junctional and extrajunctional types of receptors in mature animals. The cultured cells had two classes of intracellular α-bungarotoxin (αBT) binding components; one had the same sedimentation coefficient as that of surface receptors (9S), and the other had much smaller apparent molecular weights. Only a single major component was detected by isoelectric focusing analysis of the 9s intracellular aBT binding component, with a PI value close to that of the extra junctional receptor. These results suggest that the junctional and extrajunctional types of receptors may be synthesized through a common precursor.     

Alterations in peripheral nerves of rats treated with chlorphentermine or with iprindole

Summary This study deals with the effects of two amphiphilic lipidosis-inducing drugs (chlorphentermine, iprindole) upon the ultrastructure of peripheral nerves of rats. After prolonged drug treatment the preterminal and terminal axoplasm of motor and sensory nerves within skeletal muscles contain numerous abnormal inclusions (osmiophilic conglomerates, autophagic vacuoles, lamellated bodies). By contrast, the axons within large peripheral nerves are little affected. The present observations are tentatively interpreted as resulting from interference with catabolic processes involved in the normal turnover of axoplasmic constituents at the nerve terminal. The exact pathogenesis and the functional significance of these alterations remain to be elucidated.     

Acetylcholine Synthesis and Release in the Extensor Digitorum Longus Muscle of Mature and Aged Rats

Uptake of labeled choline and its incorporation into acetylcholine (ACh) were assayed at the neuromuscular junction of the extensor digitorum longus (EDL) muscle of rats aged 11 (mature adult) and 27 (aged) months. Under resting conditions, there were no significant differences in muscle ACh or choline levels. Following a 1-h incubation in labeled choline, however, tissue from the younger rats contained significantly greater amounts of labeled choline and labeled ACh; the specific activities of ACh and choline were nearly 10-fold higher in the 11-month-old animals, indicating reduced uptake of labeled choline in the older animals. ACh and choline efflux rates under resting conditions did not change with age, indicating an uncoupling of exogenous choline uptake and ACh efflux in EDL during aging. During nerve stimulation (1 Hz), the amount of labeled choline incorporated into ACh was 150% greater in the aged animals. The specific activity of ACh released during stimulation was correspondingly greater in the 27-month-old animals, although total ACh released did not change appreciably with age. There were no age-related differences in choline acetyltransferase activity. Contrasting results were obtained from diaphragm in previous studies; the linkage between choline uptake and ACh efflux was maintained during rest and stimulation in the diaphragm. Hypothetically, these differences between EDL and diaphragm may be related to their diverse activation patterns: EDL is recruited much less frequently and less regularly than diaphragm, a continually active vital muscle.     

Stimulated Release of Phosphatidylinositol Phosphodiesterase in an Isolated Phrenic Nerve-Diaphragm Preparation

Electrical stimulation of the phrenic nerve in an isolated nerve-diaphragm preparation resulted in the release of phosphatidylinositol phosphodiesterase into the organ bath. The released enzyme was Ca2+-dependent and exhibited two pH optima. The enzyme was released in response to nerve stimulation even in the presence of d-tubocurarine in concentrations that block neuromuscular transmission, and was not therefore released from the muscle as a consequence of its contractile activity. Phosphatidylinositol phosphodiesterase activity was determined in the soluble cytosol fractions prepared from different regions of skeletal muscles and from normal peripheral nerves and nerves that were degenerating after transection. The specific activity of the enzyme in the cytosol from the endplate-rich region of the diaphragm was significantly greater than that in cytosol from either the endplate-free region of the diaphragm or from the phrenic nerve. In degenerating nerve the activity of the enzyme was greater in the distal stump than in the proximal stump at 36 h after nerve section. Possible roles for released phosphatidylinositol phosphodiesterase at the neuromuscular junction are discussed.     

Hepatocyte growth factor is necessary for efficient outgrowth of injured peripheral axons in in vitro culture system and in vivo nerve crush mouse model

Hepatocyte growth factor (HGF) is a neurotrophic factor and its role in peripheral nerves has been relatively unknown. In this study, biological functions of HGF and its receptor c-met have been investigated in the context of regeneration of damaged peripheral nerves. Axotomy of the peripheral branch of sensory neurons from embryonic dorsal root ganglia (DRG) resulted in the increased protein levels of HGF and phosphorylated c-met. When the neuronal cultures were treated with a pharmacological inhibitor of c-met, PHA665752, the length of axotomy-induced outgrowth of neurite was significantly reduced. On the other hand, the addition of recombinant HGF proteins to the neuronal culture facilitated axon outgrowth. In the nerve crush mouse model, the protein level of HGF was increased around the injury site by almost 5.5-fold at 24 h post injury compared to control mice and was maintained at elevated levels for another 6 days. The amount of phosphorylated c-met receptor in sciatic nerve was also observed to be higher than control mice. When PHA665752 was locally applied to the injury site of sciatic nerve, axon outgrowth and injury mediated induction of cJun protein were effectively inhibited, indicating the functional involvement of HGF/c-met pathway in the nerve regeneration process. When extra HGF was exogenously provided by intramuscular injection of plasmid DNA expressing HGF, axon outgrowth from damaged sciatic nerve and cJun expression level were enhanced. Taken together, these results suggested that HGF/c-met pathway plays important roles in axon outgrowth by directly interacting with sensory neurons and thus HGF might be a useful tool for developing therapeutics for peripheral neuropathy.     

Death of an axon: studying axon loss in development and disease

The loss of axon branches is a common feature of both the developing and the diseased nervous system. Despite its fundamental importance, a clear mechanistic understanding is lacking on how axonal loss occurs. However, the first molecular inroads into post-traumatic (Wallerian) axon degeneration have recently been made. In parallel, imaging techniques that allow visualizing single axons in vivo are providing a first glimpse at the cellular mechanisms of active dismantling of superfluous or diseased axons. This gives hope that soon a clearer mechanistic understanding of axon loss will emerge: comparing different forms of axon loss will reveal the spectrum of axon loss mechanisms; studies aimed at integrating the known molecular and cellular players during axon loss will provide mechanistic insight into axon dismantling; finally—by understanding how axons are normally lost—we will hopefully find ways to protect them during neurological disease or after trauma. Note: Robert Feulgen Prize Lecture 2005 presented at the Joint meeting of The Histochemical Society and The Society for Histochemistry in Noordwijkerhout, The Netherlands. After completion of this review, a comprehensive and highly informative overview of axon loss events in development and disease was published (Luo and O’Leary 2005).     

Tonic adenosine neuromodulation is preserved in motor nerve endings of aged rats

Neuromuscular transmission is decreased in aged subject. Since endogenous adenosine is a potent neuromodulator at motor nerve endings, either inhibiting via A₁ receptors or facilitating via A_2A receptors acetylcholine release, we now investigated if the tonic effect of endogenous adenosine was modified at phrenic nerve endings of aged rats. The A_2A receptor antagonist (ZM241385, 50 nM) inhibited (77 ± 9%) and the A₁ receptor antagonist (DPCPX, 50 nM) facilitated (74 ± 13%) acetylcholine release from young adult (6 weeks old) rat preparations, indicating a simultaneous tonic activation of A_2A and A₁ receptors. Tonic modulation by adenosine was unaltered in aged (24 months old) rats, since ZM241385 (50 nM) inhibited (73 ± 8%) and DPCPX (50 nM) facilitated (91 ± 20%) acetylcholine release in aged animals similarly to young rats. This indicates that, in contrast to the central nervous system where adenosine neuromodulation is modified in aged animals, the control by adenosine of phrenic nerve function is preserved in aged animals     

Aplysia hemolymph promotes neurite outgrowth and synaptogenesis of identifiedHelix neurons in cell culture

Hemolymph of adultAplysia californica significantly affects neurite outgrowth of identified neurons of the land snailHelix pomatia. The metacerebral giant cell (MGC) and the motoneuron C3 from the cerebral ganglion and the neuron B2 from the buccal ganglion ofH. pomatia were isolated by enzymatic and mechanical dissociation and plated onto poly-l-lysine-coated dishes either containing culture medium conditioned byHelix ganglia, or pre-treated withAplysia hemolymph. To determine the extent of neuronal growth we measured the neurite elongation and the neuritic field of cultured neurons at different time points.Aplysia hemolymph enhances the extent and rate of linear outgrowth and the branching domain ofHelix neurons. With the hemolymph treatment the MGC neuron more consistently forms specific chemical synapses with its follower cell B2, and these connections are more effective than those established in the presence of the conditioned medium.     

Differential Expression of Ciliary Neurotrophic Factor Receptor in Skeletal Muscle of Chick and Rat After Nerve Injury

Abstract: The activities of ciliary neurotrophic factor (CNTF) were initially thought to be restricted to cells in the nervous system. However, the recent identification of its receptor specificity-conferring α component (CNTFRα) in skeletal muscle has provided the clue to the unexpected actions of CNTF in the periphery. In the present study, we demonstrated that the mRNA expression of CNTFRα in chick skeletal muscle was decreased by ∼10-fold after nerve transection; this finding is in sharp contrast to the dramatic up-regulation observed in denervated rat muscle. As a first step toward investigating the differential regulation of CNTFRα in chick and rat, we examined the mRNA expression of CNTFRα in different types of muscle following nerve injury in young and adult animals. Our findings demonstrated that the differential expression of CNTFRα observed in denervated skeletal muscle of the chick and rat was not dependent on age or muscle type. The temporal profile of the changes in CNTFRα expression was, however, dependent on the age of the chick as well as the types of muscle. Furthermore, the low level of CNTFRα expression observed in denervated chick muscle recovered to almost control levels in regenerating skeletal muscle. Taken together, our findings provided the first extensive analysis on the mRNA expression of CNTFRα and the α subunit of the acetylcholine receptor in various skeletal muscles of the chick following nerve injury and regeneration.     

Nitric oxide synthase activity is required for postsynaptic differentiation of the embryonic neuromuscular junction

Agrin, a synapse-organizing protein externalized by motor axons at the neuromuscular junction (NMJ), initiates a signaling cascade in muscle cells leading to aggregation of postsynaptic proteins, including acetylcholine receptors (AChRs). We examined whether nitric oxide synthase (NOS) activity is required for agrin-induced aggregation of postsynaptic AChRs at the embryonic NMJ in vivo and in cultured muscle cells. Inhibition of NOS reduced AChR aggregation at embryonic Xenopus NMJs by 50-90%, whereas overexpression of NOS increased AChR aggregate area 2- to 3-fold at these synapses. NOS inhibitors completely blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. Application of NO donors to muscle cells induced AChR clustering in the absence of agrin. Our results indicate that NOS activity is necessary for postsynaptic differentiation of embryonic NMJs and that NOS is a likely participant in the agrin-MuSK signaling pathway of skeletal muscle cells.     

Unidirectional startle responses and disrupted left–right co-ordination of motor behaviors in robo3 mutant zebrafish

The Roundabout (Robo) family of receptors and their Slit ligands play well-established roles in axonal guidance, including in humans where horizontal gaze palsy with progressive scoliosis (HGPPS) is caused by mutations in the robo3 gene. Although significant progress has been made toward understanding the mechanism by which Robo receptors establish commissural projections in the central nervous system, less is known about how these projections contribute to neural circuits mediating behavior. In this study, we report cloning of the zebrafish behavioral mutant twitch twice and show that twitch twice encodes robo3 . We show that in mutant hindbrains the axons of an identified pair of neurons, the Mauthner cells, fail to cross the midline. The Mauthner neurons are essential for the startle response, and in twitch twice / robo3 mutants misguidance of the Mauthner axons results in a unidirectional startle response. Moreover, we show that twitch twice mutants exhibit normal visual acuity but display defects in horizontal eye movements, suggesting a specific and critical role for twitch twice / robo3 in sensory-guided behavior.     

Synaptogenetic mechanisms controlling postsynaptic differentiation of the neuromuscular junction are nerve-dependent in human and nerve-independent in mouse C2C12 muscle cultures

Acetylcholinesterase (EC 3.1.1.7, AChE) is one of the components of the neuromuscular junction (NMJ). Its expression and targeting in the skeletal muscle fiber is therefore under the control of the mechanisms responsible for the formation of the highly complex structure of this synapse. Recently, it has been demonstrated that myotubes of the C2C12 mouse muscle cell line form highly differentiated pretzel-like postsynaptic accumulations of acetylcholine receptors (AChRs) in the complete absence of the nerve if they are cultured on the laminin coating. This finding questions previously stressed importance of the nerve-derived factors in NMJ synaptogenesis and therefore deserves additional testing. The aim of this paper was to test whether the reported nerve-independency can be demonstrated also in the cultured human muscle meaning that the findings on C2C12 cultures can be extrapolated also to the human muscle. In our experiments aneurally cultured human myotubes failed to form AChR clusters on its surface, no matter if they were grown on normal gelatine or laminin coating. However, when innervated by neurons extending from the rat embryonic spinal cord, human myotubes formed AChR clusters with elaborate topography but strictly on the areas contacted by the nerve. One can hypothesize that higher nerve dependency of the NMJ synaptogenesis in humans in comparison to other species reflects species-specific differences in the organization of movement. Humans have the highest "fractionation of movement" capacity which probably requests different, more nerve-controlled development of the motor system including nerve-restricted development of the neuromuscular contacts.     

Peptide influences on the development and regeneration of motor performance

ACTH 1-39 (0.2 U IP daily for up to 18 days) has a beneficial effect on the functional reorganization of regenerating motor units of the extensor digitorum longus (EDL) in the adrenalectomized adult rat following crushing of the peroneal nerve. Motor unit activity (maximum twitch tension amplitude/mean increment in twitch tension as voltage is increased by 0.1 V gradations) and nerve-muscle efficiency (tetanic tension from indirect stimulation/tetanic tension from direct stimulation of EDL) were enhanced by ACTH 1-39. Other electrophysiological and contractile parameters were unaffected by the peptide. Spontaneous motor activity in cold stressed 13 day old rats was prolonged by Org 2766, a substituted analogue of ACTH/MSH 4-9, (0.1 microgram/kg daily) but unaffected by the same dosage of ACTH/MSH 4-10. The responsiveness of developing and regenerating motor systems to neuropeptides indicates a plasticity of neuronal connections, which depends on peptide sequence, dosage and the physiological state of the animal (normal, depressed, regenerating or developing, at rest or stressed).     

Guanylate cyclase and cyclic GMP-dependent protein kinase regulate agrin signaling at the developing neuromuscular junction

During formation of the neuromuscular junction (NMJ), agrin secreted by motor axons signals the embryonic muscle cells to organize a postsynaptic apparatus including a dense aggregate of acetylcholine receptors (AChRs). Agrin signaling at the embryonic NMJ requires the activity of nitric oxide synthase (NOS). Common downstream effectors of NOS are guanylate cyclase (GC), which synthesizes cyclic GMP, and cyclic GMP-dependent protein kinase (PKG). Here we show that GC and PKG are important for agrin signaling at the embryonic NMJ of the frog, Xenopus laevis. Inhibitors of both GC and PKG reduced endogenous AChR aggregation in embryonic muscles by 50-85%, and blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. A cyclic GMP analog, 8-bromo-cyclic GMP, increased endogenous AChR aggregation in embryonic muscles to 3- to 4-fold control levels. Overexpression of either GC or PKG in embryos increased AChR aggregate area by 60-170%, whereas expression of a dominant negative form of GC inhibited endogenous aggregation by 50%. These results indicate that agrin signaling in embryonic muscle cells requires the activity of GC and PKG as well as NOS.