Ectopic Neuromuscular Junctions Research Articles

TGF-beta: A retrograde signaling molecule involved in the formation of neuromuscular junctions in C. elegans

Event Abstract Back to Event TGF-beta: A retrograde signaling molecule involved in the formation of neuromuscular junctions in C. elegans Abir Rahman1* and Bill Walthall1 1 Georgia State University, Biology Dep, United States The aim of this research is to investigate the role of Transforming Growth Factor Beta (TGF-B) in the formation of neuromuscular junctions in the nematode. Laser ablation of selected embryonic myoblasts removes guidance markers used by post-embryonic muscles (1). The absence of these guideposts results in migrating muscle losing their way and differentiating in ectopic locations. These ectopic muscle strands become innervated by processes from the DD motor neurons (mns). We postulate that a retrograde signaling molecule released by the muscles induces neurite sprouting from the DD mns, resulting in the formation of ectopic neuromuscular junctions (nmjs). An earlier report, combined with our own observations focusing on post-embryonic muscle, indicate that the transient expression pattern of TGF- B corresponds to the nmj formation in the DD mns (2). Current experiments combining laser ablations in various mutant backgrounds, with green fluorescent protein expression patterns should shed light onto th1e communication between presynaptic and the post-synaptic elements at the nmj.

Regulation of the Size and Distribution of Ectopic Neuromuscular Junctions in Adult Skeletal Muscle by Nerve-Derived Trophic Factor and Electrical Muscle Activity

Transplanted axons induced multiple, irregularly distributed acetylcholine receptor (AChR) aggregates on muscle fibers at early stages of ectopic neuromuscular junction formation in denervated adult rat soleus muscles. Subsequently, most AChR aggregates disappeared (the losers). A few aggregates survived (the winners) and, as part of the surviving junctions, reached a certain size and spatial separation along the fibers. This elimination of losers and development of winners occurred only in electrically active muscles whether the activity was elicited by intact axons or by electrical muscle stimulation after the axons had been cut early. We conclude that electrical muscle activity regulates the size and distribution of ectopic neuromuscular junctions by acting in conjunction with a nerve-derived priming influence that does not require the continued presence of nerve terminals.

Regulation of the Size and Distribution of Agrin-Induced Postsynaptic-like Apparatus in Adult Skeletal Muscle by Electrical Muscle Activity

We compared actylcholine receptor (AChR) aggregates induced by neural agrin released from transfected muscle fibers with AChR aggregates induced by transplanted axons in extrajunctional regions of denervated rat soleus muscles. Both neural agrin and transplanted axons induced multiple, irregularly distributed AChR aggregates on muscle fibers. Direct electrical muscle stimulation of transfected muscles for up to 10 weeks removed all agrin-induced AChR aggregates (the losers) except one (the winner) on many fibers. Axon-induced AChR aggregates underwent comparable selection of winners and losers. The results suggest that agrin and acetylcholine-driven muscle activity provided by transplanted axons are sufficient to elicit in a denervated adult muscle fiber processes that regulate the size and distribution of ectopic neuromuscular junctions.

Agrin-Induced Postsynaptic-like Apparatus in Skeletal Muscle Fibersin Vivo

We find that when extrajunctional regions of denervated soleus muscles in adult rats are transfected with cDNA encoding rat agrin isoform Y4Z8, which is normally secreted by motor neurons at adult neuromuscular junctions, the myofibers express and secrete the neural agrin. Muscle fibers in the vicinity of transfection form at their surface specialized areas having extracellular, plasma membrane, and cytoplasmic protein aggregates, narrow and deep plasma membrane infoldings, and an accumulation of myonuclei, all of which are characteristic of the postsynaptic apparatus at neuromuscular junctions. We conclude that at ectopic neuromuscular junctions that form in the extrajunctional region of denervated adult soleus muscles after implantation of a foreign nerve, a single neural-derived factor, agrin, is sufficient not only to cause protein aggregation in the early stages of postsynaptic apparatus formation, as predicted by the agrin hypothesis, but also to bring about changes in conformation of the muscle fiber surface and distribution of organelles which appear as the apparatus reaches maturity.

γ-AChR/ϵ-AChR Switch at Agrin-Induced Postsynaptic-like Apparatus in Skeletal Muscle

We transfected the extrajunctional region of denervated soleus muscles in adult rats with neural agrin cDNA to induce myofibers to form postsynaptic-like apparatus containing acetylcholine receptor (AChR) aggregates. By 1 week ≈30% of the AChR aggregates contained a mixture of ϵ-AChRs and γ-AChRs while ≈70% had only γ-AChRs. If the transfected muscles were reinnervated in the original junctional region, the postsynaptic-like apparatus, despite the absence of apposed axon terminals, gradually came to have only ϵ-AChRs. We conclude that at the postsynaptic apparatus of ectopic neuromuscular junctions formed by a foreign nerve implanted into the extrajunctional region of denervated muscles, agrin secreted by the axon terminal plays a direct role in the γ-AChR/ϵ-AChR switch that occurs as the apparatus reaches maturity. Our findings, together with results from other studies, indicate further that agrin and acetylcholine are the only nerve-derived factors required for this switch.

Dynamic Interactions between Nerve and Muscle inCaenorhabditis elegans

Synaptogenesis among developing motoneurons and muscles was examined in the nematodeCaenorhabditis elegans.In this animal embryonic precursor cells give rise to regionally localized, contiguous clones of muscle cells that form two dorsal and two ventral sets that run longitudinally along the body wall. Ablation of selected embryonic muscle precursors resulted in gaps in the posterior dorsal muscle quadrants. We compared the morphological development of GABAergic locomotory neurons in the presence and absence of their target muscle cells. The results led to four main conclusions: (1) target muscle cells are not required for the morphological differentiation of the motoneurons; (2) target muscle cells appear to be required for the formation of presynaptic varicosities by the motoneurons; (3) embryonic muscle cells serve as a guide for migrating postembryonic muscle cells and in the absence of these guides the postembryonic muscles often assume ectopic locations; and (4) in the presence of ectopic muscle cells, the GABAergic locomotory neurons sprouted and formed branches that contributed to ectopic neuromuscular junctions.

Release and synthesis of acetylcholine at ectopic neuromuscular junctions in the rat.

1. The ability of axons in the superficial fibular nerve to synthesize and release acetylcholine (ACh) has been studied before and after the formation of ectopic neuromuscular junctions (NMJs) with denervated soleus muscles of adult rats. 2. The central end of the severed fibular nerve was transplanted to the surface of the soleus muscle. After 3.5-5 weeks the soleus muscle was denervated in one group of rats by cutting the tibial nerve, allowing the formation of functional ectopic NMJs within a few days. In other rats the tibial nerve remained intact, preventing the formation of functional ectopic NMJs. 3. A month later the content of ACh, the levels of activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), and the amount of ACh released by depolarization by exposure to 50 mM KCl were measured in segments of isolated muscles that (i) contained normal or ectopic NMJs, (ii) were free of nerve or (iii) contained nerve that had not made NMJs. 4. Regions of muscles with ectopic nerve growth in which new NMJs had not formed contained substantial amounts of ACh and ChAT but no AChE. No detectable release of ACh could be evoked from these regions. 5. In muscles in which ectopic NMJs had formed after cutting the tibial nerve, the amounts of ACh and ChAT were about one-fifth of those in the regions of innervation of control muscles. ACh release could be evoked from the region of ectopic nerve growth in amounts nearly as great as those released from NMJs in normal and contralateral control muscles. 6. We conclude that the ability of the terminal parts of mature motor axons to synthesize and store ACh is largely independent of functional contact with muscle fibres. By contrast, the ability to release ACh in substantial amounts only develops when NMJs are formed. The possible significance of this situation for the development of synapses is discussed.

Induction of synaptic extracellular matrix molecules at ectopic neuromuscular junctions

The induction of synapse-specific molecules recognized by peanut agglutinin (PNA) was examined at ectopic neuromuscular junctions in adult frog muscles using light and electron microscopy. In normal frog muscles, PNA specifically recognizes the extracellular matrix at neuromuscular junctions but not at extrajunctional regions. This report shows binding of PNA at ectopic neuromuscular junctions which were initially extrasynaptic and hence unrecognized by PNA. Results suggest that synapse-specific extracellular matrix molecules can be induced de novo at new junctional sites.

Induction of active zones at ectopic neuromuscular junctions in the frog

To examine de novo differentiation of the active zone, ectopic neuromuscular junctions were studied in adult frog muscles. Ectopic junctions induced by excising the original endplate region and implanting the nerve to an endplate-free site were examined by light and electron microscopy 4 weeks-1 year after operations. The earliest time point at which ectopic junction formation was detected with freeze fracture was 6 weeks postoperation, when clusters of active zone particles were observed scattered across the nerve ending. Subsequently, short active zones (6-10 weeks postoperation, length mean = 0.36 +/- 0.24 microns) composed of the characteristic 2 double rows of particles are detected. Before junctional folds are observed with freeze fracture, many active zones are parallel to each other and to the long axis of the nerve. The average angle 6-10 weeks postoperation is 27 degrees +/- 23 degrees. Even during these early stages of formation, active zones are functional. As time passes, active zones attain a more typical, perpendicular orientation (12-18 weeks postoperation, mean = 62 degrees +/- 24 degrees) and also increase in length (mean = 0.69 +/- 0.45 microns). However, even after 1 year, the orientation (angle, mean = 70 degrees +/- 22 degrees) and the length (mean = 0.78 +/- 0.63 microns) of active zones at ectopic junctions are still not well correlated with active zones at normal junctions (normal active zone angle mean = 85 degrees +/- 5 degrees, length mean = 1.00 +/- 0.57 microns).(ABSTRACT TRUNCATED AT 250 WORDS)

Late reinnervation of the rat soleus muscle is differentially suppressed by chronic stimulation and by ectopic innervation.

Soleus (SOL) and extensor digitorum longus (EDL) muscles in adult rats were kept denervated for 2 months by four repeated freezes at 2-week intervals of the sciatic nerve. Reinnervation was studied in the absence or presence of chronic muscle stimulation, starting 1 month before reinnervation began. In addition, reinnervation was studied in SOL muscles where a previously transplanted fibular (FIB) nerve had formed ectopic neuromuscular junctions outside the original endplate area. After repeated freezes only, reinnervation was complete judged by tension measurements and histochemical examinations in SOL (n = 7) and EDL (n = 8) muscles. In directly stimulated muscles reinnervation was incomplete, and the force tensions evoked from indirect stimulation was on average 87 (n = 5) and 82% (n = 5) of direct muscle stimulation in SOL and EDL muscles, respectively. Of ectopically innervated SOL muscle fibres, only 26% became reinnervated in 12 muscles. Denervation and reinnervation increased the number of muscle fibres in stimulated (n = 4) and unstimulated (n = 5) EDL muscles by 18 and 15%, respectively. In stimulated (n = 4) and unstimulated (n = 7) SOL muscles, on the other hand, the number of muscle fibres remained normal. The stronger suppression of reinnervation in ectopically reinnervated compared to chronically stimulated SOL fibres indicates that reinnervation can also be suppressed by activity independent influences from the foreign nerve.

Channel gating at frog neuromuscular junctions formed by different cholinergic neurones.

1. Drug-induced membrane current fluctuations were analysed at frog ectopic neuromuscular junctions formed de novo by somatic motoneurones and by preganglionic autonomic neurones of the vagus nerve, and at denervated end-plates reinnervated by the vagus nerve. 2. At ectopic motor end-plates, the mean open time of the ion channels activated by ACh is tau ACh = 1.8 +/- 0.1 msec (S.E.) at -70 to -90 mV and 15-18 degrees C. Carbachol- and suberyldicholine-induced channels have average open times tau Carb = 1.0 +/- 0.1 msec (-80 mV, 6-8 degrees C) and tau Sub = 3.3 +/- 0.1 msec (-80 mV, 16-18 degrees C). 3. The conductances gamma of single channels induced by ACh, carbachol and suberyldicholine are: gamma ACh = 21.0 +/- 1.1 pS (+/- S.E.), gamma Carb = 14.6 +/- 1.2 pS and gamma Sub = 22.1 +/- 1.2 pS. 4. The mean open times tau of the channels is prolonged as the membrane is hyperpolarized. Their voltage dependence is similar to that of normal end-plate channels. 5. The average open time of ACh-induced channels at ectopic vagus junctions is tau = 1.6 +/- 0.2 msec (S.E.) at -70 to -80 mV and 14-15 degrees C. At denervated motor end-plates reinnervated by vagal neurones, the mean channel open time is tau = 1.5 +/- 0.1 msec at -80 mV and 14-18 degrees C. 6. At all vagus junctions, the synaptic currents are blocked by alpha-bungarotoxin. 7. Somatic motoneurons and vagal neurones induce junctional folds in the muscle membrane where they contact it to form an ectopic end-plate. Staining the ACh receptors of the motor end-plates with horseradish peroxidase-alpha-bungarotoxin conjugate shows that the receptors are restricted to the junctional region of the muscle fibre membrane.

Formation of ectopic neuromuscular junctions in adult rats.

The formation of ectopic neuromuscular junctions between a transplanted foreign nerve (the superficial fibular nerve) and the denervated soleus muscle was examined in adult rats. Formation of new junctions was induced by denervating the soleus muscle by cutting the tibial nerve. Junctional transmission began 3 days after denervation when denervation was done 3 weeks after transplantation of the foreign nerve, and progressively later when denervation was done 8, 16 and 24 weeks after transplantation. The rate at which the transmission, once started, acquired mature characteristics was approximately the same in each case. Initially, spontaneous m.e.p.p.s were infrequent, long lasting with a skewed amplitude distribution. The e.p.p.s evoked by stimulation of the foreign nerve were then commonly below threshold for eliciting action potentials and were occasionally no larger than the size of the spontaneous m.e.p.p.s. M.e.p.p. characteristics became normal in the 2nd week after transmission had started. Fully effective evoked transmission with every innervated fibre responding with overshooting action potential occurred 1-3 months after onset of transmission.

Control of junctional acetylcholinesterase by neural and muscular influences in the rat.

1. The development of AChE at ectopic neuromuscular junctions forming between a transplanted foreign nerve (the superficial fibular nerve) and the denervated soleus muscle has been studied in adult rats. 2. Junctional AChE activity began to appear in the vicinity of the fibular nerve sprouts 6-7 days after section of the soleus nerve and 3-4 days after the onset of transmission. 3. No histochemically detectable AChE appeared when the fibular nerve was cut 0-4 days after the soleus nerve had been cut. 4. Direct electrical stimulation of the denervated soleus muscle caused plaques of true AChE, as determined by inhibitor studies, to appear in muscles where the fibular nerve had been cut 2-4 days after the soleus nerve but not in muscles where the two nerves had been cut at the same time. The plaques appeared only in the vicinity of fibular nerve sprouts and coincided with newly formed but stable peaks of ACh sensitivity. Local application of Neostigmine prolonged and increased the depolarising response evoked by pulses of ACh at these sites. 5. In muscles where the fibular nerve was intact the AChE plaques changed gradually over a few weeks from an immature appearance to a mature appearance characteristic of normal end-plates. In stimulated muscles where the fibular nerve had been cut the plaques stained intensely but remained morphologically immature. 6. We conclude (1) that muscle activity is important for the appearance of AChE at developing neuromuscular junctions and (2) that AChE accumulates only at sites on the muscle surface where the nerve fibres have left a 'trace' upon contact with the muscle fibres. These traces form quickly and persist after nerve-muscle interaction of as little as 2 days. The muscle appears as a major source of junctional AChE since stimulation of the muscle induces intense AChE activity in muscles where the nerve has degenerated.