Rat Neuromuscular Junction Research Articles

Integrins at the neuromuscular junction are important for motoneuron survival

During development motoneurons depend on target contact for their survival. Following injury to the sciatic nerve in neonatal rats, a large proportion of motoneurons die. However, the same injury inflicted at 5 days of age results in no loss of motoneurons. This critical period of postnatal development coincides with the time during which there is a significant increase in the release of transmitter from the nerve terminals at the neuromuscular junction. We have proposed that the role of the target muscle cell during this period is to induce this up-regulation of transmitter release from motor nerve terminals. It has been shown that stretch-induced increase in transmitter release from frog motor nerve terminals is accomplished via an integrin-dependent mechanism. In this study we examined the role of integrins at the rat neuromuscular junction in motoneuron survival. We found that blocking integrin binding at the developing neuromuscular junction delayed the increase in choline acetyltransferase activity that normally takes place during the early postnatal period, and resulted in motoneuron death. Furthermore, the maturation of those motoneurons that survived was delayed so they remained susceptible to subsequent nerve injury. These results support the possibility that integrins, by their involvement in modulating transmitter release, can influence motoneuron survival.

Alpha-conotoxin ImI inhibits the alpha-bungarotoxin-resistant nicotinic response in bovine adrenal chromaffin cells.

The activity of alpha-conotoxin (alpha-CTX) ImI, from the vermivorous marine snail Conus imperialis, has been studied on mammalian nicotinic receptors on bovine chromaffin cells and at the rat neuromuscular junction. Synthetic alpha-CTX ImI was a potent inhibitor of the neuronal nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 microM, log IC50 = 0.4 +/- 0.07), showing competitive inhibition of nicotine-evoked catecholamine secretion. Alpha-CTX ImI also inhibited nicotine-evoked 45Ca2+ uptake but not 45Ca2+ uptake stimulated by 56 mM K+. In contrast, alpha-CTX ImI had no effect at the neuromuscular junction over the concentration range 1-20 microM. Bovine chromaffin cells are known to contain the alpha3beta4, alpha7, and (possibly) alpha3beta4alpha5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by alpha-bungarotoxin, indicating that alpha7 nicotinic receptors are not involved. We propose that alpha-CTX Iml interacts selectively with the functional (alpha3beta4 or alpha3beta4alpha5) nicotinic acetylcholine receptor to inhibit the neuronal-type nicotinic response in bovine chromaffin cells.

Localization of transforming growth factor beta in association with neuromuscular junctions in adult human muscle.

Transforming growth factors-beta 1, 2, and 3 are known for their regulatory function in embryogenesis, fibrogenesis, and tissue repair of different cell types. A trophic function of TGF-beta subclasses for motoneurons has been shown in vitro. TGF-beta 1 is a potent survival factor for cultured embryonic rat motoneurons. In addition, TGF-beta 1 stimulates proliferation of rat Schwann cells. Recently, TGF-beta 2 has been reported to be associated with the subsynaptic nuclei of mature rat neuromuscular junctions. In this study, we investigated the expression of TGF-beta 1, 2, and 3 at neuromuscular junctions in skeletal muscle of 11 adults without neuromuscular disease. On muscle biopsies, neuromuscular junctions were depicted by acetylcholine esterase reaction and acetylcholine receptor antibodies. TGF-beta 1; 2, and 3 were stained immunohistochemically with monoclonal antibodies. Some muscle fibers showed low levels of inhomogeneous immunoreactivity for both TGF-beta 1 and TGF-beta 3. Intense immunoreactivity of TGF-beta 1 and 3 was shown at the postsynaptic area of neuromuscular junctions. TGF-beta 2 was expressed in the same subcellular distribution, but less strongly. In conclusion, the colocalization of TGF-beta with neuromuscular junctions may suggest a significant function in neuromuscular communication.

Calcium channels coupled to neurotransmitter release at neonatal rat neuromuscular junctions.

1. The effects of different calcium channel blockers (omega-agatoxin IVA (omega-Aga IVA), omega-conotoxin GVIA (omega-CgTx GVIA) and dihydropyridines) were tested on spontaneous and evoked transmitter release at embryonic and newborn rat neuromuscular junctions (NMJs). 2. The nerve-evoked transmitter release quantal content (m) was strongly reduced by the P/Q-type voltage-dependent calcium channel (VDCC) blocker omega-Aga IVA (100 nM) at newly formed endplates of embryos and 0- to 11-day-old rats, in agreement with the effect of this blocker on transmitter release at mature and reinnervating muscles. 3. omega-CgTx GVIA (1-5 microM), the N-type VDCC blocker, also caused a significant reduction in m at newly formed NMJs early in development (embryos and 0- to 4-day-old rats), while it was ineffective in more mature animals (5- to 11-day-old rats). 4. L-type channel blockers, nitrendipine (1 microM) and nifedipine (1 microM), did not significantly affect neurally evoked release at developing NMJs. However, nifedipine (10 microM) was able to increase m significantly at 0- to 4-day-old rat NMJs. 5. At developing NMJs, K+-evoked transmitter release was dependent on Ca2+ entry through VDCCs of the P/Q-type family (100 nM omega-Aga IVA reduced 70 % of the K+-evoked miniature endplate potential frequency). N- and L-type VDCC blockers did not affect this type of release. 6. We conclude that at rat neuromuscular junctions the presynaptic calcium channel types involved in transmitter release undergo developmental changes during the early postnatal period.

Schwann Cells Proliferate at Rat Neuromuscular Junctions during Development and Regeneration

Terminal Schwann cells (TSCs) cover neuromuscular junctions and are important in the repair and maintenance of these synapses. We have examined how these cells are generated at developing junctions and how their number is regulated during repair of nerve injury. At birth, approximately half of the junctions in rat soleus and extensor digitorum longus muscles have one TSC soma. Somata are absent from the remainder, although Schwann cell (SC) processes arising from somata along the preterminal axon cover almost all of these synapses. By 2 months of age, junctions have gained an additional two to three TSCs. Most of this gain occurs during the first 2 postnatal weeks and largely precedes the expansion of endplate size. Although the initial addition is caused by cell migration, mitotic labeling shows extensive division of TSCs at junctions. A slower addition of TSCs occurs in adult muscles, and TSC number in the adult is correlated with endplate size. During repair of nerve injury, TSC number is regulated by a combination of signals from motor neurons and denervated tissue. As shown previously (Connor et al., 1987), denervation of adult muscles did not, in itself, cause TSC mitosis. However, TSCs became mitotic during reinnervation. Partial denervation induced division of TSCs at innervated but not denervated endplates. A disproportionate number of these mitotic cells were found at endplates contacted by TSC processes extended from nearby denervated endplates, contacts known to promote nerve sprouting. These results show an association between TSC mitotic activity and alterations in synaptic structure during development, sprouting, and reinnervation.

Prejunctional effects of the nicotinic ACh receptor agonist dimethylphenylpiperazinium at the rat neuromuscular junction

We have studied the effects of the nicotinic acetylcholine (ACh) receptor agonist dimethylphenylpiperazinium (DMPP) on the evoked release of ACh from motor terminals in the rat isolated hemidiaphragm using an electrophysiological approach. DMPP (1–4 μM) had no effect on the rate of spontaneous quantal ACh release but increased the number of quanta of ACh released per impulse during 50 Hz stimulation. The DMPP‐induced increase in evoked ACh release was dependent on the frequency of stimulation, being absent when it was reduced to 0.5 Hz, but was not Ca2+ dependent, being unaffected at 50 Hz by a 4‐fold decrease in the extracellular Ca2+ concentration. The facilitation of evoked ACh release at 50 Hz by 2 μM DMPP was abolished by 10 μM of the calmodulin antagonist W7 (N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide hydrochloride) and, in the presence of W7, 2 μM DMPP depressed evoked ACh release at 0.5 Hz. The ability of the nicotinic ACh receptor antagonist vecuronium (1 μM) to depress evoked ACh release at 50 Hz was also abolished by 10 μM W7. The present findings demonstrate, using an electrophysiological technique, that DMPP can produce changes in the evoked ACh release from rat motor nerve terminals that are consistent with the existence of facilitatory nicotinic ACh receptors on the motor nerve endings. Further, they indicate a role for calmodulin‐dependent systems in this facilitatory effect of the compound.

An early stage in sodium channel clustering at developing rat neuromuscular junctions.

Voltage-gated sodium channels (VGSCs) are concentrated in the postsynaptic membrane at the adult rat neuromuscular junction (NMJ). We have used immunolabelling to determine the pattern of initial VGSC accumulation during development. At birth, but not 3 days before, VGSC labelling is detectable at the NMJ and in the perijunctional (periJ) membrane but not elsewhere. A much higher density cluster of VGSCs forms at the NMJ itself 1-2 weeks later. If the nerve is cut 2 days after birth, VGSC labelling persists in the periJ region for at least 4 weeks but the clustering of VGSCs at the NMJ fails to develop. Thus an early, stable accumulation of VGSCs develops near the NMJ at least a week before high density postsynaptic VGSC clusters form.

Differential susceptibility of young and old rat neuromuscular junctions to antibody-mediated AChR degradation in experimental autoimmune myasthenia gravis.

Differential susceptibility of young and old rat neuromuscular junctions to antibody-mediated AChR degradation in experimental autoimmune myasthenia gravis.

Stimulated single-fiber electromyography in the rat.

We constructed an animal model of stimulated single-fiber electromyography (SFEMG) by testing Wistar rats under anesthesia. Stimuli of 1 Hz were applied to the sciatic nerve through an insulated monopolar needle electrode. Single-fiber action potentials were acquired from the gastrocnemius muscle. Jitter was assessed by the mean consecutive difference (MCD). Eighty-seven fibers were obtained from 12 rats. Their MCDs ranged from 2 to 72 micros (17.7+/-13.4). Seven of these values were less than or equal to 5 micros, and three exceeded 50 micros. Neuromuscular blocking agents injected into some of the rats induced considerable increases in jitter and blocking. A rat with one fiber with an MCD less than 5 micros also received an injection of curare. The jitter showed the same pattern of increment, evidence that the small jitter was not attributable to direct muscle stimulation. These results show that SFEMG can be used on rats. In addition, jitter reflects the changes in motor end-plate function. The findings also suggest the presence of an extremely high safety factor in rat neuromuscular junctions.

Beta-Spectrin is colocalized with both voltage-gated sodium channels and ankyrinG at the adult rat neuromuscular junction.

Voltage-gated sodium channels (VGSCs) are concentrated in the depths of the postsynaptic folds at mammalian neuromuscular junctions (NMJs) where they facilitate action potential generation during neuromuscular transmission. At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, beta-spectrin and ankyrin, which may help to maintain the high local density of VGSCs. Here we show in skeletal muscle, using immunofluorescence, that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG, the nodal isoform of ankyrin. In en face views of rat NMJs, acetylcholine receptors (AChRs), and utrophin immunolabeling are organized in distinctive linear arrays corresponding to the crests of the postsynaptic folds. In contrast, beta-spectrin, VGSCs, and ankyrinG have a punctate distribution that extends laterally beyond the AChRs, consistent with a localization in the depths of the folds. Double antibody labeling shows that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG at the NMJ. Furthermore, quantification of immunofluorescence in labeled transverse sections reveals that beta-spectrin is also concentrated in perijunctional regions, in parallel with an increase in labeling of VGSCs and ankyrinG, but not of dystrophin. These observations suggest that interactions with beta-spectrin and ankyrinG help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density.

Nitric oxide synthase in rat neuromuscular junctions and in nerve terminals of Torpedo electric organ: its role as regulator of acetylcholine release.

The distribution of nitric oxide synthase on peripheral motor system was studied using a specific antibody against the neuronal isoform of nitric oxide synthase (nNOS). The immunoreactivity for nNOS was detected on the sarcolemmal surface of muscle cells, in intramuscular axons and in neuromuscular synapses. At the neuromuscular junctions, ultrastructural immunolabeling demonstrated that nNOS immunoreactivity was localized mainly into the presynaptic nerve terminals as well as adjacent postsynaptic muscle membrane. Similar immunostaining pattern was present in frog muscles and Torpedo electric organs. After chronic muscle denervation, nNOS immunoreactity at endplate level decreased during the first week but it was upregulated after 30 days of denervation. In denervated endplates, nNOS immunoreactivity was localized in the terminal Schwann cells covering the degenerated neuromuscular junctions whereas nNOS was not detected in Schwann cells under normal conditions. In Torpedo synaptosomes, acetylcholine (ACh) release elicited by potassium depolarization was inhibited by NO donors such as sodium nitroprusside. In contrast, application of inhibitors of NOS activity, aminoguanidine (AMG) and N(omega)-Nitro-L-arginine methyl esther (L-NAME) increased acetylcholine release. These results indicate that nNOS is present at the motor nerve terminals in a variety of vertebrates and that it may be involved in the physiological modulation of ACh release and in the regulation of muscle response to nerve injury.

Spontaneous acetylcholine release in mammalian neuromuscular junctions.

Spontaneous secretion of the neurotransmitter acetylcholine in mammalian neuromuscular synapsis depends on the Ca2+ content of nerve terminals. The Ca2+ electrochemical gradient favors the entry of this cation. We investigated the possible involvement of three voltage-dependent Ca2+ channels (VDCC) (L-, N-, and P/Q-types) on spontaneous transmitter, release at the rat neuromuscular junction. Miniature end-plate potential (MEPP) frequency was clearly reduced by 5 microM nifedipine, a blocker of the L-type VDCC, and to a lesser extent by the N-type VDCC blocker, omega-conotoxin GVIA (omega-CgTx, 5 microM). On the other hand, nifedipine and omega-CgTx had no effect on K(+)-induced transmitter secretion. omega-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, prevents acetylcholine release induced by K+ depolarization but failed to affect MEPP frequency in basal conditions. These results suggest that in the mammalian neuromuscular junction Ca2+ enters nerve terminals through at least three different channels, two of them (L- and N-types) mainly related to spontaneous acetylcholine release and the other (P/Q-type) mostly involved in depolarization-induced neurotransmitter release. Ca(2+)-binding molecule-related spontaneous release apparently binds Ca2+ very rapidly and would probably be located very close to Ca2+ channels, since the fast Ca2+ chelator (BAPTA-AM) significantly reduced MEPP frequency, whereas EGTA-AM, exhibiting slower kinetics, had a lower effect. The increase in MEPP frequency induced by exposing the preparation to hypertonic solutions was affected by neither external Ca2+ concentration nor L-, N-, and P/Q-type VDCC blockers, indicating that extracellular Ca2+ is not necessary to produce hyperosmotic neurosecretion. On the other hand, MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the same extent, supporting the view that hypertonic response is promoted by "bulk" intracellular Ca2+ concentration increases.

Immunolocalization of nitric oxide synthases at the postsynaptic domain of human and rat neuromuscular junctions--light and electron microscopic studies.

Neuronal (n) and inducible (i) nitric oxide synthase (NOS) were immunolocalized at the postsynaptic domain of human and rat neuromuscular junctions (NMJs) by light and electron microscopy. We applied polyclonal and monoclonal antibodies for colocalization with three other synaptic proteins, utilizing double and triple fluorescence labeling, and gold and peroxidase for immunoelectron microscopy. By light microscopy, nNOS and iNOS colocalized with desmin and dystrophin, known postsynaptic components, but not with neurofilament protein, a presynaptic component. By electronmicroscopy, nNOS, but not iNOS, colocalized postsynaptically on the same structures as desmin; iNOS was also postsynaptic, but did not colocalize with desmin immunoreactivity. At the NMJs of Duchenne muscular dystrophy patients, both nNOS and iNOS were strongly immunoreactive. At the NMJs of a patient with myasthenia gravis, nNOS was weaker than in controls. Total denervation of rat sciatic nerve did not cause any decrease of nNOS or iNOS immunoreactivity 7 days thereafter. At 15 days after denervation, there was a gradual decrease of immunoreactivity, and immunoreactivity disappeared 30 days after denervation, corresponding to the ultrastructurally detectable disorganization of the postsynaptic region. This seems to be the first combined light and electron microscopic description of the postsynaptic localization of nNOS and iNOS at human and rat NMJs.

Nerve Terminal Withdrawal from Rat Neuromuscular Junctions Induced by Neuregulin and Schwann Cells

Schwann cells (SCs) that cap neuromuscular junctions (nmjs) play roles in guiding nerve terminal growth in paralyzed and partially denervated muscles; however, the role of these cells in the day-to-day maintenance of this synapse is obscure. Neuregulins, alternatively spliced ligands for several erbB receptor tyrosine kinases, are thought to play important roles in cell-cell communication at the nmj, affecting synapse-specific gene expression in muscle fibers and the survival of terminal SCs during development. Here we show that application of a soluble neuregulin isoform, glial growth factor II (GGF2), to developing rat muscles alters terminal SCs, nerve terminals, and muscle fibers. SCs extend processes and migrate from the synapse. Nerve terminals retract from acetylcholine receptor-rich synaptic sites, and their axons grow, in association with SCs, to the ends of the muscle. These axons make effective synapses only after withdrawal of GGF2. These synaptic alterations appear to be induced by the actions of neuregulin on SCs, because SC transplants growing into contact with synaptic sites also caused withdrawal of nerve terminal branches. These results show that SCs can alter synaptic structure at the nmj and implicate these cells in the maintenance of this synapse.

Adenosine depresses transmitter release but is not the basis for `tetanic fade' at the neuromuscular junction of the rat

It has been suggested that during repetitive neural stimulation adenosine accumulates at the neuromuscular junction and the resulting negative feedback action of adenosine is the major basis for tetanic fade (decline in action of adenosine during repetitive stimulation) This hypothesis was examined at the rat neuromuscular junction by examining the effects of blocking adenosine A 1-receptors. Intracellular recording techniques were used to monitor end-plate potentials and miniature end-plate potentials. The data suggest that while adenosine serves a role in depressing transmitter release, adenosine accumulation during brief periods of stimulation is minimal and adenosine is not the cause for tetanic fade.

The contribution of postsynaptic folds to the safety factor for neuromuscular transmission in rat fast‐ and slow‐twitch muscles.

1. At the rat neuromuscular junction, the postsynaptic folds and the voltage-gated sodium channels (VGSCs) within them are thought to amplify the effects of postsynaptic currents. In this study, the contribution of this effect to the safety factor for neuromuscular transmission, the ratio of the normal quantal content to the number of quanta required to reach threshold, has been estimated. 2. Normal quantal content was determined in isolated nerve-muscle preparations of rat soleus and extensor digitorum longus (EDL) muscles in which muscle action potentials were blocked by mu-conotoxin. The quantal content estimated from voltage recordings was 61.8 and 79.4 in soleus and EDL, respectively, and from charge measurements derived from current recordings was 46.3 (soleus) and 65.1 (EDL). 3. The threshold for action potential generation in response to nerve stimulation was determined from endplate potentials (EPPs) and endplate currents (EPCs) in preparations partially blocked with d-tubocurarine. The number of quanta required to reach threshold was estimated from voltage recordings to be 19.7 (soleus) and 23.2 (EDL) and from charge measurements derived from current recordings to be 13.3 (soleus) and 13.0 (EDL). 4. When intracellular electrodes were used to inject current into the muscle fibre, the total charge required to reach threshold was approximately twice that of the nerve-evoked threshold EPC. 5. The safety factor for nerve-evoked responses at the junction was 3.5 (soleus) and 5.0 (EDL). In the extrajunctional region the safety factor estimated from injected currents was 1.7 (soleus) and 2.5 (EDL). 6. It is concluded that the effect of the postsynaptic folds and the VGSCs within them is to double the safety factor. At normal frequencies of nerve impulse activity in vivo, this effect is likely to be crucial for ensuring effective neuromuscular transmission.

In vitro neuromuscular effects of valproic acid

The contribution of the anticonvulsant agent, valproic acid, to the interaction between neuromuscular blockers and anticonvulsants remains unclear. Therefore, this study was undertaken to examine the acute neuromuscular effects of valproic acid and its potential for interaction with neuromuscular blocking drugs. Phrenic nerve-hemidiaphragm preparations from rats were mounted in modified Krebs buffer, maintained at 37 degrees C and aerated with a 5% carbon dioxide-95% oxygen gas mixture. Phrenic nerves were stimulated with 0.1 Hz supramaximal pulses of 0.2-ms duration and the elicited tension of the hemidiaphragm was recorded in seven preparations in the presence of valproic acid 100, 500 and 1000 mumol litre-1. In another six preparations, neuromuscular transmission was blocked completely with the non-depolarizing blocker tubocurarine 3 mumol litre-1 and the hemidiaphragm muscle was stimulated directly (2 Hz and 2 ms duration). The effect of the anticholinesterase drug, neostigmine, on an established block induced by valproic acid 1000 mumol litre-1 was also evaluated in five phrenic nerve stimulated preparations. The effect of valproic acid 100, 500 or 1000 mumol litre-1 on suxamethonium- or atracurium-induced neuromuscular block was also evaluated. Valproic acid produced a mean of 29.7 (SEM 1.7)% and 24.7 (1.7) % block of indirectly or directly elicited muscle twitches. The concentration of valproic acid that caused half maximal paralysis did not differ between indirect (460 (59) mumol litre-1) and direct (329 (35) mumol litre-1) stimulation. Neostigmine 1-3 mumol litre-1 failed to significantly alter the block of 19.8% induced by valproic acid 1000 mumol litre-1. Valproic acid 100, 500 or 1000 mumol litre-1 did not alter the concentrations of suxamethonium or atracurium needed to produce paralysis. These findings suggest that valproic acid does not produce significant block at the rat neuromuscular junction. The partial block that is produced is caused predominantly by a direct inhibitory effect on the muscle itself.

Purinoceptors and synaptic plasticity

Adenosine is considered an important modulator of the nervous system acting at pre-, post-, and nonsynaptic levels. In models (e.g. the rat neuromuscular junction) where adenosine has only presynaptic (inhibitory, which are A 1 -receptor mediated; and excitatory, which are A 2A -receptor mediated) effects, the action of adenosine on transmitter release depends on the intensity of stimulation. It is inhibitory with low frequencies of stimulation and excitatory with high frequencies of stimulation. This finding suggests that the presynaptic adenosine A 1 /A 2A receptor activation balance depends on the pattern of stimulation. At the rat hippocampal slices, endogenous adenosine inhibits the neurophysiological basis of learning and memory: long-term potentiation (LTP), long-term depression (LTD), and depotentiation. Adenosine A 2A agonists facilitate LTP. Because the adenosine analogue 2-chloroadenosine in low concentrations (100 nM) inhibits N-methyl-D-aspartate (NMDA) currents in pyramidal cells, it is likely that the inhibitory effects of adenosine on LTP and LTD/depotentiation are a consequence of its ability to inhibit NMDA currents. The modulatory actions of adenosine on synaptic plasticity support the role of this nucleoside in cognition, and therefore, A 1 antagonists and A 2A agonists could be considered as potential cognitive enhancers. The adaptation of adenosine to activate presynaptic A 1 or A 2A receptors at low or high frequencies of stimulation anticipates the potential interest of A 1 antagonists and A 2A agonists as enhancers of neuromuscular transmission.

Nitric oxide synthase is concentrated at the skeletal muscle endplate

NO performs a wide array of cell signaling functions. Neuronal NO synthase (nNOS) immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase (NDP) activity, a marker of nNOS, were concentrated at adult rat neuromuscular junctions and persisted in denervated muscle indicating the localization of the enzyme to the postsynaptic surface. The concentration of nNOS at the muscle endplate suggests NO could serve as a messenger pre- and postsynapticly.

Nitric oxide synthase is concentrated at the skeletal muscle endplate

NO performs a wide array of cell signaling functions. Neuronal NO synthase (nNOS) immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase (NDP) activity, a marker of nNOS, were concentrated at adult rat neuromuscular junctions and persisted in denervated muscle indicating the localization of the enzyme to the postsynaptic surface. The concentration of nNOS at the muscle endplate suggests NO could serve as a messenger pre- and postsynapticly.