Postnatal development of rat motor nerve terminals

Summary The nerve terminals of neuromuscular junctions in the rat diaphragm, extensor digitorum longus muscle and soleus muscle have been studied in animals between 3 weeks and 2.5 years of age using methylene blue stain and light microscopy. Dimensions, structure and organization of the nerve terminals were shown to change during life at various rates in different muscles and postnatal periods. The area and length of the terminals increase in all three muscles until young adult age. Later these dimensions continue to increase in the extensor digitorum longus and soleus muscles. In the diaphragm only the length increases, and this occurs late in adult life. The area also increases in relation to the diameter of the corresponding muscle fiber. Adult soleus terminals are more elongated than terminals in the diaphragm and extensor digitorum longus muscle. During adult life the extension of nerve terminals in relation to muscle fiber length increases in the extensor digitorum longus and soleus muscles, but is almost unchanged in the diaphragm. The nerve terminal branches are mainly coarse and irregular in young animals, but possess varying numbers of varicosities in adult animals. The number of varicosities is high in the extensor digitorum longus muscle and low in the diaphragm. In old animals the number of varicosities tends to be reduced. With increasing age the nerve terminal branches become organized in distinct groups with increasing distance between the groups. This is prominent in the soleus.     

Three potassium currents in mouse motor nerve terminals

A study of the K conductance of the presynaptic membrane has been performed in thetriangularis sterni muscle of the mouse. External currents generated in the presynaptic terminals upon invasion by action potentials have been recorded using microelectrodes inserted into the perineurium of preterminal nerve bundles. The voltage-dependent K current could be pharmacologically dissected into fast (IK_f) and slow (IK_s) components. While both are sensitive to 3,4-diaminopyridine (3,4-DAP), only IK_f is sensitive to tetraethylammonium (TEA). Uranyl (100–200 M) and guanidine (5–10 mM) produced a near complete block of IK_f and IK_s, which can explain their facilitatory effect upon transmitter release. The third K current of presynaptic terminals is Ca²⁺-dependent, but was activated also by Sr²⁺. This current could be suppressed by nanomolar doses of charybdotoxin; it is also sensitive to TEA but not to 3,4-DAP, uranyl or guanidine.     

Two different presynaptic calcium currents in mouse motor nerve terminals

Extracellular recordings of potential changes under the perineural sheath of nerve bundles close to some of the nerve terminals were performed using the M. triangularis sterni of the mouse. The nerve signals consisted of a predominant double-peaked negativity which was often preceded by a small positive deflection. While the first negative peak is related to the propagating nerve action potential, the second negative deflection can be attributed to a potassium conductance since it was selectively blocked by tetraethylammonium (TEA) or 3,4-diaminopyridine (3,4-DAP). Combined application of TEA and 3,4-DAP gave rise to a prolonged positive-going wave which was blocked by Cd2+, thus, indicating its underlying cause to be a Ca current. Ionophoretic application of TEA and Cd2+ to the endplates affected potassium and calcium components of the subendothelial signals, respectively, thus indicating their presynaptic origin. This finding is supported by the decrease of the amplitude of these components with increasing distance from the endplate region. Maximal effects on K conductance attainable with 3,4-DAP could still be potentiated by TEA, indicating the presence of at least two distinct sets of K channels. The prolonged positive potential induced by TEA and 3,4-DAP consisted of a fast and slow component, both of which can be attributed to Ca conductances with different characteristics. The fast positive signal component is attributed to the voltage-dependent Ca channel, responsible for the initiation of transmitter release. Its amplitude and duration depend on extracellular Ca2+ -concentration. The fast component is still present when Ca2+ is substituted by Sr2+ or Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of lead on transmitter release at mouse motor nerve terminals

The actions of lead (Pb²⁺) on transmitter release were studied at neuromuscular junctions in mouse diaphragm in vitro. The quantal content of end-plate potentials (EPPs) was reduced by Pb²⁺ in a dose-related manner consistent with inhibition of Ca²⁺ entry into nerve terminals, with a half-maximal effect at 1.4 M (in 0.5 mM Ca²⁺ and 2 mM Mg²⁺). Pb²⁺ also inhibited the increased frequency of MEPPs (f _MEPP where MEPPs denotes miniature EPPs) produced by Ba²⁺ in the presence of raised K⁺, blocking the calculated Ba²⁺ entry half-maximally at 170 M. However, at concentrations of 50–200 nM, Pb²⁺ often increased f _MEPP in 20 mM K⁺ in the presence of Ca²⁺ and acted to promote the irreversible effect of lanthanum (La³⁺) to raise f _MEPP. In nominally Ca²⁺-free solution with 20 mM K⁺, brief (1 min) application of Pb²⁺ (20–320 M) caused rapid dose-dependent reversible rises in f _MEPP. With prolonged exposure to Pb²⁺,f _MEPP rose and then slowly declined; after removal of Pb²⁺, once f _MEPP had fallen to low levels, f _MEPP responded nearly normally to Ca²⁺ or ethanol, but not to Pb²⁺ itself. In 5 mM K⁺, 0 mM Ca²⁺ and varied [Pb²⁺] (where [ ] denotes concentration), nerve stimulation caused no EPPs, but prolonged tetanic stimulation produced increases in f _MEPP graded with [Pb²⁺] that persisted as a tail; results were consistent with growth f _MEPP with the 4th power of intracellular Pb²⁺ and removal of intracellular Pb²⁺ with a time constant of about 30 s. These results suggest that Pb²⁺ acts to block the entry of Ca²⁺ and Ba²⁺ into the terminal via voltage-gated Ca²⁺ channels through which Pb²⁺, at higher concentrations, also penetrates and then acts as an agonist at intracellular sites that govern transmitter release.     

Transduction mechanism involving the presynaptic adenosine receptor at mouse motor nerve terminals

The inhibitory effect of 2-chloroadenosine on spontaneous quantal release of transmitter at the mouse neuromuscular junction was abolished after pretreating tissues either with pertussis toxin (PTX), or with H7, a protein kinase inhibitor. H7 alone caused a fall in miniature endplate potential (MEPP) frequency, but PTX did not. The results are consistent with the hypothesis that rates of neurotransmitter release are directly related to intraterminal cyclic AMP levels, and that these can be reduced by A1 adenosine receptor agonists through the mediation of a Gi protein.     

Electrotonic properties of motor nerve terminals

To obtain information about the electric membrane properties of frog motor nerve terminals we examined how depolarizing or hyperpolarizing current pulses of 2–8 ms duration to the preterminal, by electrotonic spread of potential, affected depolarization induced transmitter release. Sodium channels were blocked by tetrodotoxin. Under this condition a hyperpolarizing current pulse produced inhibition of release, followed by poten-tiation of release. Inhibition lasted more than 100 ms with a time constant of %I50 ms. When, in addition, potassium channels were blocked by 3,4-diaminopyridine or tetra-ethylammonium a depolarizing current pulse potentiated transmitter release for a period up to 50 ms. The results imply that inward currents in the nerve terminal are carried mainly by sodium and calcium ions and outward currents by potassium ions while “leak” conductances are negligible. A low “leak” conductance and therefore a high specific membrane resistance facilitates the spread of electrotonic potentials and thereby explains the long-lasting effects on transmitter release of brief current pulses to the preterminal.     

Omega-conotoxin does not block the verapamil-sensitive calcium channels at mouse motor nerve terminals

Release of acetylcholine at the neuromuscular junctions of skeletal muscle is not sensitive to organic Ca2+ channel blockers. However, in mouse motor nerve endings, extracellular recording reveals that a verapamil-sensitive Ca2+ current can be induced after block of K+ channels. Recordings of extracellular action potentials from inside the perineural sheaths of nerves innervating mouse triangularis sterni muscles reveal that this verapamil-sensitive current is not blocked by omega-conotoxin, and hence, it does not involve channels similar to the L-channels of neuronal cell bodies.     

Anion permeability of motor nerve terminals

Motor nerve terminals in mouse and frog display behavior consistent with an appreciable permeability of the nerve terminal membrane to chloride. In mouse diaphragm, in the presence of 15 mM K⁺ and 2 mM or 8 mM Ca²⁺, replacement of Cl^– by NO ₃ ^– , Br^– or acetate causes a transient increase in the quantal release of acetylcholine, measured as the frequency of spontaneously occurring miniature end plate potentials (FMEPP); a rapid rise in FMEPP is followed by a slow decline, with a half-time of about 4 min, to an equilibration level close to the control level. After equilibration in a solution in which the Cl^– is replaced by another anion, return to Cl^–-containing solution causes a transient decrease in FMEPP with a subsequent slow recovery. The data are consistent with transient nerve terminal depolarization or hyperpolarization, reflecting a nerve terminal permeability to anions in the sequence Cl^–>Br^–>NO ₃ ^– >acetate. In 5 mM K⁺, changes in nerve terminal excitability, determined using focal stimulation, are also consistent with alteration of nerve terminal membrane potential as a consequence of anion substitution. The time course of relaxation of FMEPP after a change from Cl^– to an anion of lower permeability, or vice versa, is considerably slower than that expected if Cl^– permeability of nerve terminals is similar to that of skeletal muscle fibres, and if the nerve terminal behaves as a single compartment. In frog cutaneous pectoris, transient changes in FMEPP produced by substitution of anions in the bathing solution were similar to those produced in mouse diaphragm, but more rapid in time course.This work was supported by grants from the Muscular DystrophyAssociation of Canada and the Medical Research Council of Canada     

Postnatal loss of synaptic terminals in the normal mouse soleus muscle

Using conventional physiological techniques for measuring unitary contractions and end-plate potentials (epps), the number, size and segmental properties of motor units (MUs) in the soleus muscle of the mouse during postnatal development have been examined. The number of MUs remains constant after birth, and there is no evidence of segmental preferences in the innervation pattern, before, during or after the postnatal elimination of redundant terminals. In neonates, MU size estimates based on twitch contractions are 30% smaller than tetanic estimates. Intracellular recording of epps shows that this is caused by facilitation of epps on repetitive stimulation. The frequency of occurrence of epps in the muscle from a few, isolated motor axons shows that the average motoneuron contacts 36% of the fibres in the muscle neonatally. A substantial fraction of the contacts is subthreshold for twitch activation of their fibre. The MU size remains constant up to day 5. During the next 10 days, the MU size is reduced to the mature value of 5% of the fibres in the muscle. It is concluded that the neonatal loss of synaptic terminals in this muscle takes place without concomitant loss of entire motor neurons, and that it is independent of possible segmental preferences in the innervation of the muscle.     

Coupling of L-type calcium channels to neurotransmitter release at mouse motor nerve terminals

Previously, we have presented evidence for the presence of L-type voltage-dependent Ca2+ channels (VDCC) in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (BAPTA-AM)-incubated motor nerve terminals (MNTs) of the levator auris muscle of mature mice. The aim of the present work was to study the coupling of these L-type VDCC to neurotransmitter release by inhibiting protein phosphatases. We thus studied the effects of the protein phosphatase inhibitors okadaic acid (OA) and pervanadate on quantal content (QC) of transmitter release with the P/Q-type channels fully blocked. The QC was not significantly different under the three experimental conditions tested: incubation with dimethylsulphoxide (DMSO), ethylene-glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (EGTA-AM) and BAPTA-AM. After preincubation with OA (1 microM), but not with pervanadate, QC increased substantially in the BAPTA-AM-incubated (up to 400%) MNT, but not in those incubated with DMSO or EGTA-AM. The OA-induced increment of QC was attenuated greatly (approximately 95% reduction) by preincubation with either nitrendipine (10 microM) or calciseptine (300 nM). The effect of OA (1 microM) and pervanadate (0.1 mM) on spontaneous neurotransmitter release was also studied. After preincubation with OA, but not per-vanadate, miniature end-plate potential (MEPP) frequency increased only in the BAPTA-AM-incubated MNT (up to 700% increment). This response was attenuated (by approximately 80%) by nitrendipine (10 microM) or calciseptine (300 nM). In contrast, neither omega-agatoxin IVA (120 nM) nor omega-conotoxin GVIA (1 microM) affected this OA-induced increment significantly. We also evaluated the relationship between QC and extracellular [Ca2+] ([Ca2+]o) in BAPTA-AM-incubated MNT. Under conditions in which only P/Q-type VDCC were available to participate in neurotransmitter release, QC increased as [Ca2+]o was raised from 0.5 to 2 mM. However, when only L-type VDCC were available, QC increased when [Ca2+]o increased from 0.5 to 1 mM, but decreased significantly at 2 mM. The mean latency for P/Q-type VDCC-mediated EPP was 1.7-1.9 ms; for L-type VDCC-mediated EPP, 1.9-2.5 ms. The rise time of the L-type VDCC mediated EPP was significantly slower than that mediated by P/Q-type VDCC. Preincubation with H-7 (100 microM), a potent inhibitor of protein kinase C (PKC) and adenosine 3',5'cyclic monophosphate (cAMP)-dependent protein kinase (PKA), attenuated the OA-induced increment of both QC and MEPP frequency (50% and 70% decrement, respectively), suggesting the participation of at least these two protein kinases in the coupling of L-type VDCC. In summary, our results show coupling of L-type VDCC to neurotransmitter release when protein phosphatases are inhibited and intracellular [Ca2+] is buffered by the fast chelator BAPTA.     

Postnatal loss of synaptic terminals in the partially denervated mouse soleus muscle

The present work aims to distinguish between processes that lead to neonatal synapse elimination. We have partially denervated the mouse soleus muscle just after birth by cutting one (L5) of the two (L4 and L5) spinal nerves which supply its innervation. After 4-6 weeks' survival times, we determined the number of remaining motor units (MUs) and the number of innervated fibres in the muscle by conventional physiological and histological techniques. There was no significant overlap between the remaining MUs. Their average size was reduced from about 230 muscle fibres at birth to about 80 after 4-6 weeks, compared to only 30 in normal animals of the same age. We conclude that two processes are required to explain synapse elimination in the muscle: a non-competitive process, inherent to the immature motor neurons and leading to a substantial reduction in their field of innervation; a competitive process between axon terminals innervating the same muscle fibre.     

Intramuscular nerve sprouting induced by CNTF is associated with increases in CGRP content in mouse motor nerve terminals

It is known that motor nerve terminal sprouting induced by either nerve injury or muscle paralysis is associated with an increase in calcitonin gene-related peptide (CGRP) content in the soma of motoneurons and in motor endplates. In the present study, CGRP-like immunoreactivity (CGRP-LI) was determined in motor endplates of animals in which nerve terminal sprouting had been induced by exogenous application of ciliary neurotrophic factor (CNTF). After 18 days of CNTF treatment we observed a significant increase in CGRP-LI in motor endplates. The results indicate that CGRP is upregulated when motor nerve outgrowth is induced, even in the absence of muscle paralysis or nerve lesion.     

Neurotransmitter release in motor nerve terminals of a mouse model of mild spinal muscular atrophy

Spinal muscular atrophy is a genetic disease which severity depends on the amount of SMN protein, the product of the genes SMN1 and SMN2. Symptomatology goes from severe neuromuscular impairment leading to early death in infants to slow progressing motor deficits during adulthood. Much of the knowledge about the pathophysiology of SMA comes from studies using genetically engineered animal models of the disease. Here we investigated one of the milder models, the homozygous A2G SMA mice, in which the level of the protein is restored to almost normal levels by the addition of a mutated transgene to the severe SMN-deficient background. We examined neuromuscular function and found that calcium-dependent neurotransmitter release was significantly decreased. In addition, the amplitude of spontaneous endplate potentials was decreased, the morphology of NMJ altered, and slight changes in short-term synaptic plasticity were found. In spite of these defects, excitation contraction coupling was well preserved, possibly due to the safety factor of this synapse. These data further support that the quasi-normal restoration of SMN levels in severe cases preserves neuromuscular function, even when neurotransmitter release is significantly decreased at motor nerve terminals. Nevertheless, this deficit could represent a greater risk of motor impairment during aging or after injuries.     

The spatial pattern of exocytosis and post-exocytic mobility of synaptopHluorin in mouse motor nerve terminals

We monitored the spatial distribution of exo- and endocytosis at 37°C in mouse motor nerve terminals expressing synaptopHluorin (spH), confirming and extending earlier work at room temperature, which had revealed fluorescent 'hot spots' appearing in repeatable locations during tetanic stimulation. We also tested whether hot spots appeared during mild stimulation. Averaged responses from single shocks showed a clear fluorescence jump, but revealed no sign of hot spots; instead, fluorescence rose uniformly across the terminal. Only after 5–25 stimuli given at high frequency did hot spots appear, suggesting a novel initiation mechanism. Experiments showed that about half of the surface spH molecules were mobile, and that spH movement occurred out of hot spots, demonstrating their origin as exocytic sources, not endocytic sinks. Taken together, our results suggest that synaptic vesicles exocytose equally throughout the terminal with mild stimulation, but preferentially exocytose at specific, repeatable locations during tetanic stimulation.     

A regenerating release of acetylcholine from mouse motor nerve terminals treated with anticholinesterase agents

It was found by intracellular recording with glass microelectrodes that train stimulation (50-200 Hz) of the phrenic nerve of intact or cut mouse diaphragm induced an accumulative depolarization of the endplate and triggered after a few pulses an 'all-or-none' regenerative depolarization lasting for 300-900 ms when acetylcholinesterase was inhibited by neostigmine or diisopropylfluorophosphate. This depolarization was associated with a noise of the membrane potential and a failure of the end plate potential. Low Ca2+ prolonged whereas high Ca2+ shortened the duration of regenerative depolarization which needed no further stimulation once triggered. d-Tubocurarine abolished the depolarization while restoring the end plate potential. A regenerative release of acetylcholine due to an activation of presynaptic cholinoceptors is speculated.     

Cross-innervation of fast and slow-twitch muscles by motor axons of the sural nerve in the mouse

The extensor digitorum longus (EDL) or soleus muscles of adult mice were cross-innervated by the sural nerve (SN) and deprived of their original innervation. The number and sizes of motor units and the location of endplates in these muscles were studied 1.5 to 16 months later. In the EDL muscle, the SN cross-innervated the original endplates. Very few ectopic endplates were seen, even when the nerve was implanted well outside of the original endplate area. Only 3% of the fibres were polyneuronally innervated. In the soleus muscle, however, the SN formed large numbers of ectopic endplates whether the nerve was implanted in the original endplate zone or outside of it. In addition, 20% of the muscle fibres were polyneuronally innervated. The SN cross-innervated both EDL and soleus muscles completely. There was no preference for a particular group of the SN motoneurones since all the cross-innervated muscles were innervated by all SN motor axons and the motor unit sizes of the SN were similar in the cross-innervated EDL and soleus muscles. It is concluded that intrinsic properties of a muscle determine the ability to form ectopic synapses. The distribution of the motor unit sizes is determined by the particular pool of motoneurones which innervates the muscle.