Synaptic competition and the persistence of polyneuronal innervation at frog neuromuscular junctions

Mechanisms governing the elimination of polyneuronal innervation were examined by correlating the morphology and physiology of competing nerve terminals at identified dually innervated neuromuscular junctions in sartorius muscles of adult frogs (Rana pipiens). Synaptic efficacy (endplate potential amplitude per unit nerve terminal length) was presumed to reflect the ability of a terminal to compete for synaptic space. The synaptic efficacies of two terminals at the same synaptic site were found to be surprisingly equal, with a median difference of 33%. Much more variation would be expected if dually innervated junctions were randomly innervated by pairs of terminals having the same range of synaptic efficacy as that found at singly innervated junctions in the same muscle. This finding supports the hypothesis that the weaker input is eliminated from dually innervated junctions when there is a large discrepancy in competitive efficacy, and that both inputs may persist if competitive efficacies are relatively equal. We also tested but failed to find support for the hypothesis that spatial proximity between competing terminals intensifies competition for synaptic space during synapse elimination.     

Agrin and acetylcholine receptors are removed from abandoned synaptic sites at reinnervated frog neuromuscular junctions

Changes in the distribution of agrin and acetylcholine receptors (AChRs) were examined during reinnervation and following permanent denervation as a means of understanding mechanisms controlling the distribution of these molecules. Following nerve damage in the peripheral nervous system, regenerating nerve terminals preferentially return to previous synaptic sites leading to the restoration of synaptic activity. However, not all portions of original synaptic sites are reoccupied: Some of the synaptic sites are abandoned by both the nerve terminal and the Schwann cell. Abandoned synaptic sites contain agrin, AChRs, and acetylcholinesterase (AChE) without an overlying nerve terminal or Schwann cell providing a unique location to observe changes in the distribution of these synapse-specific molecules. The distribution of anti-agrin and AChR staining at abandoned synaptic sites was altered during the process of reinnervation, changing from a dense, wide distribution to a punctate, pale pattern, and finally becoming entirely absent. Agrin and AChRs were removed from abandoned synaptic sites in reinnervated frog neuromuscular junctions, while in contralateral muscles which were permanently denervated, anti-agrin and AChR staining remained at abandoned synaptic sites. Decreasing synaptic activity during reinnervation delayed the removal of agrin and AChRs from abandoned synaptic sites. Altogether, these results support the hypothesis that synaptic activity controls a cellular mechanism that directs the removal of agrin from synaptic basal lamina and the loss of agrin leads to the dispersal of AChRs. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 999–1018, 1997     

Synaptic competition and the elimination of polyneuronal innervation follwoing reinnervation of adult frog sartorius muscles

The elimination of polyneuronal innervation (synapse elimination) that occurs following reinnervation was studied in sartorius muscles of adult Rana pipiens. The percentage of neuromuscular junctions that were polyneuronally innervated declined from 47% at 40–80 days after nerve crush to 22% at greater than 250 days after nerve crush. We measured the size, synaptic strength, and position of competing nerve terminals at identified dually innervated neuromuscular junctions at these two different periods of synapse elimination. Our goal was to determine if any of these parameters play a role in the competition between nerve terminals that ultimately results in the elimination of polyneuronal innervation. Our data support the hypothesis that polyneuronal innervation will persist if competing nerve terminals are of similar synaptic efficacies but will be eliminated if the competing terminals are of different synaptic efficacies. We also tested, but failed to find any evidence, that the spatial proximity of competing nerve terminals at the same synaptic site influences the elimination of polyneuronal innervation.     

Synaptic competition and the elimination of polyneuronal innervation following reinnervation of adult frog sartorius muscles

The elimination of polyneuronal innervation (synapse elimination) that occurs following reinnervation was studied in sartorius muscles of adult Rana pipiens. The percentage of neuromuscular junctions that were polyneuronally innervated declined from 47% at 40-80 days after nerve crush to 22% at greater than 250 days after nerve crush. We measured the size, synaptic strength, and position of competing nerve terminals at identified dually innervated neuromuscular junctions at these two different periods of synapse elimination. Our goal was to determine if any of these parameters play a role in the competition between nerve terminals that ultimately results in the elimination of polyneuronal innervation. Our data support the hypothesis that polyneuronal innervation will persist if competing nerve terminals are of similar synaptic efficacies but will be eliminated if the competing terminals are of different synaptic efficacies. We also tested, but failed to find any evidence, that the spatial proximity of competing nerve terminals at the same synaptic site influences the elimination of polyneuronal innervation.     

Postmetamorphic development of neuromuscular junctions and muscle fibers in the frog cutaneous pectoris

Synaptic size, synaptic remodelling, polyneuronal innervation, and synaptic efficacy of neuromuscular junctions were studied as a function of growth in cutaneous pectoris muscles of postmetamorphic Rana pipiens. Recently metamorphosed frogs grew rapidly, and this growth was accompanied by hypertrophy of muscle fibers, myogenesis, and increases in the size and complexity of neuromuscular junctions. There were pronounced gradients in pre- and postsynaptic size across the width of the muscle, with neuromuscular junctions and muscle fibers near the medial edge being smaller than in more lateral regions. The incidence of polyneuronal innervation, measured physiologically and histologically, was also higher near the medial edge. Growth-associated declines in all measures of polyneuronal innervation indicated that synapse elimination occurs throughout life. Electrophysiology also demonstrated regional differences in synaptic efficacy and showed that doubly innervated junctions have lower synaptic efficacy than singly innervated junctions. Repeated, in vivo observations revealed extensive growth and remodelling of motor nerve terminals and confirmed that synapse elimination is a slow process. It was concluded that some processes normally associated with embryonic development persist long into adulthood in frog muscles.     

Innervation and membrane specialization at neuromuscular junctions in submaxillaris muscle of the frog

The submaxillaris muscle of the frog after zinc iodide-osmium staining reveals the presence of polyneural innervation. Cholinesterase staining shows that the longer terminals have postsynaptic folds whereas the smaller terminals (up to 5 micron) lack them. Thin-section electron microscopy shows that muscle fibers with or without an M line have terminals with and without postsynaptic folds. The terminals with postsynaptic folds have presynaptic membrane outpocketings above folds. These outpocketings are rudimentary or absent in the terminals without postsynaptic folds. In longer junctions, the P face of the presynaptic membrane has double rows of paired particles on active zone ridges perpendicular to the axis of the muscle. In smaller junctions active zone ridges are rudimentary or absent and double rows of particles form various patterns. Postsynaptic active zones in longer junctions consist of clusters of particles leaving gaps in between, whereas in the smaller junctions they lack gaps. The polyneural innervation and different deployment of membrane particles at neuromuscluar junctions could be a factor responsible for different physiological properties of this muscle.     

In vivo observations of terminal Schwann cells at normal, denervated, and reinnervated mouse neuromuscular junctions

We found a low-molecular-mass, fluorescent dye, Calcein blue am ester (CB), that labels terminal Schwann cells at neuromuscular junctions in vivo without damaging them. This dye was used to follow terminal Schwann cells at neuromuscular junctions in the mouse sternomastoid muscle over periods of days to months. Terminal Schwann cell bodies and processes were stable in their spatial distribution over these intervals, with processes that in most junctions were precisely aligned with motor nerve terminal branches. Three days after nerve cut, the extensive processes elaborated by terminal Schwann cells in denervated muscle were labeled by CB. The number and length of CB-labeled terminal Schwann cell processes decreased between 3 days and 1 month after denervation, suggesting that terminal Schwann cell processes are only transiently maintained in the absence of innervation. During reinnervation after nerve crush, however, terminal Schwann cell processes were extended in advance of axon sprouts, and these processes persisted until reinnervation was completed. By viewing the same junctions twice during reinnervation, we directly observed that axon sprouts used existing Schwann cell processes and chains of cell bodies as substrates for outgrowth. Thus, CB can be used to monitor the dynamic behavior of terminal Schwann cells, whose interactions with motor axons and their terminals are important for junction homeostasis and repair.     

Anti-agrin staining is absent at abandoned synaptic sites of frog neuromuscular junctions

The neuromuscular junction is a plastic structure and is constantly undergoing changes as the nerve terminals that innervate the muscle fiber extend and retract their processes. In vivo observations on developing mouse neuromuscular junctions revealed that prior to the retraction of a nerve terminal the acetylcholine receptors (AChRs) under that nerve terminal disperse. Agrin is a protein released by nerve terminals that binds to synaptic basal lamina and directs the aggregation of AChRs and acetylcholinesterase (AChE) in and on the surface of the myotube. Thus, if the AChRs under a nerve terminal disperse, then the cellular signaling mechanism by which agrin maintains the aggregation of those AChRs must have been disrupted. Two possibilities that could lead to the disruption of the agrin induced aggregation are that agrin is present at the synaptic basal lamina but is unable to direct the aggregation of AChRs, or that agrin has been removed from the synaptic basal lamina. Thus, if agrin were blocked, one would expect to see anti-agrin staining at abandoned synaptic sites; whereas if agrin were removed, anti-agrin staining would be absent at abandoned synaptic sites. We find that anti-agrin staining and α-bungarotoxin staining are absent at abandoned synaptic sites. Further, in vivo observations of retracting nerve terminals confirm that agrin is removed from the synaptic basal lamina within 7 days. Thus, while agrin will remain bound to synaptic basal lamina for months following denervation, it is removed within days following synaptic retraction. © 1996 John Wiley & Sons, Inc.     

Mechanisms of elimination, remodeling, and competition at frog neuromuscular junctions

Mechanisms governing synapse elimination, synaptic remodeling, and polyneuronal innervation were examined in anatomical and electrophysiological studies of frog neuromuscular junctions. There was a substantial level of polyneuronal innervation in adult junctions and this varied seasonally. Nerve terminal retraction and synapse elimination occurred during normal growth and following reinnervation. Synapse elimination was not inevitable, however. Repeated in vivo observations of some identified junctions showed that polyneuronal innervation could persist for over a year, while at other junctions it arose de novo by terminal sprouting. We concluded that polyneuronal innervation in adult muscles was governed by an equilibrium between processes of retraction and elimination on one hand, and sprouting and synaptogenesis on the other. Other observations revealed that structural remodeling was a common feature of adult junctions. Most often, remodeling involved the simultaneous growth and retraction of different parts of the same junction. The net result was usually junctional growth that, in small frogs, appeared to provide a good match between synaptic size and the electrical demands of transmission. In larger animals, pre- and postsynaptic sizes were not as well matched, providing morphological evidence for a growth-associated decline in synaptic efficacy. Finally, electrophysiology was used to describe some of the functional correlates and consequences of competitive interactions between the terminals of different axons. These results are explained by a hypothetical mechanism that involves trophic support provided by the muscle to the motoneuron, the overall level of nerve-muscle activity, and the synchrony of pre- and postsynaptic activity.     

Numerical analysis of the voltage-clamp technique applied to frog neuromuscular junctions.

The nonlinear cable equation was solved numerically by means of an implicit procedure. The correlation between end-plate length and fiber diameter was determined in frog (Rana pipiens) sartorius muscles stained with gold chloride (Löwit, 1875). The diameter of the fibers stained by the Löwit method was 80 (74-85) micron (median and its 95% confidence interval for 52 fibers), the length of the end plates in the same fibers was 382 (353-417) micron. The fibers simulated were 80 micron in diameter. To solve the equation the muscle fibers were represented by 500 segments 20 micron long, and the equation was solved in steps of 10 microseconds; a double exponential function was incorporated to the first seven segments to represent the neuromuscular junction. The potential of the first segment of the cable was set to the clamping level and the membrane potential of the remaining segments calculated. The current needed to hold the first segment was estimated by adding the current flowing through the first segment to the current flowing from it to the second segment. Our results indicate that the lack of space clamp in the point voltage-clamp studies of the frog neuromuscular junction introduces serious errors in the estimates of the end-plate conductance value, the kinetics of the conductance changes, and the reversal potential of the end-plate currents. The possibility of an efficient voltage-clamp technique is also explored. Our calculations suggest that the study of end-plate current and conductance is possible with little error if the end-plate potential is controlled at both ends of the synaptic area simultaneously.