Activity-dependent switch from synapse formation to synapse elimination during development of neuromuscular junctions

The embryonic development of neuromuscular junctions consists of two successive epochs, an early period marked by exuberant synapse formation and a later period marked by synapse elimination. In the frog muscles we have studied, myogenesis is protracted and overlaps the periods of synapse formation and elimination. Thus, the formative and regressive events of synaptic development do not occur in synchrony across different fibers in the muscle. We propose that local activity orchestrates a shift from synaptogenesis to synapse elimination at the level of single muscle fibers. We also present evidence that perisynaptic Schwann cells and the expression of ion channels in the sarcolemma play important roles in the development of neuromuscular junctions. Questions for future study are outlined.     

Activity and synapse elimination at the neuromuscular junction

The neuromuscular junction undergoes a loss of synaptic connections during early development. This loss converts the innervation of each muscle fiber from polyneuronal to single. During this change the number of motor neurons remains constant but the number of muscle fibers innervated by each motor neuron is reduced. Evidence indicates that a local competition among the inputs on each muscle fiber determines which inputs are eliminated. The role of synapse elimination in the development of neuromuscular circuits, other than ensuring a single innervation of each fiber, is unclear. Most evidence suggests that the elimination plays little or no role in correcting for errant connections. Rather, it seems that connections are initially highly specific, in terms of both which motor neurons connect to which muscles and which neurons connect to which particular fibers within these muscles. A number of attempts have been made to determine the importance of neuromuscular activity during early development for this rearrangement of synaptic connections. Experiments reducing neuromuscular activity by muscle tenotomy, deafferentation and spinal cord section, block of nerve impulse conduction with tetrodotoxin, and the use of postsynaptic and presynaptic blocking agents have all shown that normal activity is required for normal synapse elimination. Most experiments in which complete muscle paralysis has been achieved show that activity may be essential for the occurrence of synapse elimination. Furthermore, experiments in which neuromuscular activity has been augmented by external stimulation show that synapse elimination is accelerated. A plausible hypothesis to explain the activity dependence of neuromuscular synapse elimination is that a neuromuscular trophic agent is produced by the muscle fibers and that this production is controlled by muscle-fiber activity. The terminals on each fiber compete for the substance produced by that fiber. Inactive fibers produce large quantities of this substance; on the other hand, muscle activity suppresses the level of synthesis of this agent to the point where only a single synaptic terminal can be maintained. Inactive muscle fibers would be expected to be able to maintain more nerve terminals. The attractiveness of this scheme is that it provides a simple feedback mechanism to ensure that each fiber retains a single effective input.     

Building a bilayer model of the neuromuscular synapse

Progress over the past 10 years has made it possible to construct a simple model of neurotransmitter release. Currently, some models use artificially formed vesicles to represent synaptic vesicles and a planar lipid bilayer as a presynaptic membrane. Fusion of vesicles with the bilayer is via channel proteins in the vesicle membrane and an osmotic gradient. In this paper, a framework is presented for the successful construction of a more complete model of synaptic transmission. This model includes real synaptic vesicles that fuse with a planar bilayer. The bilayer contains acetylcholine receptor (AChR) channels which function as autoreceptors in the membrane. Vesicle fusion is initiated following a Ca²⁺ flux through voltage-gated Ca²⁺ channels. Key steps in the plan are validated by mathematical modeling. Specifically, the probability that a reconstituted AChR channel opens following the release of ACh from a fusing vesicle, is calculated as a function of time, quantal content, and number of reconstituted AChRs. Experimentally obtainable parameters for construction of a working synapse are given. The inevitable construction of a full working model will mean that the minimal structures necessary for synaptic transmission are identified. This will open the door in determining regulatory and modulatory factors of transmitter release.     

Critical period for the androgenic block of neuromuscular synapse elimination

Juvenile androgen treatment during developmental synapse elimination changes the pattern of innervation in the adult levator ani (LA), an androgen-sensitive muscle (Jordan, Letinsky, and Arnold, 1989b). Most notably, such adult muscles contain an unusually high number of muscle fibers that are innervated by two or more axons indicating that these fibers are multiply innervated. Juvenile androgen treatment also increases the adult level of preterminal branching, the number of junctional sites per adult fiber, and the size of adult LA muscle fibers and motoneurons in the spinal nucleus of the bulbocavernosus (SNB). The present study was designed to determine when in development androgen treatment is most effective in maintaining multiple innervation in adulthood and whether there are different critical periods for the different effects of juvenile androgen treatment. Male rats were castrated on 7, 21, or 34 days after birth (roughly corresponding to the beginning, middle, and end of synapse elimination in the LA muscle) and treated daily with testosterone propionate for the next 2 weeks. All rats were sacrificed at 9 weeks and their spinal cords and LA muscles were stained and analyzed. Only during the first treatment period (7-20) did androgen treatment result in increased levels of multiple innervation at 9 weeks. During this period, androgen also increased the number of junctional sites per fiber and the size of SNB somata but did not influence the adult level of preterminal branching or the diameter of adult LA muscle fibers. Androgen treatment during the two later periods increased the level of preterminal branching and the size of LA muscle fibers without influencing the level of multiple innervation.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-resolution localization of acetylcholinesterase at the rat neuromuscular junction

To overcome the limited ultrastructural resolution of conventional acetylcholinesterase (AChE) ultrahistochemistry, acetylcholine (ATCh) was used to reduce the rate of enzymic thiocholine liberation. The conventionally limited resolution is mainly due to the high focal activity of the enzyme in neural structures, because cleavage of substrate is faster than histochemical trapping reactions. Therefore, using the copper-thiocholine method, we investigated the reduction of thiocholine liberation by acetylcholine (ACh). As examined biochemically, the apparent Ki for ACh was close to the Km for ATCh. The ACh/ATCh ratio, therefore, determined the reduction of thiocholine production in histochemical experiments. In addition, the morphological appearance of the precipitated reaction product after its changes during the histochemical procedure was monitored using electric eel AChE immobilized on Sepharose 4B. The improved fine structural resolution at 40- to 100-fold excess of ACh over ATCh is demonstrated at the neuromuscular junction of rat lumbricalis muscle. The highest focal enzyme activity is found at the presynaptic membrane and in the secondary cleft, but not on top of the junctional folds, indicating the separation of esterase and nicotinic receptors. The physiological events during neuromuscular transmission are discussed on the basis of the new "gradient switch hypothesis" suggested in this report.     

Effect of armin on the ultrastructure of the neuromuscular synapse]

Alterations induced by the cholinesterase inhibitor armin (5.10(-7) g/ml) in the ultrastructure of motor nerve endings of the rat phrenic diaphragmal preparations at rest or electric stimulation of the nerve were studied. It was shown that armin at rest induced ultrastructural lesions in the endings similar to those in the control preparations during nerve stimulation. Electric stimulation did not produce additional changes in the ultrastructure of the neuromuscular junction under armin action. It is suggested that the disorder of the nerve ending function may be of importance in the mechanism of the blocking action of armin on the neuromuscular transmission.     

The role of synapse elimination in the establishment of neuromuscular compartments

To investigate the specificity of development of initial neuromuscular connections, we examined the compartmental distribution of synapses in neonatal rat lateral gastrocnemius (LG) muscle. Initial neuromuscular connections might be restricted to the compartmental territories present in adults; alternatively, synapse elimination could establish the compartments from a less precise pattern of innervation. We examined 46 pups of ages 0 to 14 postnatal days using a variety of techniques. The principle method was evoked electromyographic (EMG) activity in response to nerve stimulation. The nerve branch to one neuromuscular compartment was cut and the remainder of the nerve was stimulated. The presence of EMG activity was used to identify the areas of muscle contracting in response to nerve stimulation. After cutting a particular branch, EMG activity generally could not be recorded from the denervated compartment. These results indicate that the pattern of innervation at birth is essentially compartment-specific, and that neuromuscular compartments are not shaped from some less precise pattern by postnatal synapse elimination. The factors which operate prenatally to determine this high degree of specificity in neuromuscular connectivity seen at the time of birth, however, remain unknown.     

Ankyrin-dependent Na+ channel clustering prevents neuromuscular synapse fatigue

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Cell adhesion molecules: signalling functions at the synapse

Many cell adhesion molecules are localized at synaptic sites in neuronal axons and dendrites. These molecules bridge pre- and postsynaptic specializations but do far more than simply provide a mechanical link between cells. In this review, we will discuss the roles these proteins have during development and at mature synapses. Synaptic adhesion proteins participate in the formation, maturation, function and plasticity of synaptic connections. Together with conventional synaptic transmission mechanisms, these molecules are an important element in the trans-cellular communication mediated by synapses.     

A novel pathway for MuSK to induce key genes in neuromuscular synapse formation

At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChR epsilon. We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.     

In vivo regulation of acetylcholinesterase insertion at the neuromuscular junction

The efficiency of synaptic transmission between nerve and muscle depends on the number and density of acetylcholinesterase molecules (AChE) at the neuromuscular junction. However, little is known about the way this density is maintained and regulated in vivo. By using time lapse and quantitative fluorescence imaging assays in living mice, we demonstrated that insertion of new AChEs occurs within hours of saturating pre-existing AChEs with fasciculin2, a snake toxin that selectively labels AChE. In the absence of muscle postsynaptic activity or evoked nerve presynaptic neurotransmitter release, AChE insertion was decreased significantly, whereas direct stimulation of the muscle completely restored AChE insertion to control levels. This activity-dependent AChE insertion is mediated by intracellular calcium. In muscle stimulated in the presence of a Ca2+ channel blocker or calcium-permeable Ca2+ chelator, AChE insertion into synapses was significantly decreased, whereas ryanodine or ionophore A12387 treatment of blocked and unstimulated synapses significantly increased AChE insertion. These results demonstrated that synaptic activity is critical for AChE insertion and indicated that a rise in intracellular calcium either through voltage-gated calcium channels or from intracellular stores is critical for proper AChE insertion into the adult synapse.