Neuromuscular Synapse Elimination Research Articles

Lose it to use it.

In this issue, Wang et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.201911114) describe a phenomenon in which neuromuscular junction synapse elimination triggers myelination of terminal motor axon branches. They propose a mechanism initiated by synaptic pruning that depends on synaptic activity, cytoskeletal maturation, and the associated anterograde transport of trophic factors including Neuregulin 1-III.

Opposed Actions of PKA Isozymes (RI and RII) and PKC Isoforms (cPKCβI and nPKCε) in Neuromuscular Developmental Synapse Elimination.

Background: During neuromuscular junction (NMJ) development, synapses are produced in excess. By sensing the activity-dependent release of ACh, adenosine, and neurotrophins, presynaptic receptors prompt axonal competition and loss of the unnecessary axons. The receptor action is mediated by synergistic and antagonistic relations when they couple to downstream kinases (mainly protein kinases A and C (PKA and PKC)), which phosphorylate targets involved in axonal disconnection. Here, we directly investigated the involvement of PKA subunits and PKC isoforms in synapse elimination. Methods: Selective PKA and PKC peptide modulators were applied daily to the Levator auris longus (LAL) muscle surface of P5–P8 transgenic B6.Cg-Tg (Thy1-YFP) 16 Jrs/J (and also C57BL/6J) mice, and the number of axons and the postsynaptic receptor cluster morphology were evaluated in P9 NMJ. Results: PKA (PKA-I and PKA-II isozymes) acts at the pre- and postsynaptic sites to delay both axonal elimination and nAChR cluster differentiation, PKC activity promotes both axonal loss (a cPKCβI and nPKCε isoform action), and postsynaptic nAChR cluster maturation (a possible role for PKCθ). Moreover, PKC-induced changes in axon number indirectly influence postsynaptic maturation. Conclusions: PKC and PKA have opposed actions, which suggests that changes in the balance of these kinases may play a major role in the mechanism of developmental synapse elimination.

Branch-Specific Microtubule Destabilization Mediates Axon Branch Loss during Neuromuscular Synapse Elimination

SummaryDevelopmental axon remodeling is characterized by the selective removal of branches from axon arbors. The mechanisms that underlie such branch loss are largely unknown. Additionally, how neuronal resources are specifically assigned to the branches of remodeling arbors is not understood. Here we show that axon branch loss at the developing mouse neuromuscular junction is mediated by branch-specific microtubule severing, which results in local disassembly of the microtubule cytoskeleton and loss of axonal transport in branches that will subsequently dismantle. Accordingly, pharmacological microtubule stabilization delays neuromuscular synapse elimination. This branch-specific disassembly of the cytoskeleton appears to be mediated by the microtubule-severing enzyme spastin, which is dysfunctional in some forms of upper motor neuron disease. Our results demonstrate a physiological role for a neurodegeneration-associated modulator of the cytoskeleton, reveal unexpected cell biology of branch-specific axon plasticity and underscore the mechanistic similarities of axon loss in development and disease.

MHCI promotes developmental synapse elimination and aging-related synapse loss at the vertebrate neuromuscular junction

Synapse elimination at the developing neuromuscular junction (NMJ) sculpts motor circuits, and synapse loss at the aging NMJ drives motor impairments that are a major cause of loss of independence in the elderly. Here we provide evidence that at the NMJ, both developmental synapse elimination and aging-related synapse loss are promoted by specific immune proteins, members of the major histocompatibility complex class I (MHCI). MHCI is expressed at the developing NMJ, and three different methods of reducing MHCI function all disrupt synapse elimination during the second postnatal week, leaving some muscle fibers multiply-innervated, despite otherwise outwardly normal synapse formation and maturation. Conversely, overexpressing MHCI modestly accelerates developmental synapse elimination. MHCI levels at the NMJ rise with aging, and reducing MHCI levels ameliorates muscle denervation in aged mice. These findings identify an unexpected role for MHCI in the elimination of neuromuscular synapses during development, and indicate that reducing MHCI levels can preserve youthful innervation of aging muscle.

Decreased phosphorylation of delta and epsilon subunits of the acetylcholine receptor coincides with delayed postsynaptic maturation in PKC theta deficient mouse

Protein kinase C (PKC) activity is involved in the nicotinic acetylcholine receptor (nAChR) redistribution at the neuromuscular junction in vivo during postnatal maturation. Here we studied, in PKC theta (PKCθ) deficient mice (KO), how the theta isoform of PKC is involved in the nAChR cluster maturation that is accompanied by the developmental activity-dependent neuromuscular synapse elimination process. We found that axonal elimination and dispersion of nAChR from the postsynaptic plaques and its redistribution to form the mature postsynaptic apparatus were delayed but not totally suppressed in PKCθ deficient mice. Moreover, the delay in the maturation of the morphology of the nAChR clusters during the early postnatal synapse elimination period in the PKCθ deficient mice coincides with a reduction in the PKCθ-mediated phosphorylation on the delta subunit of the nAChR. In addition, we show evidence for PKCθ regulation of PKA in normally phosphorylating the epsilon subunit of nAChR. We have also found that the θ isoform of PKC is located on the postsynaptic component of the neuromuscular junction but is also expressed by motoneurons in the spinal cord and in the motor nerve terminals. The results allow us to hypothesize that a spatially specific and opposing action of PKCθ and PKA may result in activity-dependent alterations to synaptic connectivity at both the nerve inputs and the postsynaptic nAChR clusters.

Reduced gap junctional coupling leads to uncorrelated motor neuron firing and precocious neuromuscular synapse elimination

During late embryonic and early postnatal life, neuromuscular junctions undergo synapse elimination that is modulated by patterns of motor neuron activity. Here, we test the hypothesis that reduced spinal neuron gap junctional coupling decreases temporally correlated motor neuron activity that, in turn, modulates neuromuscular synapse elimination, by using mutant mice lacking connexin 40 (Cx40), a developmentally regulated gap junction protein expressed in motor and other spinal neurons. In Cx40-/- mice, electrical coupling among lumbar motor neurons, measured by whole-cell recordings, was reduced, and single motor unit recordings in awake, behaving neonates showed that temporally correlated motor neuron activity was also reduced. Immunostaining and intracellular recording showed that the neuromuscular synapse elimination was accelerated in muscles from Cx40-/- mice compared with WT littermates. Our work shows that gap junctional coupling modulates neuronal activity patterns that, in turn, mediate synaptic competition, a process that shapes synaptic circuitry in the developing brain.

Activity-dependent synaptic competition at mammalian neuromuscular junctions.

Synapse elimination is a widespread developmental process in the peripheral and central nervous system that brings about refinement of neural connections through epigenetic mechanisms. Here we describe recent advances concerning the role of the pattern of motoneuronal firing, synchronous or asynchronous, in neuromuscular synapse elimination.

The relationship of neuromuscular synapse elimination to synaptic degeneration and pathology: Insights from WldSand other mutant mice

Neuromuscular synapse elimination, Wallerian degeneration and peripheral neuropathies are not normally considered as related phenomena. However, recent studies of mutant and transgenic mice, particularly the Wld(S) mutant-in which orthograde degeneration is delayed following axotomy-suggest that re-evaluation of possible links between natural, traumatic and pathogenic regression of synapses may be warranted. During developmental synapse elimination from polyneuronally innervated junctions, some motor nerve terminals progressively and asynchronously vacate motor endplates. A form of asynchronous synapse withdrawal, strongly resembling synapse elimination, also occurs from mononeuronally-innervated motor endplates following axotomy in young adult Wld(S) mutant mice. A similar pattern is observed in skeletal muscles of several neuropathic mutants, including mouse models of dying-back neuropathies, motor neuron disease and-remarkably-models of neurodegenerative diseases such as Huntington's and Alzheimer's diseases. Taken together with recent analysis of synaptic remodelling at neuromuscular junctions in Drosophila, a strong candidate for a common regulatory mechanism in these diverse conditions is one based on protein ubiquitination/deubiquitination. Axotomised neuromuscular junctions in Wld(S) mutant mice offer favourable experimental opportunities for examining developmental mechanisms of synaptic regression, that may also benefit our understanding of how degeneration in the synaptic compartment of a neuron is initiated, and its role in progressive, whole-cell neuronal degeneration.

Disparity in Neurotransmitter Release Probability among Competing Inputs during Neuromuscular Synapse Elimination

Competition among the several motor axons transiently innervating neonatal muscle fibers results in an increasing disparity in the quantal content and synaptic territory of each competitor, culminating in the permanent loss of all but one axon from neuromuscular junctions. We asked whether differences in the probability of neurotransmitter release also contribute to the increasing disparity in quantal content among competing inputs, and when in the process of competition changes in release probability become apparent. To address these questions, intracellular recordings were made from dually innervated neonatal mouse soleus muscle fibers, and quantal content and paired-pulse facilitation were evaluated for each input. At short interpulse intervals, paired-pulse facilitation was significantly higher for the weaker input with the smaller quantal content than the stronger input with the larger quantal content. Because neurotransmitter release probability across all release sites is inversely related to the extent of facilitation observed after paired-pulse stimulation, this result suggests that release probability is lower for weak compared with strong inputs innervating the same junction. A disparity in the probability of neurotransmitter release thus contributes to the disparity in quantal content that occurs during synaptic competition. Together, this work suggests that an input incapable of sustaining a high release probability may be at a competitive disadvantage for synaptic maintenance.

Morphological effects of ciliary neurotrophic factor treatment during neuromuscular synapse elimination.

In adult skeletal muscles, exogenous ciliary neurotrophic factor (CNTF) induces axons and their nerve terminals to sprout. CNTF also regulates the amount of multiple innervation in developing skeletal muscles during synapse elimination, maintaining multiple innervation of muscle fibers. While CNTF may maintain multiple innervation by regulating developmental synapse elimination, it is also possible that CNTF induces the formation of new multiple innervation through a sprouting response. In this study I examined morphologically the effects of CNTF during synapse elimination in the extensor digitorum longus (EDL) muscle. Rat pups received injections of CNTF in one leg and vehicle in the other either early [postnatal day 7 (P7)-P13] or late (P14-P20) in development. The early treatment period corresponds to that time when the pattern of innervation in the EDL is converted from predominantly multiple to single innervation. The late treatment period is at the end of synapse elimination for the EDL but corresponds to the major period of synapse elimination in the levator ani (LA), allowing a comparison of effects on these two muscles from the same animals. On the day after the final injection, EDL muscles were dissected and stained with tetranitroblue tetrazolium and the resulting pattern of innervation was assessed. The present findings indicate that only the early CNTF treatment regulates the level of multiple innervation in the EDL. Moreover, the effect o early CNTF treatment was local, affecting multiple innervation only in the EDL from the CNTF-treated leg. CNTF injected during the late treatment period had no apparent effects on the EDL but had a potent effect on the pattern of innervation in the LA, significantly increasing the level of multiple innervation in this muscle. Thus, CNTF affected multiple innervation in these two muscles only if provided during the period when single innervation normally develops. There was no evidence to indicate that CNTF induced axons or their terminals to sprout during either treatment period. In conclusion, CNTF increases the level of multiple innervation, probably by regulating synapse elimination, and skeletal muscles themselves may be an important target site for CNTF action. Presumably, the sprouting response to CNTF found in adult muscle develops sometime after P21.

Morphological effects of ciliary neurotrophic factor treatment during neuromuscular synapse elimination

Morphological effects of ciliary neurotrophic factor treatment during neuromuscular synapse elimination

Ciliary neurotrophic factor may act in target musculature to regulate developmental synapse elimination.

During development of mammalian skeletal muscles, synaptic contacts from different motoneurons are made on to individual muscle fibers but then are progressively lost until the adult pattern of single innervation is established. Although this process of synapse elimination occurs throughout the developing nervous system, little is known about the underlying mechanisms that drive this process. Competition for target-derived trophic substances has been proposed as one mechanism whereby synapses are selectively maintained or eliminated. To directly test whether exogenous trophic substance could alter neuromuscular synapse elimination, levator ani (LA) muscles of male rats were treated during the period of synapse elimination with either human recombinant ciliary neurotrophic factor (CNTF-a putative motoneuronal survival factor), or vehicle. LA muscles were stained with tetranitroblue tetrazolium at the end of treatment and the number of axonal inputs per muscle fiber was quantified. Effects of CNTF on other parameters such as body weight, axonal sprouting, muscle fiber and motoneuronal growth were also assessed. CNTF-treated muscles contained 3 times more multiple innervation than did vehicle-treated muscles, suggesting that CNTF can regulate synapse elimination in the LA. Moreover, CNTF delivered near the LA was more potent in blocking synapse elimination than the same dose of CNTF delivered at a site distant from the LA, suggesting that the target muscle is an important site, either for direct CNTF action on synapse elimination, and/or for directed transport of CNTF to motoneuronal cell bodies.

Both Basic Fibroblast Growth Factor and Ciliary Neurotrophic Factor Promote the Retention of Polyneuronal Innervation of Developing Skeletal Muscle Fibers

At birth, nearly all rat muscle fibers receive synaptic inputs from more than one motoneuron at a single end-plate site. By the end of the third postnatal week all but one of these inputs has been eliminated. During this loss of polyneuronal innervation, developing neuromuscular synapses compete with one another. Although the nature of this competition is not known, it is commonly assumed that it is mediated through differential activity of the competing inputs. One means by which such differential activity might be translated into a biological signal would be if the synapses compete in an activity-dependent manner for a scarce supply of neurotrophic molecules. A prediction of this hypothesis is that excess quantities of such trophic molecules will reduce competition and thereby slow down or abolish the normal loss of polyneuronal innervation. In newborn rats, the effects of injection of either basic fibroblast growth factor (bFGF) or ciliary neurotrophic factor (CNTF) on the outcome of neuromuscular synapse elimination were investigated. Daily injections of either bFGF or CNTF were made for 1 week into the lateral gastrocnemius muscle beginning at the postnatal age of 2 days. The amount of polyneuronal innervation of fibers in trophic molecule-injected muscles and saline-injected contralateral muscles was assayed using intracellular recording methods. For both bFGF- and CNTF-injected muscles, an increase in the percentage of polyneuronally innervated fibers relative to saline-injected muscles was noted. For bFGF-injected muscles, the amount of polyneuronal innervation remained at nearly 60% as late as the postnatal age of 14 days (P14). This is the amount of polyneuronal innervation found at age 6 days in normal animals. Nearly 40% of the fibers of CNTF-injected muscles remained polyneuronally innervated at age P14, the amount expected at age 9 days. These results indicate that both bFGF and CNTF exert powerful and long-lasting effects on developing neuromuscular synapses.

Retrograde interactions during formation and elimination of neuromuscular synapses

Maturation of neuromuscular synapses depends on dynamic interactions between presynaptic motor neurons and postsynaptic muscle cells. Recent studies have addressed the cellular mechanisms underlying these interactions in cell cultures and in developing animals. Retrograde signals from the postsynaptic muscle cells appear to play critical roles in all stages of synapse development, from the initial synaptogenesis to the stabilization or elimination of the synapse.

200 kD neurofilament protein and synapse elimination in the rat soleus muscle

We studied the distribution and appearance of the phosphorylated form of the 200 kD neurofilament protein in the rat soleus muscle during the period of postnatal synapse elimination. Unlike many muscles, the appearance of singly innervated muscle cells in soleus occurs well after myogenesis has been completed, so that synapses are eliminated from a stable population of muscle cells. Immunoreactivity to the 200 kD neurofilament protein is present in the terminals of neuromuscular synapses of animals at all postnatal ages from 0 to 21 days. Before postnatal day 10, when physiological studies indicate that all soleus muscle cells receive more than one synaptic input, as many as 30% of soleus muscle cells contain phosphorylated 200 kD neurofilament protein immunoreactivity in only one synaptic terminal. At older ages the number of polyneuronally innervated muscle cells observed using immunostaining is similar to that observed physiologically. These findings suggest that not all developing neuromuscular synapses contain phosphorylated 200 kD neurofilament protein, and that those terminals lacking it comprise most of those eliminated early in the postnatal period. We conclude that the presence of phosphorylated 200 kD neurofilament protein might be highly correlated with the survival of motor nerve terminals during postnatal neuromuscular synapse elimination.

Ontogeny of steroid accumulation in spinal lumbar motoneurons of the rat: Implications for androgen's site of action during synapse elimination

Androgens influence the postnatal development of motoneurons in the spinal nucleus of the bulbocavernosus (SNB) by regulating neuromuscular synapse elimination, the process through which multiple axonal inputs are retracted from each muscle fiber until single innervation is established. In the rat levator ani (LA), one of the target muscles for SNB motoneurons, much of this loss of multiple innervation can be prevented by prepubertal androgen treatment. We used steroid autoradiography to measure the ontogeny of steroid accumulation in the SNB and the retrodorsolateral nucleus (RDLN), two motoneuronal groups thought to differ in their sensitivity to androgens. Spinal cord tissue was analyzed from castrated male rats at 7, 14, 21, and 60 days of age after injection of radiolabelled testosterone. SNB and RDLN motoneurons differ in the ontogeny of androgen accumulation. Over 80% of SNB motoneurons develop the capacity to accumulate androgen during the second week after birth, during the period when androgen regulates synapse elimination in the LA. In contrast, androgen accumulation in RDLN motoneurons develops much later (after 21 days). These data suggest that androgen may act directly on SNB motoneurons to influence synapse elimination.

Development of compartmentalized innervation of the rat gluteus maximus muscle

The innervation territories of the nerves to the gluteus maximus muscle were investigated in neonatal rats by using evoked electromyographic mapping techniques. Similar methods were employed to determine the terminal fields of individual motoneurons. Both at birth and after the period of neuromuscular synapse elimination, rat gluteus maximus is partitioned by its two muscle nerves. Each of these partitions is further divided into neuromuscular compartments by the primary branches of the muscle nerves. The terminal fields of individual motoneurons are about as well localized in early neonates as in 26 day-old animals. Intracellular records from gluteal muscle cells reveal that some myotubes in a narrow region near the center of the muscle receive inputs from both of the muscle nerves during the period of polyneuronal innervation. Such cross-compartmental innervation could be demonstrated only until the eighth postnatal day, even though polyneuronally innervated muscle cells were found as late as postnatal day 18. Analysis of the trajectories of individual axons filled with horseradish peroxidase indicates that they branch into only one primary branch of the nerves to gluteus maximus, both in newborn and 14 day-old pups. The innervation of gluteus maximus at birth is essentially restricted to neuromuscular compartments. Synapse elimination probably plays a minor role in establishing neuromuscular compartments.

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)

Competitive mechanisms underlying synapse elimination in the lumbrical muscle of the rat

The process of neuromuscular synapse elimination has been studied in the fourth deep lumbrical (4DL) muscle of the rat, a preparation which offers technical advantages for some types of experimental work. Studies have been performed both during development and in adult denervated muscles undergoing reinnervation. Results indicate that synapse elimination is dependent upon competition between motoneurons. Cellular mechanisms underlying this competition have also been explored. Both neuromuscular activity and muscle fiber type recognition appear to play a role, but positional cues appear unimportant in this small muscle.

Differential loss of neuromuscular connections according to activity level and spinal position of neonatal rabbit soleus motor neurons

We have tested whether the ability of synapses to compete for occupancy of endplates during neuromuscular synapse elimination is affected by differences in the spinal position or in the activity level of the parent motor neuron. To test the role of spinal position, the relative sizes of motor units for motor neurons from middle and extreme (rostral/caudal) positions in the rabbit soleus motor pool were determined at 3 postnatal ages: 4-5 d ("early" ages, when the soleus is heavily polyinnervated), 8-9 d ("intermediate"), and 11-15 d ("late," when the soleus has just reached singly innervated state). Average motor unit sizes from extreme ventral roots were similar to those from middle ventral roots in early-aged soleus muscles but were significantly smaller (by 18-27%) for both intermediate and late muscles. Thus, motor neurons from extreme positions evidently compete less effectively for retention of synapses than those from middle positions. To test the role of differential activity, inactive and active synapses were pitted directly against one another by implanting Silastic plugs laden with tetrodotoxin (TTX) into one of the spinal nerves containing a minority of the soleus motor axons. Differential activity was maintained during a period of extensive synapse loss, from the time of the implant at day 4 or 5 until the intermediate age (day 8-9). Motor unit twitch tensions were subsequently measured to determine the relative number of synapses retained by individual active and inactive motor neurons. The inactivated motor units were on average significantly larger (by more than 50%) than the corresponding group from normal and control-implanted animals. The abnormally large size of inactivated motor units persisted in animals allowed to recover from the TTX block and examined after multiple innervation had disappeared. Hence, the effect of the TTX block cannot be attributed to a simple slowing of synapse elimination specifically among the inactive motor neurons. We conclude that complete presynaptic inactivity improves the chances of survival relative to that for normal activity during synapse elimination in the neonatal rabbit soleus muscle. This difference in competitive ability may contribute to the development of an important characteristic of adult muscles, the correlation between motor unit size and recruitment threshold.