The relationship of neuromuscular synapse elimination to synaptic degeneration and pathology: insights from WldS and 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.     

Early and transient increase in spontaneous synaptic inputs to the rat facial motoneurons after axotomy in isolated brainstem slices of rats

Section of motor nerve fibers (axotomy) elicits a variety of morphofunctional responses in the motoneurons in the motor nuclei. Later than the fifth post-operational day after section of the facial nerve, synapse elimination occurs in the facial motoneuron pool, leading to gradual abolishment of synaptic input-driven activities of the axotomized motoneurons. However, it remains unknown how the amount of synaptic input changes during this period between the axotomy and the synaptic elimination. Here we examined a hypothesis that axotomy of the motoneurons itself modifies the synaptic inputs to the motoneurons. One day after axotomy, the postsynaptic currents, mostly mediated by non-N-methyl-D-aspartic acid (non-NMDA) receptors, recorded from the axotomized facial motoneurons in the acute slice preparations of the rats were of higher frequency and larger amplitude than those in the intact motoneurons. This difference was not observed after the third post-operational day and appeared earlier than the changes in the electrophysiological properties and increase in the number of dead neurons in the axotomized motor nucleus. The larger postsynaptic current frequency of the axotomized motoneurons was observed both in the absence and in the presence of tetrodotoxin citrate, suggesting that increased excitability and facilitated release underlie the postsynaptic current frequency increase. These results suggest that synaptic re-organization occurs in the synapses of motoneurons at an early stage following axotomy.     

Effects of evoked and spontaneous motoneuronal firing on synapse competition and elimination in skeletal muscle

Synapse elimination is a general feature of the development of neural connections, including the connections of motoneurons to skeletal muscle fibers. Our work addressed two questions: (1) how the action potentials generated in the set of motoneurons innervating an individual muscle ( i.e., in a motor pool) are correlated in time during development in vivo; (2) what influence different firing patterns exert on the processes of polyneuronal innervation and synapse elimination which characterize the establishment of muscle innervation.We recorded the spontaneous electromyographic activity of the tibialis anterior and soleus muscles of late embryonic and neonatal rats, identifying the firing of at least two single motor unit signals in each record. We found that a striking switch occurs a few days after birth from a highly synchronous type of firing to an asynchronous one, the first thus characterizing embryonic while the second one adult motoneurons. We also investigated the effects of an evoked synchronous type of discharge on neuromuscular synapse formation, measuring polyneuronal innervation and synapse elimination. This was done in an adult in vivo model of de novo synapse formation, while a chronic TTX nerve conduction block, placed centrally with respect to the stimulating electrodes, eliminated the natural activity of motoneurons. We found that the imposed synchronous activity greatly inhibits synapse elimination, causing polyneuronal innervation to persist. We conclude that the early synchronous firing, favors the establishment of polyneuronal innervation while the subsequent switch to an asynchronous one promotes synapse elimination.     

Ultrastructural observations on synapse elimination in neonatal rabbit skeletal muscle

Summary Electron microscopic techniques were used to investigate two main questions about mammalian neuromuscular development. One, does neonatal synapse elimination proceed by the degeneration of synaptic terminals and preterminal axons, or are the terminals retracted into the parent axon, in a process analogous to the resorption of axonal growth cones? Two, is there any discernible relationship between the elimination of supernumerary synapses and the myelination of preterminal axons? Examination of several hundred sections through endplates fixed at the peak time of synapse elimination revealed no signs of degeneration. This result is not consistent with the proposal that the major mechanism of synapse elimination is terminal degeneration, according to calculations based on the time course of terminal degeneration following neonatal nerve transection.Serial and semi-serial reconstruction of terminals and preterminal axons suggest that myelination of intramuscular axons lags behind synapse elimination and that elimination can proceed while axons bear an immature relationship to Schwann cells. In addition, reconstruction of serial sections through neonatal synapses revealed that their three-dimensional configuration is more complex than that of mature neuromuscular synapses; this feature may be indicative of a dynamic relationship between nerve and muscle at early stages.     

Activity-dependent plasticity of developing climbing fiber–Purkinje cell synapses

Elimination of redundant synapses and strengthening of the surviving ones are crucial steps in the development of the nervous system. Both processes can be readily followed at the climbing fiber to Purkinje cell synapse in the cerebellum. Shortly after birth, around five equally strong climbing fiber synapses are established. Subsequently, one of these five synaptic connections starts to grow in size and synaptic strength, while the others degenerate and eventually disappear. Both the elimination of the redundant climbing fiber synapses and the strengthening of the surviving one depend on a combination of a genetically coded blueprint and synaptic activity. Recently, it has been shown that synaptic activity affects the synaptic strength of developing climbing fibers. Remarkably, the same pattern of paired activity of the presynaptic climbing fiber and the postsynaptic Purkinje cell resulted in strengthening of already “large” climbing fibers and weakening of already “weak” climbing fibers. In this review, we will integrate the current knowledge of synaptic plasticity of climbing fibers with that of other processes affecting climbing fiber development.     

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.     

Presynaptic efficacy directs normalization of synaptic strength in layer 2/3 rat neocortex after paired activity

Paired neuronal activity is known to induce changes in synaptic strength that result in the synapse in question having different properties to unmodified synapses. Here we show that in layer 2/3 excitatory connections in young adult rat cortex paired activity acts to normalize the strength and quantal parameters of connections. Paired action potential firing produces long-term potentiation in only a third of connections, whereas a third remain with their amplitude unchanged and a third exhibit long-term depression. Furthermore, the direction of plasticity can be predicted by the initial strength of the connection: weak connections potentiate and strong connections depress. A quantal analysis reveals that changes in synaptic efficacy were predominantly presynaptic in locus and that the key determinant of the direction and magnitude of synaptic modification was the initial release probability (P(r)) of the synapse, which correlated inversely with change in P(r) after pairing. Furthermore, distal synapses also exhibited larger potentiations including postsynaptic increases in efficacy, whereas more proximal inputs did not. This may represent a means by which distal synapses preferentially increase their efficacy to achieve equal weighting at the soma. Paired activity thus acts to normalize synaptic strength, by both pre- and postsynaptic mechanisms.     

Age-Dependent Synapse Withdrawal at Axotomised Neuromuscular Junctions in Wlds Mutant and Ube4b/Nmnat Transgenic Mice

Axons in Wld^S mutant mice are protected from Wallerian degeneration by overexpression of a chimeric Ube4b/Nmnat (Wld) gene. Expression of Wld protein was independent of age in these mice. However we identified two distinct neuromuscular synaptic responses to axotomy. In young adult Wld^s mice, axotomy induced progressive, asynchronous synapse withdrawal from motor endplates, strongly resembling neonatal synapse elimination. Thus, five days after axotomy, 50–90 % of endplates were still partially or fully occupied and expressed endplate potentials (EPPs). By 10 days, fewer than 20 % of endplates still showed evidence of synaptic activity. Recordings from partially occupied junctions indicated a progressive decrease in quantal content in inverse proportion to endplate occupancy. In Wld^s mice aged > 7 months, axons were still protected from axotomy but synapses degenerated rapidly, in wild-type fashion: within three days less than 5 % of endplates contained vestiges of nerve terminals. The axotomy-induced synaptic withdrawal phenotype decayed with a time constant of ∼30 days. Regenerated synapses in mature Wld^s mice recapitulated the juvenile phenotype. Within 4–6 days of axotomy 30–50 % of regenerated nerve terminals still occupied motor endplates. Age-dependent synapse withdrawal was also seen in transgenic mice expressing the Wld gene. Co-expression of Wld protein and cyan fluorescent protein (CFP) in axons and neuromuscular synapses did not interfere with the protection from axotomy conferred by the Wld gene. Thus, Wld expression unmasks age-dependent, compartmentally organised programmes of synapse withdrawal and degeneration.     

Some reminiscences on studies of age‐dependent and activity‐dependent degeneration of sensory and motor endings in mammalian skeletal muscle

I present here an overview of research on the biology of neuromuscular sensory and motor endings that was inspired and influenced partly by my educational experience in the Department of Zoology at the University of Durham, from 1971 to 1974. I allude briefly to neuromuscular synaptic structure and function in dystrophic mice, influences of activity on synapse elimination in development and regeneration, and activity‐dependent protection and degeneration of neuromuscular junctions in Wld^S mice.     

Synaptic modifications at the CA3–CA1 synapse after chronic AMPA receptor blockade in rat hippocampal slices

Maintenance of dendritic spines, the postsynaptic elements of most glutamatergic synapses in the central nervous system, requires continued activation of AMPA receptors. In organotypic hippocampal slice cultures, chronic blockade of AMPA receptors for 14 days induces a substantial loss of dendritic spines on CA1 pyramidal neurons. Here, using serial section electron microscopy, we show that loss of dendritic spines is paralleled by a significant reduction in synapse density. In contrast, we observed an increased number of asymmetric synapses onto the dendritic shaft, suggesting that spine retraction does not inevitably lead to synapse elimination. Functional analysis of the remaining synapses revealed that hippocampal circuitry compensates for the anatomical loss of synapses by increasing synaptic efficacy. Moreover, we found that the observed morphological and functional changes were associated with altered bidirectional synaptic plasticity. We conclude that continued activation of AMPA receptors is necessary for maintaining structure and function of central glutamatergic synapses.     

Synaptic dynamics on different time scales in a parallel fiber feedback pathway of the weakly electric fish

Synaptic dynamics comprise a variety of interacting processes acting on a wide range of time scales. This enables a synapse to perform a large array of computations, from temporal and spatial filtering to associative learning. In this study, we describe how changing synaptic gain via long-term plasticity can act to shape the temporal filtering of a synapse through modulation of short-term plasticity. In the weakly electric fish, parallel fibers from cerebellar granule cells provide massive feedback inputs to the pyramidal neurons of the electrosensory lateral line lobe. We demonstrate a long-term synaptic enhancement (LTE) of these synapses that is biochemically similar to the presynaptic long-term potentiation expressed by parallel fibers in the mammalian cerebellum. Using a novel stimulation protocol and a simple modeling paradigm, we then quantify the changes in short-term plasticity during the induction of LTE and show that these changes can be explained by gradual changes in only one model parameter, that which is associated with the baseline probability of transmitter release. These changes lead to a shift in the spike frequency preference of the synapse, suggesting that long-term plasticity is not only involved in controlling the gain of the parallel fiber synapse, but also provides a means of controlling synaptic filtering over multiple time scales.     

Phosphorylation reactions in activity-dependent synapse modification at the neuromuscular junction during development

We have studied developmental activity-dependent synapse diminution in both an in vitro tissue culture chamber system and at the intact rodent neuromuscular junction (nmj). In both types of preparations, pre- and postsynaptic alterations in synapse structure and function are produced by manipulations of thrombin (Thr) and protein kinase C (PKC) activity. An opposing postsynaptic effect of PKC and protein kinase A (PKA) action on the acetycholine receptor (AChR) can be shown in vitro with PKA stabilizing and PKC destabilizing the nmj synapses. In vivo studies of normal junctional maturation show that changes in axonal inputs and postsynaptic receptor cluster morphology occur, to a substantial degree, independently of one another. Presynaptic actions of PKA are involved in the activity dependent synapse modulation that can be demonstrated in vitro. Late in the elimination process, (>12 days in vivo ) the process becomes independent of PKC, implying that diverse, redundant mechanisms are involved in this important developmental process.     

Comparison of synaptic changes in the precentral and postcentral cerebral cortex of aging humans: a quantitative ultrastructural study

Ultrastructural quantitative analysis was undertaken to determine whether any age-related synaptic changes occur in cortical layer 1 of the human precentral motor gyrus (Brodmann's area 4) and postcentral somatosensory gyrus (Brodmann's area 3). Immersion fixed, osmicated, uranyl acetate/lead citrate stained (OsUL) preparations of autopsied brains were taken from patients aged 45 to 84 years, with no prior history of neurological or intellectual abnormalities. In the precentral gyrus there was a significant decrease in the number of synapses, which was primarily due to a decrease in asymmetrical axospinous synapses. Symmetrical synapses remained constant in number, while axodendritic synapses showed a small increase with age. Accompanying the decline in synapse number was an increase in mean length of the postsynaptic contact zone. In the postcentral gyrus there were no significant changes in synaptic number or in any of the synaptic parameters measured. The results suggest that the motor cortex of the human brain is capable of synaptic plasticity in response to aging-induced synaptic loss. This plasticity is not apparent in the somatosensory cortex, where there is no age-related synapse loss.     

The Drosophila NSF protein, dNSF1, plays a similar role at neuromuscular and some central synapses.

The N-ethylmaleimide sensitive fusion protein (NSF) was originally identified as a cytosolic factor required for constitutive vesicular transport and later implicated in synaptic vesicle trafficking as well. Our previous work at neuromuscular synapses in the temperature-sensitive NSF mutant, comatose (comt), has shown that the comt gene product, dNSF1, functions after synaptic vesicle docking in the priming of vesicles for fast calcium-triggered fusion. Here we investigate whether dNSF1 performs a similar function at central synapses associated with the well-characterized giant fiber neural pathway. These include a synapse within the giant fiber pathway, made by the peripherally synapsing interneuron (PSI), as well as synapses providing input to the giant fiber pathway. The latency (delay) between stimulation and a resulting muscle action potential was used to assess the function of each class of synapses. Repetitive stimulation of the giant fiber pathway in comt produced wild-type responses at both 20 and 36 degrees C, exhibiting a characteristic and constant latency between stimulation and the muscle response. In contrast, stimulation of presynaptic inputs to the giant fiber (referred to as the "long latency pathway") revealed a striking difference between wild type and comt at 36 degrees C. Repetitive stimulation of the long latency pathway led to a progressive, activity-dependent increase in the response latency in comt, but not in wild type. Thus the giant fiber pathway, including the PSI synapse, appears to function normally in comt, whereas the presynaptic inputs to the giant fiber pathway are disrupted. Several aspects of the progressive latency increase observed in the long latency pathway can be understood in the context of the activity-dependent reduction in neurotransmitter release we observed previously at neuromuscular synapses. These results suggest that repetitive stimulation causes a progressive reduction in neurotransmitter release by presynaptic inputs to the giant fiber neuron, resulting in an increased latency preceding a giant fiber action potential. Thus synapses presynaptic to the giant fiber appear to utilize dNSF1 in a manner similar to the neuromuscular synapse, whereas the PSI chemical synapse may differ with respect to the expression or activity of dNSF1.     

Impulse activity of a crayfish motoneuron regulated its neuromuscular synaptic properties

1. Previous studies have demonstrated that initial transmitter release, fatigability, and the morphology of identified crayfish neuromuscular synapses adapt to long-term changes in motoneuron impulse activity. 2. Experiments were performed to determine whether these long-term, adaptive alterations in neuromuscular synaptic physiology are triggered by changes in neuromuscular synaptic activity, muscle activity, or neuronal impulse activity. The fast closer excitor of the crayfish claw, a phasic motoneuron, was studied. Either the central or the peripheral region of the motoneuron was selectively stimulated in vivo by blocking impulse activity midway along the motor axon with localized application of tetrodotoxin and stimulating either central or distal to the blocked region. 3. Neither muscle activity nor transmitter release from the neuromuscular synapses was required to trigger the changes in synaptic physiology. Stimulation central to the block induced changes in neuromuscular transmission that included a long-lasting decrease in initial transmitter release and increased fatique resistance. 4. Because peripheral stimulation also produced decreased initial transmitter release, it appears that increased impulse activity in either region of the motoneuron can produce the synaptic changes. These results along with earlier findings suggest that neuronal depolarization induces adaptive, long-term changes in synapses. 5. These results are discussed in relation to findings at vertebrate and invertebrate synapses.     

Dendritic excitability and synaptic plasticity

Most synaptic inputs are made onto the dendritic tree. Recent work has shown that dendrites play an active role in transforming synaptic input into neuronal output and in defining the relationships between active synapses. In this review, we discuss how these dendritic properties influence the rules governing the induction of synaptic plasticity. We argue that the location of synapses in the dendritic tree, and the type of dendritic excitability associated with each synapse, play decisive roles in determining the plastic properties of that synapse. Furthermore, since the electrical properties of the dendritic tree are not static, but can be altered by neuromodulators and by synaptic activity itself, we discuss how learning rules may be dynamically shaped by tuning dendritic function. We conclude by describing how this reciprocal relationship between plasticity of dendritic excitability and synaptic plasticity has changed our view of information processing and memory storage in neuronal networks.     

Competition at silent synapses in reinnervated skeletal muscle

Synaptic connections are made and broken in an activity-dependent manner in diverse regions of the nervous system. However, whether activity is strictly necessary for synapse elimination has not been resolved directly. Here we report that synaptic terminals occupying motor endplates made electrically silent by tetrodotoxin and alpha-bungarotoxin block were frequently displaced by regenerating axons that were also both inactive and synaptically ineffective. Thus, neither evoked nor spontaneous activation of acetylcholine receptors is required for competitive reoccupation of neuromuscular synaptic sites by regenerating motor axons.     

突触形成调控蛋白的研究与进展

背景：干细胞在体外也被证实可以分化为神经元细胞,然而干细胞分化成神经细胞后如何形成突触连接而实现信息传递功能,以及突触形成的调控机制是什么,这些尚未可知。 目的：通过对2000年以来影响突触形成调控蛋白的实验研究检索,总结这些蛋白在突触研究中的作用。 方法：以“synapse、synaptogenesis”为检索词,应用计算机检索中国知网和pubmed数据库相关文章,排除重复性研究,保留39篇文章做进一步分析。 结果与结论：突触形成的过程主要包括3个方面的相关内容：①突触结构的形成。②一些突触由无活性到有活性的转换。③非必要突触的消除。而与之相关的蛋白及其功能得以肯定并得到初步研究的主要有血小板反应蛋白、突触分化诱导基因产物、突触细胞黏附分子、主要组织相容性复合物Ⅰ型、肌细胞增强因子2、脆性X智力低下蛋白等。这些蛋白在突触的形成,发育和成熟过程中发挥着重要作用。     

Neonatal synapse elimination in the rat submandibular ganglion: effect of retarded target growth

1. We have studied synapse elimination in the submandibular ganglion of neonatal rats to determine the effects of retarded target growth on synaptic development. Neurons of this ganglion provide parasympathetic innervation to the submandibular and sublingual salivary glands. 2. Ligating the main salivary ducts 2-4 days after birth at a point where nerve fibers were not damaged reduces gland weight by 55% during the 2nd wk after birth and 80% by adulthood. 3. In control animals, the average number of preganglionic inputs/neuron normally declines steadily during the first few weeks after birth, before stabilizing during the 5th wk at the control adult level. Between birth and adulthood, the number of ganglionic neurons increases by 150%. 4. Ganglia from duct-ligated animals showed an acceleration in the process of synapse elimination. Input number in experimental ganglia reached the control adult level during the 3rd wk after birth. This acceleration is confined solely to ganglia that innervate the underdeveloped glands. 5. The loss of inputs was not further enhanced by prolonged target atrophy. Thus average input numbers to neurons of 5th wk or adult experimental ganglia were not different from age-matched control values. 6. No differences from control values were seen in most cases for resting potentials, input resistances, or cell size. However, the increase in neuron number was retarded in experimental animals, and the number of synapses/neuronal profile was reduced in the adult animals. 7. Thus subnormal target growth leads to an acceleration in the process of synaptic elimination in neonatal rats. This acceleration may be mediated by alterations in the level of trophic factors emanating from the target.     

Influence of parallel fiber–Purkinje cell synapse formation on postnatal development of climbing fiber–Purkinje cell synapses in the cerebellum

The climbing fiber (CF) to Purkinje cell (PC) synapse in the cerebellum provides an ideal model for the study of developmental rearrangements of neural circuits. At birth, each PC is innervated by multiple CFs. These surplus CFs are eliminated during postnatal development, and mono innervation is attained by postnatal day 20 (P20) in mice. Earlier studies on spontaneous mutant mice and animals with “hypogranular” cerebella indicate that regression of surplus CFs requires normal generation of granule cells and their axons, parallel fibers (PFs), and normal formation of PF–PC synapses. Our understanding of how PF–PC synapse formation affects development of CF–PC synapse has been greatly advanced by analyses of mutant mice deficient in glutamate receptor δ2 subunit (GluRδ2), an orphan receptor expressed selectively in PCs. Deletion of GluRδ2 results in impairment of PF–PC synapse formation, which leads to defects in development of CF–PC synapses. In this article, we review how impaired PF–PC synapse formation affects wiring of CFs to PCs based mostly on our data on GluRδ2 knockout mice. We propose a new scheme that CF–PC synapses are shaped by the three consecutive events, namely functional differentiation of multiple CFs into one strong and a few weak inputs from P3 to P7, “early phase” of CF synapse elimination from P7 to around P11, and “late phase” of CF synapse elimination from around P12. Normal PF–PC synapse formation is required for the “late phase” of CF synapse elimination.