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.     

Ultrastructural correlates of synapse withdrawal at axotomized neuromuscular junctions in mutant and transgenic mice expressing the Wld gene

We carried out an ultrastructural analysis of axotomized synaptic terminals in Wld(s) and Ube4b/Nmnat (Wld) transgenic mice, in which severed distal axons are protected from Wallerian degeneration. Previous studies have suggested that axotomy in juvenile (< 2 months) Wld mice induced a progressive nerve terminal withdrawal from motor endplates. In this study we confirm that axotomy-induced terminal withdrawal occurs in the absence of all major ultrastructural characteristics of Wallerian degeneration. Pre- and post-synaptic membranes showed no signs of disruption or fragmentation, synaptic vesicle densities remained at pre-axotomy levels, the numbers of synaptic vesicles clustered towards presynaptic active zones did not diminish, and mitochondria retained their membranes and cristae. However, motor nerve terminal ultrastructure was measurably different following axotomy in Wld transgenic 4836 line mice, which strongly express Wld protein: axotomized presynaptic terminals were retained, but many were significantly depleted of synaptic vesicles. These findings suggest that the Wld gene interacts with the mechanisms regulating transmitter release and vesicle recycling.     

Compartmental neurodegeneration and synaptic plasticity in the Wlds mutant mouse

This review focuses on recent developments in our understanding of neurodegeneration at the mammalian neuromuscular junction. We provide evidence to support a hypothesis of compartmental neurodegeneration, whereby synaptic degeneration occurs by a separate, distinct mechanism from cell body and axonal degeneration. Studies of the spontaneous mutant Wld^s mouse, in which Wallerian degeneration is characteristically slow, provide key evidence in support of this hypothesis. Some features of synaptic degeneration in the absence of Wallerian degeneration resemble synapse elimination in neonatal muscle. This and other forms of synaptic plasticity may be accessible to further investigations, exploiting advantages afforded by the Wld^s mutant, or transgenic mice that express the Wld^s gene.     

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.     

Rapid loss of motor nerve terminals following hypoxia-reperfusion injury occurs via mechanisms distinct from classic Wallerian degeneration

Motor nerve terminals are known to be vulnerable to a wide range of pathological stimuli. To further characterize this vulnerability, we have developed a novel model system to examine the response of mouse motor nerve terminals in ex vivo nerve/muscle preparations to 2 h hypoxia followed by 2 h reperfusion. This insult induced a rapid loss of neurofilament and synaptic vesicle protein immunoreactivity at pre-synaptic motor nerve terminals but did not appear to affect post-synaptic endplates or muscle fibres. The severity of nerve terminal loss was dependent on the age of the mouse and muscle type: in 8–12-week-old mice the predominantly fast-twitch lumbrical muscles showed an 82.5% loss, whereas the predominantly slow-twitch muscles transversus abdominis and triangularis sterni showed a 57.8% and 27.2% loss, respectively. This was contrasted with a > 97% loss in the predominantly slow-twitch muscles from 5–6-week-old mice. We have also demonstrated that nerve terminal loss occurs by a mechanism distinct from Wallerian degeneration, as the slow Wallerian degeneration ( Wld^s ) gene did not modify the extent of nerve terminal pathology. Together, these data show that our new model of hypoxia–reperfusion injury is robust and repeatable, that it induces rapid, quantitative changes in motor nerve terminals and that it can be used to further examine the mechanisms regulating nerve terminal vulnerability in response to hypoxia–reperfusion injury.     

Morphological Features of Nerve Terminal Degeneration as Part of the Remodeling Process in the Motor Endplate in Adult Muscles

With the exception of signs of retraction and withdrawal, there have been few morphological data concerning degenerated neural profiles in adult motor endplates. Here, investigation into the ultrastructure of the soleus motor endplates of adult rats (4 months old) turned up particular axonal degeneration in approximately 3% of the subjects. These axons occur as synaptic debris in the synaptic matrix of the motor endplate, adjacent to thin processes of the perisynaptic cells occupying the outer most layer of the motor endplate and were devoid of basal lamina. They often possessed dense-cored vesicles (50-80 nm). Axonal debris released from Schwann cell processes occurred during the period of acute sciatic neurectomy, when nerve terminals progressively disrupted within the motor endplate associated Schwann cells. Finally, immunohistochemical staining for antibodies to label macrophages (ED1 or ED2) has shown that nerve fiber-associated macrophages are located near the motor endplate. The results suggest that during the course of endplate remodeling, a few parts of the terminal branches are disposed of through spontaneous collapse, subsequent release from the Schwann cell investment, and eventual ingestion by macrophages in the perisynaptic space.     

Neuromuscular plasticity following limb immobilization

Summary The effects of immobilization on the ultrastructure of the rat neuromuscular junction of type I and type II muscle fibres were studied both qualitatively and quantitatively. Muscle fibre areas were measured as well The plantaris muscle was immobilized in a shortened position by applying a plaster cast for three weeks. Immobilized muscles were then compared to normal litter mates. Both type I and type II immobilized muscle fibres atrophied. Endplates from type II muscle fibres exhibited greater amounts of degeneration than type I endplates. Degeneration consisted of nerve terminal disruption, exposed junctional folds, postsynaptic areas which contained little or no postjunctional folds, and subjunctional sarcoplasmic masses. In addition to degeneration, the type II endplates also exhibited regeneration in the same endplate consisting of small terminals associated with large expanses of junctional folds, several small terminals occurring within the same primary synaptic cleft, and several axons wrapped by the same Schwann cell. These observations suggest terminal axonal regeneration. Our results demonstrate that limb immobilization produces muscle atrophy as well as denervation-like changes at the neuromuscular junctions which leads to terminal axonal sprouting and an ultrastructural remodelling.     

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.     

Morphological features of nerve terminal degeneration as part of the remodeling process in the motor endplate in adult muscles

With the exception of signs of retraction and withdrawal, there have been few morphological data concerning degenerated neural profiles in adult motor endplates. Here, investigation into the ultrastructure of the soleus motor endplates of adult rats (4 months old) turned up particular axonal degeneration in approximately 3% of the subjects. These axons occur as synaptic debris in the synaptic matrix of the motor endplate, adjacent to thin processes of the perisynaptic cells occupying the outer most layer of the motor endplate and were devoid of basal lamina. They often possessed dense-cored vesicles (50-80 nm). Axonal debris released from Schwann cell processes occurred during the period of acute sciatic neurectomy, when nerve terminals progressively disrupted within the motor endplate associated Schwann cells. Finally, immunohistochemical staining for antibodies to label macrophages (ED1 or ED2) has shown that nerve fiber-associated macrophages are located near the motor endplate. The results suggest that during the course of endplate remodeling, a few parts of the terminal branches are disposed of through spontaneous collapse, subsequent release from the Schwann cell investment, and eventual ingestion by macrophages in the perisynaptic space.     

Cholinergic regulation of the evoked quantal release at frog neuromuscular junction

The effects of cholinergic drugs on the quantal contents of the nerve-evoked endplate currents (EPCs) and the parameters of the time course of quantal release (minimal synaptic latency, main modal value of latency histogram and variability of synaptic latencies) were studied at proximal, central and distal regions of the frog neuromuscular synapse. Acetylcholine (ACh, 5 × 10⁻⁴ m ), carbachol (CCh, 1 × 10⁻⁵ m ) or nicotine (5 × 10⁻⁶ m ) increased the numbers of EPCs with long release latencies mainly in the distal region of the endplate (90–120 μm from the last node of Ranvier), where the synchronization of transmitter release was the most pronounced. The parameters of focally recorded motor nerve action potentials were not changed by either ACh or CCh. The effects of CCh and nicotine on quantal dispersion were reduced substantially by 5 × 10⁻⁷ m (+)tubocurarine (TC). The muscarinic agonists, oxotremorine and the propargyl ester of arecaidine, as well as antagonists such as pirenzepine, AF-DX 116 and methoctramine, alone or in combination, did not affect the dispersion of the release. Muscarinic antagonists did not block the dispersion action of CCh. Cholinergic drugs either decreased the quantal content m _o (muscarinic agonist, oxotremorine M, and nicotinic antagonist, TC), or decreased m _o and dispersed the release (ACh, CCh and nicotine). The effects on m _o were not related either to the endplate region or to the initial level of release dispersion. It follows that the mechanisms regulating the amount and the time course of transmitter release are different and that, among other factors, they are altered by presynaptic nicotinic receptors.     

Enhanced G protein-dependent modulation of excitatory synaptic transmission in the cerebellum of the Ca2+ channel-mutant mouse, tottering

Tottering , a mouse model for absence epilepsy and cerebellar ataxia, carries a mutation in the gene encoding class A (P/Q-type) Ca²⁺ channels, the dominant exocytotic Ca²⁺ channel at most synapses in the mammalian central nervous system. Comparing tottering to wild-type mice, we have studied glutamatergic transmission between parallel fibres and Purkinje cells in cerebellar slices. Results from biochemical assays and electrical field recordings demonstrate that glutamate release from parallel fibre terminals of the tottering mouse is controlled largely by class B Ca²⁺ channels (N-type), in contrast to the P/Q-channels that dominate release from wild-type terminals. Since N-channels, in a variety of assays, are more effectively inhibited by G proteins than are P/Q-channels, we tested whether synaptic transmission between parallel fibres and Purkinje cells in tottering mice was more susceptible to inhibitory modulation by G protein-coupled receptors than in their wild-type counterparts. GABA_B receptors and α₂-adrenergic receptors (activated by bath application of transmitters) produced a three- to fivefold more potent inhibition of transmission in tottering than in wild-type synapses. This increased modulation is likely to be important for cerebellar transmission in vivo , since heterosynaptic depression, produced by activating GABAergic interneurones, greatly prolonged GABA_B receptor-mediated presynaptic inhibition in tottering as compared to wild-type slices. We propose that this enhanced modulation shifts the balance of synaptic input to Purkinje cells in favour of inhibition, reducing Purkinje cell output from the cerebellum, and may contribute to the aberrant motor phenotype that is characteristic of this mutant animal.     

Presynaptic N-type and P/Q-type Ca2+ channels mediating synaptic transmission at the calyx of Held of mice

At the nerve terminal, both N- and P/Q-type Ca²⁺ channels mediate synaptic transmission, with their relative contribution varying between synapses and with postnatal age. To clarify functional significance of different presynaptic Ca²⁺ channel subtypes, we recorded N-type and P/Q-type Ca²⁺ currents directly from calyces of Held nerve terminals in α_1A-subunit-deficient mice and wild-type (WT) mice, respectively. The most prominent feature of P/Q-type Ca²⁺ currents was activity-dependent facilitation, which was absent for N-type Ca²⁺ currents. EPSCs mediated by P/Q-type Ca²⁺ currents showed less depression during high-frequency stimulation compared with those mediated by N-type Ca²⁺ currents. In addition, the maximal inhibition by the GABA_B receptor agonist baclofen was greater for EPSCs mediated by N-type channels than for those mediated by P/Q-type channels. These results suggest that the developmental switch of presynaptic Ca²⁺ channels from N- to P/Q-type may serve to increase synaptic efficacy at high frequencies of activity, securing high-fidelity synaptic transmission.     

Involvement of neurotrophin-3 (NT-3) in the functional elimination of synaptic contacts during neuromuscular development

Confocal immunohistochemistry shows that neurotrophin-3 (NT-3) and its receptor tropomyosin-related tyrosin kinase C (trkC) are present in both neonatal (P6) and adult (P45) mouse motor nerve terminals in neuromuscular junctions (NMJ) colocalized with several synaptic proteins. NT-3 incubation (1–3 h, in the range 10–200 ng/ml) does not change the size of the evoked and spontaneous endplate potentials at P45. However, NT-3 (1 h, 100 ng/ml) strongly potentiates evoked ACh release from the weak (70%) and the strong (50%) axonal inputs on dually innervated postnatal endplates (P6) but not in the most developed postnatal singly innervated synapses at P6. The present results indicate that NT-3 has a role in the developmental mechanism that eliminates redundant synapses though it cannot modulate synaptic transmission locally as the NMJ matures.     

Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene.

Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/WldS mouse are protected. The degenerative and protective mechanisms are unknown. We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration. Transected distal axons survived for two weeks, and neuromuscular junctions were also protected. Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism. Nmnat enzyme activity, but not NAD+ content, was increased fourfold in WldS tissues. Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.     

Inhibition of acetylcholinesterase accelerates axon terminal withdrawal at the developing rat neuromuscular junction

Summary In developing skeletal muscles, the rate at which superfluous innervation is lost from the endplates depends on the general level of neuromuscular activity. Whether it is activity of the presynaptic or postsynaptic structures (or both) that is critical is not well established. In this work, we transitorily inhibited the AChE of soleus muscle in postnatal rats, in order to increase postsynaptic activity, without directly altering activity of the nerve terminals. We then followed the time course of disappearance of axon terminals from the endplates of treated and normal muscles, using electron-microscope techniques. Three hours after inhibition of AChE, the muscle fibres exhibited local supercontracture and ultrastructural damage in the region of the endplate, consistent with local elevation of Ca²⁺ levels. At the same time, small electron-opaque vesicles, apparently of muscular origin, appeared in the synaptic cleft. The nerve terminals, however, were entirely normal in number and appearance. One day after treatment, endplates of esteraseinhibited muscles showed accelerated loss of nerve terminals, compared to endplates of normally developing muscles. No further loss of nerve terminals occurred, once AChE activity returned at the endplate. These results suggest that the rate at which superfluous nerve terminals retract from the developing neuromuscular junction is regulated by the level of activation of the muscle. It seems likely that activity of postsynaptic sites may similarly regulate changes in innervation patterns, in other developing or adapting neuro-neuronal or neuro-effector systems.     

Scanning and light microscopic study of age changes at a neuromuscular junction in the mouse

Summary From previous work, it appears that synaptic transmission is well preserved at aging mouse neuromuscular junctions despite profound ultrastructural changes. Scanning and light microscopy have been used to determine whether expansion or sprouting of nerve terminals or post-synaptic reorganization play a role in this apparent compensatory mechanism. The number and length of nerve terminal branches in the extensor digitorum longus of young (7 months) and old (29 months) mice were studied with a combined silver-cholinesterase method. In aged animals, there were increases in nerve terminal length and number of intrasynaptic branches, with no change in muscle fibre diameter or numbers of axons entering the junction. Neither collateral sprouting nor collateral innervation, hallmarks of partial denervation, were present.Motor endplates visualized by scanning electron microscopy appeared as slightly elevated, elliptical plateaux (raised areas) with smooth surfaces into which the synaptic clefts were etched. In the aged endplates more than in young endplates, the primary clefts were often interrupted by narrow short outpouchings approximately perpendicular to the long axis of the primary cleft. In addition, oval primary cleft islets were more frequent and there was increased randomness and branching of secondary clefts.Both light and scanning microscopy gave concordant quantitative evidence that nerve terminals and the underlying postsynaptic cleft are longer and more branched in aged mice. The increased length of synaptic nerve terminal approximately balances the loss of girth previously reported leaving nerve terminal volume unchanged. The observed expansion of the synaptic area in the aged neuromuscular junction may be compensatory, preserving neuromuscular function. The data also point to plasticity of adult neuromuscular synaptic structure.     

The neuroprotective factor Wld fails to mitigate distal axonal and neuromuscular junction (NMJ) defects in mouse models of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a common autosomal recessive neurodegenerative disorder in humans. Amongst the earliest signs of neurodegeneration are severe and progressive defects of the neuromuscular synapse. These defects, characterized by poor terminal arborization and immature motor endplates, presumably result in a loss of functional synapses. The slow Wallerian degeneration (Wld^s) mutation in rodents has been shown to have a protective effect on mouse models of motor neuron disease by retarding axonal die-back and preventing neuromuscular synapse loss. In this study we tested the effects of the Wld^s mutation on the disease phenotype of SMA model mice. Consistent with previous reports, the mutation slows axon and neuromuscular synapse loss following nerve injury in wild-type as well as in SMA mice. However, the synaptic defects found in severely affected SMA patients and model mice persist in the double (Wld^s;SMA) mutants. No delay in disease onset was observed and survival was not significantly altered. Finally, Wld^s had no effect on the striking phrenic nerve projection defects that we discovered in SMA model mice. Our results indicate that the reported protective effects of Wld^s are insufficient to mitigate the neuromuscular phenotype due to reduced SMN protein, and that the mechanisms responsible for distal defects of the motor unit in SMA are unlikely to be similar to those causing neurodegeneration in genetic mutants such as the pmn mouse which is partially rescued by the Wld^s protein.     

Developmental changes in potassium currents at the rat calyx of Held presynaptic terminal

During early postnatal development, the calyx of Held synapse in the auditory brainstem of rodents undergoes a variety of morphological and functional changes. Among ionic channels expressed in the calyx, voltage-dependent K⁺ channels regulate transmitter release by repolarizing the nerve terminal. Here we asked whether voltage-dependent K⁺ channels in calyceal terminals undergo developmental changes, and whether they contribute to functional maturation of this auditory synapse. From postnatal day (P) 7 to P14, K⁺ currents became larger and faster in activation kinetics, but did not change any further to P21. Likewise, presynaptic action potentials became shorter in duration from P7 to P14 and remained stable thereafter. The density of presynaptic K⁺ currents, assessed from excised patch recording and whole-cell recordings with reduced [K⁺]_i, increased by 2–3-fold during the second postnatal week. Pharmacological isolation of K⁺ current subtypes using tetraethylammonium (1 m m ) and margatoxin (10 n m ) revealed that the density of Kv3 and Kv1 currents underwent a parallel increase, and their activation kinetics became accelerated by 2–3-fold. In contrast, BK currents, isolated using iberiotoxin (100 n m ), showed no significant change during the second postnatal week. Pharmacological block of Kv3 or Kv1 channels at P7 and P14 calyceal terminals indicated that the developmental changes of Kv3 channels contribute to the establishment of reliable action potential generation at high frequency, whereas those of Kv1 channels contribute to stabilizing the nerve terminal. We conclude that developmental changes in K⁺ currents in the nerve terminal contribute to maturation of high-fidelity fast synaptic transmission at this auditory relay synapse.     

Morphology of diaphragm neuromuscular junctions on different fibre types

Summary We hypothesize that the morphology of the neuromuscular junction on different muscle fibre types varies, reflecting differences in activation history. In the rat diaphragm muscle, we used a three-colour fluorescent immunocytochemical technique to simultaneously visualize (1) innervating axons and presynaptic nerve terminals, (2) motor endplates and (3) myosin heavy chain isoform expression (muscle fibre type). Laser-scanning confocal microscopy was then used to optically section the triple-labelled muscle fibres, and create three-dimensional views of the neuromuscular junction. Type I fibres were innervated by the smallest axons, and type IIa, IIx and IIb fibres by progressively larger axons. Absolute planar areas of nerve terminals and endplates progressively increased from type I, IIa, IIx to IIb fibres. When normalized for fibre diameter planar areas of nerve terminals were largest on type I fibres, with no difference among type II fibres. The normalized planar area of endplates were larger for type I and IIb fibres, compared to type IIa and IIx fibres. The three-dimensional surface area of endplates was largest on type I fibres, with no differences across type II fibres. When normalized for fibre diameter, endplate surface areas increase progressively from type I, IIa, IIx to IIb fibres. The branches increased progressively from type I, IIa, IIx to IIb fibres. Conversely, individual branch length was longest on type I fibres, and shortest on type IIb fibres. The extent of overlap of pre- and postsynaptic elements of the neuromuscular junction decreased progressively on type I, IIa, IIx and IIb fibres. We conclude that these morphological differences at the neuromuscular function of different fibre types reflect differences in activation history and may underlie phenotypic differences in neuromuscular transmission.     

High throughput protein expression screening in the nervous system – needs and limitations

The cellular complexity of the brain (some estimate that there are up to 10³ different cell types) is exceeded by the synaptic complexity, with each of the ∼10¹¹ neurons in the brain having around 10³–10⁴ synapses. Proteomic studies of the synapse have revealed that the postsynaptic density is the most complex multiprotein structure yet identified, with ∼10³ different proteins. Such studies, however, use brain tissue with many different regions and therefore different cell types, and there is clear potential for heterogeneity of protein content at different synapses within and between brain regions. Although large-scale mRNA-based assays are in progress to map this sort of complexity at the cellular level, and indeed all brain-expressed genes, analysis of protein distribution (at synapses and other structures) is still in the very early stages. We review existing large-scale protein expression studies and the specific technical obstacles that need to be overcome before applying the scaling used in nucleic acid based approaches.