Energy-optimal controls in the mammalian neuromuscular system

The hypothesis is advanced that the specific patterns of motor unit recruitment and stimulation frequencies observed in mammalian skeletal muscle under static isometric contractions are determined by a minimum-energy principle. By performing a constrained energy optimization based on a control model of skeletal muscle comprising three different fibre types, and appropriate expressions for the energy rates, it is indeed possible to obtain detailed predictions of recruitment and stimulation frequency patterns which agree well with the experimentally observed functions, thereby providing strong support for the minimum-energy hypothesis. Since the orderly recruitment sequence determined by the size principle is also, independently, predicted by the minimum-energy principle, it is concluded that there exists a relationship between motor unit size and the myoenergetic properties of the recruited unit. It is suggested that this relationship, together with the possibility of adjusting the relative proportions of the fibre types present in a muscle, constitutes an optimal adaptation of the neuromuscular system for practically all types of muscular performances normally encountered. For various types of muscles, the energy rates as functions of the force output are also discussed.     

Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction

Frog nerve-muscle preparations were quick-frozen at various times after a single electrical stimulus in the presence of 4-aminopyridine (4-AP), after which motor nerve terminals were visualized by freeze-fracture. Previous studies have shown that such stimulation causes prompt discharge of 3,000-6,000 synaptic vesicles from each nerve terminal and, as a result, adds a large amount of synaptic vesicle membrane to its plasmalemma. In the current experiments, we sought to visualize the endocytic retrieval of this vesicle membrane back into the terminal, during the interval between 1 s and 2 min after stimulation. Two distinct types of endocytosis were observed. The first appeared to be rapid and nonselective. Within the first few seconds after stimulation, relatively large vacuoles (approximately 0.1 micron) pinched off from the plasma membrane, both near to and far away from the active zones. Previous thin-section studies have shown that such vacuoles are not coated with clathrin at any stage during their formation. The second endocytic process was slower and appeared to be selective, because it internalized large intramembrane particles. This process was manifest first by the formation of relatively small (approximately 0.05 micron) indentations in the plasma membrane, which occurred everywhere except at the active zones. These indentations first appeared at 1 s, reached a peak abundance of 5.5/micron2 by 30 s after the stimulus, and disappeared almost completely by 90 s. Previous thin-section studies indicate that these indentations correspond to clathrin-coated pits. Their total abundance is comparable with the number of vesicles that were discharged initially. These endocytic structures could be classified into four intermediate forms, whose relative abundance over time suggests that, at this type of nerve terminal, endocytosis of coated vesicles has the following characteristics: (a) the single endocytotic event is short lived relative to the time scale of two minutes; (b) earlier forms last longer than later forms; and (c) a single event spends a smaller portion of its lifetime in the flat configuration soon after the stimulus than it does later on.     

Neuregulin: An activity-dependent synaptic modulator at the neuromuscular junction

Synaptic activity in the form of neurotransmitter release and postsynaptic depolarization is a prime motive force that guides synaptic development throughout the nervous system. The molecular basis of how synaptic activity is converted into structural changes that build and maintain synapses is a key question that has recently become focused on regulatory factors that act on tyrosine kinase receptors on both sides of the synaptic interface. The neuregulins are such a family of growth and differentiation factors that exist as both membrane-bound and soluble forms through alternatively splicing. Neuregulin functions to promote the local expression of acetylcholine receptors at neuromuscular synapses and therefore has the potential to strengthen specific synaptic connections. Recent evidence suggests that synaptic activity at the neuromuscular junction is coupled to presynaptic neuregulin release through an indirect mechanism acting through the postsynaptic expression of neurotrophic factors. At early stages of development, this could potentiate the stability of more active synapses. Later in development, heparin-binding forms of neuregulin accumulate to high levels in the synaptic basal lamina through the developmentally programmed expression of heparan sulfate proteoglycans, thus providing a sustained source of neuregulin to the most active synapses.     

Transient synaptic ribbons in the mammalian retina at unusual sites

In the vertebrate retina the presence of synaptic ribbons (SRs) is well documented in two sites only, viz., in photoreceptor axon terminals in the outer plexiform layer and in bipolar cell axons in the inner plexiform layer. The present paper reports the presence of non-photoreceptor SRs in the outer plexiform layer of cattle and mouse, where they were seen in small numbers in thin cell processes near cone pedicles of light-adapted animals. They were never seen near rod spherules. Quantitative data obtained in mice killed at different time-points revealed that the SRs under consideration increased in number during day time and were absent during the dark phase. Moreover, under high light intensity of 10000 lux they were more frequent in number compared to 100-lux-exposed animals. It is concluded that the cell processes revealing the temporary presence of SRs are processes of flat bipolar cells which may provide a feedback to cones during the light phase.     

Discrepancies between spontaneous and evoked synaptic potentials at normal, regenerating and botulinum toxin poisoned mammalian neuromuscular junctions

Amplitudes and times to peak of spontaneous miniature endplate potentials (m.e.p.ps) and evoked quantal endplate potentials (e.p.ps) were compared at normal, regenerating and botulinum toxin poisoned neuromuscular junctions of the extensor digitorum longus muscle of the rat. At normal junctions the mean time to peak of m.e.p.ps was longer and more variable than that of similar-sized e.p.ps. At endplates where nerve regeneration was induced by mechanical crushing of the motor nerve the frequency of m.e.p.ps was reduced and their amplitude distribution was broader than normal. The distribution of times to peak of m.e.p.ps was considerably broader than that of quantal e.p.ps recorded at the same endplates. At neuromuscular junctions poisoned with botulinum toxin type A, spontaneous and evoked transmitter release were greatly reduced. The amplitude distribution of m.e.p.ps was wider than that of e.p.ps and the time to peak of e.p.ps was about twice as fast as and less variable than that of m.e.p.ps. To explain the observed differences in time to peak among m.e.p.ps and between m.e.p.ps and quantal e.p.ps we suggest that some m.e.p.ps, but not e.p.ps, originate from transmitter quanta released from sites at a greater distance from postsynaptic receptors or that the release or diffusion process for acetylcholine is more prolonged when producing some of the m.e.p.ps. Such mechanisms produce at normal junctions a small population of m.e.p.ps with prolonged times to peak, at regenerating junctions a greater proportion of such m.e.p.ps and in botulinum toxin poisoning a majority.     

Pervasive adaptive evolution in mammalian fertilization proteins

Mammalian fertilization exhibits species specificity, and the proteins mediating sperm-egg interactions evolve rapidly between species. In this study, we demonstrate that the evolution of seven genes involved in mammalian fertilization is promoted by positive Darwinian selection by using likelihood ratio tests (LRTs). Several of these proteins are sperm proteins that have been implicated in binding the mammalian egg coat zona pellucida glycoproteins, which were shown previously to be subjected to positive selection. Taken together, these represent the major candidates involved in mammalian fertilization, indicating positive selection is pervasive amongst mammalian reproductive proteins. A new LRT is implemented to determine if the d(N)/d(S) ratio is significantly greater than one. This is a more refined test of positive selection than the previous LRTs which only identified if there was a class of sites with a d(N)/d(S) ratio >1 but did not test if that ratio was significantly greater than one.     

Glial processes at the Drosophila larval neuromuscular junction match synaptic growth

Glia are integral participants in synaptic physiology, remodeling and maturation from blowflies to humans, yet how glial structure is coordinated with synaptic growth is unknown. To investigate the dynamics of glial development at the Drosophila larval neuromuscular junction (NMJ), we developed a live imaging system to establish the relationship between glia, neuronal boutons, and the muscle subsynaptic reticulum. Using this system we observed processes from two classes of peripheral glia present at the NMJ. Processes from the subperineurial glia formed a blood-nerve barrier around the axon proximal to the first bouton. Processes from the perineurial glial extended beyond the end of the blood-nerve barrier into the NMJ where they contacted synapses and extended across non-synaptic muscle. Growth of the glial processes was coordinated with NMJ growth and synaptic activity. Increasing synaptic size through elevated temperature or the highwire mutation increased the extent of glial processes at the NMJ and conversely blocking synaptic activity and size decreased the presence and size of glial processes. We found that elevated temperature was required during embryogenesis in order to increase glial expansion at the nmj. Therefore, in our live imaging system, glial processes at the NMJ are likely indirectly regulated by synaptic changes to ensure the coordinated growth of all components of the tripartite larval NMJ.     

Moesin helps to restrain synaptic growth at the Drosophila neuromuscular junction

The precise role of actin and actin-binding proteins in synaptic development is unclear. In Drosophila, overexpression of a dominant-negative NSF2 construct perturbs filamentous actin, which is associated with overgrowth of the NMJ, while co-expression of moesin, which encodes an actin binding protein, suppresses this overgrowth phenotype. These data suggest that Moesin may play a role in synaptic development at the Drosophila NMJ. To further investigate this possibility, we examined the influence of loss-of-function moesin alleles on the NSF2-induced overgrowth phenotype. We found that flies carrying P-element insertions that reduce moesin expression enhanced the NMJ overgrowth phenotype, indicating a role for Moesin in normal NMJ morphology. In addition to the NMJ overgrowth phenotype, expression of dominant-negative NSF2 is known to reduce the frequency of miniature excitatory junctional potentials and the amplitude of excitatory junctional potentials. We found that moesin coexpression did not restore the physiology of the mutant NSF2 phenotype. Together, our results demonstrate a role for moesin in regulating synaptic growth in the Drosophila NMJ and suggest that the effect of dominant-negative NSF2 on NMJ morphology and physiology may have different underlying molecular origins.     

Rapid introduction of long-lasting synaptic changes at crustacean neuromuscular junctions

In this review we present recent evidence implicating second-messenger systems in two forms of long-lasting synaptic change seen at crustacean neuromuscular junctions. Crustacean motor axons are endowed with numerous terminals, each possessing many individual synapses. Some synapses appear to be quiescent or impotent, but can be recruited in response to imposed functional demands. Supernormal impulse activity leads to long-term facilitation (LTF) which persists for many hours. During the persistent phase, additional synapses are physiologically effective, and morphological changes in synapses are seen at the ultrastructural level. Pulsatile application of serotonin, a neuromodulator, also enhances synaptic transmission, but this enhancement declines more rapidly than LTF. Elevation of intraterminal Ca2+ is neither necessary nor sufficient for long-lasting enhancement of transmission, but activation of A-kinase is necessary. LTF is set in motion by an unknown depolarization-dependent mechanism leading to A-kinase activation, whereas serotonin facilitation depends for its initiation on the phosphatidylinositol system. The initial phase of serotonin facilitation may be accounted for by production of inositol triphosphate, whereas the secondary long-lasting phase appears to require participation of both C kinase and A kinase. Neither LTF nor serotonin facilitation requires an intact neuron; both are presynaptic phenomena expressed by the nerve terminals. Brief comparison is made with long-lasting synaptic changes in other systems.     

Structural determinants of the reliability of synaptic transmission at the vertebrate neuromuscular junction

Brain Cell Biology - The reliability of neuromuscular transmission depends on the size and molecular organization of the neuromuscular junction. Comparative studies show that the quantal release...     

Axon branch removal at developing synapses by axosome shedding

In many parts of the developing nervous system, the number of axonal inputs to each postsynaptic cell is dramatically reduced. This synapse elimination has been extensively studied at the neuromuscular junction, but how axons are lost is unknown. Here, we combine time-lapse imaging of fluorescently labeled axons and serial electron microscopy to show that axons at neuromuscular junctions are removed by an unusual cellular mechanism. As axons disappear, they shed numerous membrane bound remnants. These "axosomes" contain a high density of synaptic organelles and are formed by engulfment of axon tips by Schwann cells. After this engulfment, the axosome's contents mix with the cytoplasm of the glial cell. Axosome shedding might underlie other forms of axon loss and may provide a pathway for interactions between axons and glia.     

Essential role of endophilin A in synaptic vesicle budding at the Drosophila neuromuscular junction

We characterized Drosophila endophilin A (D-endoA), and generated and analysed D-endoA mutants. Like its mammalian homologue, D-endoA exhibits lysophosphatidic acid acyl transferase activity and contains a functional SH3 domain. D-endoA is recruited to the sites of endocytosis, as revealed by immunocytochemistry of the neuromuscular junction (NMJ) of mutant L3 larvae carrying the temperature-sensitive allele of dynamin, shibire. D-endoA null mutants show severe defects in motility and die at the early L2 larval stage. Mutants with reduced D-endoA levels exhibit a range of defects of synaptic vesicle endocytosis, as observed at L3 larvae NMJs using FM1-43 uptake and electron microscopy. NMJs with an almost complete loss of synaptic vesicles did not show an accumulation of intermediates of the budding process, whereas NMJs with only slightly reduced levels of synaptic vesicles showed a striking increase in early-stage, but not late-stage, budding intermediates at the plasma membrane. Together with results of previous studies, these observations indicate that endophilin A is essential for synaptic vesicle endocytosis, being required from the onset of budding until fission.     

Retrograde control of synaptic transmission by postsynaptic CaMKII at the Drosophila neuromuscular junction

Retrograde signaling plays an important role in synaptic homeostasis, growth, and plasticity. A retrograde signal at the neuromuscular junction (NMJ) of Drosophila controls the homeostasis of neurotransmitter release. Here, we show that this retrograde signal is regulated by the postsynaptic activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Reducing CaMKII activity in muscles enhances the signal and increases neurotransmitter release, while constitutive activation of CaMKII in muscles inhibits the signal and decreases neurotransmitter release. Postsynaptic inhibition of CaMKII increases the number of presynaptic, vesicle-associated T bars at the active zones. Consistently, we show that glutamate receptor mutants also have a higher number of T bars; this increase is suppressed by postsynaptic activation of CaMKII. Furthermore, we demonstrate that presynaptic BMP receptor wishful thinking is required for the retrograde signal to function. Our results indicate that CaMKII plays a key role in the retrograde control of homeostasis of synaptic transmission at the NMJ of Drosophila.     

The Drosophila miR-310 cluster negatively regulates synaptic strength at the neuromuscular junction

Emerging data implicate microRNAs (miRNAs) in the regulation of synaptic structure and function, but we know little about their role in the regulation of neurotransmission in presynaptic neurons. Here, we demonstrate that the miR-310-313 cluster is required for normal synaptic transmission at the Drosophila larval neuromuscular junction. Loss of miR-310-313 cluster leads to a significant enhancement of neurotransmitter release, which can be rescued with temporally restricted expression of mir-310-313 in larval presynaptic neurons. Kinesin family member, Khc-73 is a functional target for miR-310-313 as its expression is increased in mir-310-313 mutants and reducing it restores normal synaptic function. Cluster mutants show an increase in the active zone protein Bruchpilot accompanied by an increase in electron dense T bars. Finally, we show that repression of Khc-73 by miR-310-313 cluster influences the establishment of normal synaptic homeostasis. Our findings establish a role for miRNAs in the regulation of neurotransmitter release.