Mitochondrial clustering at the vertebrate neuromuscular junction during presynaptic differentiation

During vertebrate neuromuscular junction (NMJ) development, presynaptic motor axons differentiate into nerve termini enriched in synaptic vesicles (SVs). At the nerve terminal, mitochondria are also concentrated, but how mitochondria become localized at these specialized domains is poorly understood. This process was studied in cultured Xenopus spinal neurons with mitochondrion-specific probe MitoTracker and SV markers. In nerve-muscle cocultures, mitochondria were concentrated stably at sites where neurites and muscle cells formed NMJs, and mitochondria coclustered with SVs where neurites were focally stimulated by beads coated with growth factors. Labeling with a mitochondrial membrane potential-dependent probe JC-1 revealed that these synaptic mitochondria were with higher membrane potential than the extrasynaptic ones. At early stages of bead-stimulation, actin-based protrusions and microtubule fragmentation were observed in neurites at bead contact sites, suggesting the involvement of cytoskeletal dynamics and rearrangement during presynaptic differentiation. Treating the cultures with an actin polymerization blocker, latrunculin A (Ltn A), almost completely abolished the formation of actin-based protrusions and partially inhibited bead-induced mitochondrial and SV clustering, whereas the microtubule disrupting agent nocodazole was ineffective in inhibiting the clustering of mitochondria and SVs. Lastly, in contrast to Ltn A, which blocked bead-induced clustering of both mitochondria and SVs, the ser/thr phosphatase inhibitor okadaic acid inhibited SV clustering but not mitochondrial clustering. These results suggest that at developing NMJs, synaptogenic stimuli induce the clustering of mitochondria together with SVs at presynaptic terminals in an actin cytoskeleton-dependent manner and involving different intracellular signaling molecules.     

Presynaptic mitochondria and the temporal pattern of neurotransmitter release

Mitochondria are critical for the function of nerve terminals as the cycling of synaptic vesicle membrane requires an efficient supply of ATP. In addition, the presynaptic mitochondria take part in functions such as Ca2+ buffering and neurotransmitter synthesis. To learn more about presynaptic mitochondria, we have examined their organization in two types of synapse in the lamprey, both of which are glutamatergic but are adapted to different temporal patterns of activity. The first is the giant lamprey reticulospinal synapse, which is specialized to transmit phasic signals (i.e. bursts of impulses). The second is the synapse established by sensory dorsal column axons, which is adapted to tonic activity. In both cases, the presynaptic axons were found to contain two distinct types of mitochondria; small 'synaptic' mitochondria, located near release sites, and larger mitochondria located in more central parts of the axon. The size of the synapse-associated mitochondria was similar in both types of synapse. However, their number differed considerably. Whereas the reticulospinal synapses contained only single mitochondria within 1 micron distance from the edge of the active zone (on average 1.2 per active zone, range of 1-3), the tonic dorsal column synapses were surrounded by clusters of mitochondria (4.5 per active zone, range of 3-6), with individual mitochondria sometimes apparently connected by intermitochondrial contacts. In conjunction with studies of crustacean neuromuscular junctions, these observations indicate that the temporal pattern of transmitter release is an important determinant of the organization of presynaptic mitochondria.     

Regulation of synaptic vesicle accumulation and axon terminal remodeling during synapse formation by distinct Ca2+ signaling

The synaptic vesicle accumulation and subsequent morphological remodeling of axon terminals are characteristic features of presynaptic differentiation of zebrafish olfactory sensory neurons. The synaptic vesicle accumulation and axon terminal remodeling are regulated by protein kinase A and calcineurin signaling, respectively. To investigate upstream signals of presynaptic differentiation, we focused on Ca²⁺ signaling as Ca²⁺/calmodulin is required for the activation of both calcineurin and some adenylyl cyclases. We here showed that application of Ca²⁺/calmodulin inhibitor or olfactory sensory neuron-specific expression of calmodulin inhibitory peptide suppressed both synaptic vesicle accumulation and axon terminal remodeling. Thus, the trigger of presynaptic differentiation could be Ca²⁺ release from intracellular stores or Ca²⁺ influx. Application of a phospholipase C inhibitor or olfactory sensory neuron-specific expression of inositol 1,4,5-trisphosphate (IP₃) 5-phosphatase suppressed synaptic vesicle accumulation, but not morphological remodeling. In contrast, application of a voltage-gated Ca²⁺ channel blocker or expression of Kir2.1 inward rectifying potassium channel prevented the morphological remodeling. We also provided evidence that IP₃ signaling acted upstream of protein kinase A signaling. Our results suggest that IP₃-mediated Ca²⁺/calmodulin signaling stimulates synaptic vesicle accumulation and subsequent neuronal activity-dependent Ca²⁺/calmodulin signaling induces the morphological remodeling of axon terminals.     

Dense cored vesicles in presynaptic profiles of the rabbit dorsal lateral geniculate nucleus

We are carrying out a study about the synaptic relations between identified synaptic profiles in the dorsal lateral geniculate nucleus (dLGN) of the rabbit. Here, the types of synaptic vesicle containing profiles of the dLGN are described. There are presynaptic large profiles containing round vesicles and pale mitochondria (RLP terminals) and small profiles that contain round vesicles and dark mitochondria (RSD terminals) which respectively arise from the retina and the visual cortex. Another type of presynaptic profile contains elliptical vesicles (F-boutons) which can be subdivided according to their cytoplasmic content. These F-boutons arise from dLGN interneurons. We have found different sized vesicles that have a dense core within RLP, and F terminals and a possible RSD terminal. The significance of the coexistance of pale and dense cored vesicles in the presynaptic profiles of the rabbit dLGN is discussed.     

The synaptic organization of visual interneurons in the lobula complex of flies

Summary The synaptic organization of three classes of cobalt-filled and silver-intensified visual interneurons in the lobula complex of the blowfly Calliphora (Col A cells, horizontal cells and vertical cells) was studied electron microscopically. The Col A cells are regularly spaced, columnar, small field neurons of the lobula, which constitute a plexus of arborizations at the posterior surface of the neuropil and the axons of which terminate in the ventrolateral protocerebrum. They show postsynaptic specializations in the distal layer of their lobula-arborizations and additional presynaptic sites in a more proximal layer; their axon terminals are presynaptic to large descending neurons projecting into the thoracic ganglion. The horizontal and vertical cells are giant tangential neurons, the arborizations of which cover the anterior and posterior surface of the lobula plate, respectively, and which terminate in the perioesophageal region of the protocerebrum. Both classes of these giant neurons were found to be postsynaptic in the lobula plate and pre- and postsynaptic at their axon terminals and axon collaterals. The significance of these findings with respect to the functional properties of the neurons investigated is discussed.     

The axosomatic contacts on the bursting neuron of the snailHelix pomatia. I. ultrastructural features of the axosomatic contacts

The analysis of serial ultrathin sections of the RPAI bursting neuron of the snail Helix pomatia reveals the presence of axosomatic contacts on its surface membrane. These contacts have a number of specific features: the presynaptic axon contains synaptic vesicles and electron-dense granules, typical of peptidergic terminals; the terminal part of the axon forms many finger-like processes which invaginate the neuronal soma; the width of the cleft (80 nm) in the area of the contact is larger than that in usual synaptic contacts; and there is a system of lacoons in the region of the axosomatic contact; this system is formed by protrusions of the soma and it accompanies the contact along its extent. It is suggested that the system of lacoons which communicates with the space between the terminal and the soma may serve as a ramified synaptic cleft into which the secretion from the terminal is released. This system may contribute to a considerable prolongation of the time of action of the secretory product on the membrane of the RPAI neuron.     

Central Presynaptic Terminals Are Enriched in ATP but the Majority Lack Mitochondria

Synaptic neurotransmission is known to be an energy demanding process. At the presynapse, ATP is required for loading neurotransmitters into synaptic vesicles, for priming synaptic vesicles before release, and as a substrate for various kinases and ATPases. Although it is assumed that presynaptic sites usually harbor local mitochondria, which may serve as energy powerhouse to generate ATP as well as a presynaptic calcium depot, a clear role of presynaptic mitochondria in biochemical functioning of the presynapse is not well-defined. Besides a few synaptic subtypes like the mossy fibers and the Calyx of Held, most central presynaptic sites are either en passant or tiny axonal terminals that have little space to accommodate a large mitochondrion. Here, we have used imaging studies to demonstrate that mitochondrial antigens poorly co-localize with the synaptic vesicle clusters and active zone marker in the cerebral cortex, hippocampus and the cerebellum. Confocal imaging analysis on neuronal cultures revealed that most neuronal mitochondria are either somatic or distributed in the proximal part of major dendrites. A large number of synapses in culture are devoid of any mitochondria. Electron micrographs from neuronal cultures further confirm our finding that the majority of presynapses may not harbor resident mitochondria. We corroborated our ultrastructural findings using serial block face scanning electron microscopy (SBFSEM) and found that more than 60% of the presynaptic terminals lacked discernible mitochondria in the wild-type mice hippocampus. Biochemical fractionation of crude synaptosomes into mitochondria and pure synaptosomes also revealed a sparse presence of mitochondrial antigen at the presynaptic boutons. Despite a low abundance of mitochondria, the synaptosomal membranes were found to be highly enriched in ATP suggesting that the presynapse may possess alternative mechanism/s for concentrating ATP for its function. The potential mechanisms including local glycolysis and the possible roles of ATP-binding synaptic proteins such as synapsins, are discussed.     

Regulation of Synaptic Transmission by Mitochondrial Ion Channels

Mitochondria are abundant within neuronal presynaptic terminals, where they provide energy for sustained neurotransmitter secretion. Injection of Bcl-xL protein into squid giant presynaptic terminal potentiates neurotransmitter release, while a naturally occurring, proteolytic fragment of BCL-xL causes rundown of synaptic function. The cleaved form of BCL-xL generates large, multiconductance ion channel activity in synaptic mitochondrial outer membranes. A rapid onset of synaptic rundown can also be produced by depriving the synapse of oxygen, and hypoxia also induces large channel activity in mitochondrial outer membranes. Channel activity induced by cleaved BCL-xL or by hypoxia is attenuated by NADH, an inhibitor of the voltage-dependent anion channel (VDAC) of mitochondrial outer membranes. Finally, the large conductances elicited by hypoxia are prevented by the addition of a protease inhibitor that prevents cleavage of BCL-xL. The opposing activities of BCL-xL and its proteolytic fragment may regulate the release of ATP from mitochondria during synaptic transmission.     

Terminal Axonal Arborization and Synaptic Bouton Formation Critically Rely on Abp1 and the Arp2/3 Complex

Neuronal network formation depends on properly timed and localized generation of presynaptic as well as postsynaptic structures. Although of utmost importance for understanding development and plasticity of the nervous system and neurodegenerative diseases, the molecular mechanisms that ensure the fine-control needed for coordinated establishment of pre- and postsynapses are still largely unknown. We show that the F-actin-binding protein Abp1 is prominently expressed in the Drosophila nervous system and reveal that Abp1 is an important regulator in shaping glutamatergic neuromuscular junctions (NMJs) of flies. STED microscopy shows that Abp1 accumulations can be found in close proximity of synaptic vesicles and at the cell cortex in nerve terminals. Abp1 knock-out larvae have locomotion defects and underdeveloped NMJs that are characterized by a reduced number of both type Ib synaptic boutons and branches of motornerve terminals. Abp1 is able to indirectly trigger Arp2/3 complex-mediated actin nucleation and interacts with both WASP and Scar. Consistently, Arp2 and Arp3 loss-of-function also resulted in impairments of bouton formation and arborization at NMJs, i.e. fully phenocopied abp1 knock-out. Interestingly, neuron- and muscle-specific rescue experiments revealed that synaptic bouton formation critically depends on presynaptic Abp1, whereas the NMJ branching defects can be compensated for by restoring Abp1 functions at either side. In line with this presynaptic importance of Abp1, also presynaptic Arp2 and Arp3 are crucial for the formation of type Ib synaptic boutons. Interestingly, presynaptic Abp1 functions in NMJ formation were fully dependent on the Arp2/3 complex, as revealed by suppression of Abp1-induced synaptic bouton formation and branching of axon terminals upon presynaptic Arp2 RNAi. These data reveal that Abp1 and Arp2/3 complex-mediated actin cytoskeletal dynamics drive both synaptic bouton formation and NMJ branching. Our data furthermore shed light on an intense bidirectional functional crosstalk between pre- and postsynapses during the development of synaptic contacts.     

The fine structure of the nerve cord of Myxicola infundibulum (annelida,polychaeta)

Summary Ultrastructural observations of the giant axon of Myxicola infundibulum reveal that the axoplasm contains neurofilaments, a few neurotubules and mitochondria. Finger-like projections issuing from the glial cells of the sheath encircle the giant axon at various angles. The space between the axolemma and sheath is 125 Å. Branches of the giant axon are also surrounded by a glial sheath as they course through the neuropil. Some branches of the giant axon seem to fuse with certain neurons, creating a syncytial arrangement between the giant axon and these neurons.Many small nerve fibers course longitudinally in the neuropil of the nerve cord. Most of these axons are separated from each other by a space of 200 Å without intervening glial processes. Synapses in the neuropil have both clear 600 Å vesicles and larger dense core vesicles suggesting chemical transmission. Some, but not all, of the synaptic areas show thickened membranes and dense material in the synaptic cleft.This study was supported in part by PHS NS-07740 to R.L.P., J.A.B. is a NDEA Predoctoral Fellow in the Department of Physiology.     

大白鼠脑干听觉中枢的研究下丘中心核内神经突触的特点

1.大白鼠下丘中心核(the Central Nucleus of the Inferior Colliculus,ICCN)内神经末稍以群体的形式有在,神经突触排列的类型主要为系列突触.2.末稍群体(Clustered ending)中轴突终末内含有多种类型的突触小泡.3.ICCN内具有不对称突触与对称突触两种类型的突触结构.4.在ICCN内,突触前终末有大量的突触小泡聚集,并且在突触后常有1—2个大线粒体靠近突触后膜.5.以上结果表明了脑干听觉中枢下丘中心核的结构及其突触连结的模式;突触的结构及其特点,这是频有意义的.     

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.

Neuromuscular junctions (NMJs) are a favorite model system to study the development, maintenance, and function of neuronal synapses because of their accessibility, size, and simplicity. Although many synaptic mechanisms discovered at the peripheral NMJ have provided important insights into synaptic mechanisms in the central nervous system (CNS), the phenomena of synapse elimination and refinement remain poorly understood in both. In the peripheral nervous system (PNS), synapse elimination is an essential developmental step that removes redundant presynaptic inputs to the muscle fiber. In addition, peripheral motor axon terminals must become myelinated to facilitate rapid and synchronized acetylcholine release to the muscle fiber. However, whether these two essential events during PNS development are coordinately regulated remains unknown.The immature rodent NMJ is first innervated by many axons which are then removed until the synapse reaches a dually innervated state (1). These two axons then further compete for synaptic territory, leaving one “winner” that eventually occupies the motor endplate by the end of the second postnatal week. To determine the relationship between synapse elimination and myelination, Wang et al. (2) used the formation of paranodal junctions between axons and Schwann cells as a surrogate for myelination and then determined whether axons that occupied NMJs in a singly or dually innervated state were more or less likely to be myelinated. They found that when the NMJ is dually innervated, myelination of the terminal axon branch is inhibited; neither synaptic occupancy of the competing axons nor axon diameter influenced myelination. However, once synapse elimination at the NMJ is complete, i.e., a single axon terminal innervates the motor endplate, the winner branch becomes myelinated. Thus, synapse elimination precedes myelination of the terminal axon branch, and competition between dually innervated NMJs restricts myelination.What mechanisms regulate the coordinated maturation of the motor neuron, Schwann cell, and muscle circuit? Since previous studies showed that synapse elimination at the NMJ depends on muscle activity (3), Wang et al. (2) inhibited synapse elimination by blocking acetylcholine receptors with α-bungarotoxin (α-Btx). This inhibition of motor endplate and muscle activity increased not only the number of dually innervated NMJs, but also significantly decreased myelination of terminal axon branches of singly innervated NMJs. Thus, neuromuscular activity must induce retrograde signaling mechanisms that promote not only synapse elimination but also myelination.During synapse elimination, the microtubule cytoskeleton of retracting axons is degraded and reduced (4). In contrast, axons that singly innervate NMJs have a higher microtubule content. α-Btx–dependent block of neuromuscular transmission reduced microtubule content in axons that singly innervate NMJs. Thus, α-Btx treatment simultaneously reduces both microtubule content and myelination.To determine if a mature microtubule-based cytoskeleton is causally related to myelination, Wang et al. used spastin knockout (spastin^KO) mice to artificially stabilize microtubules. Although spastin^KO mice had delayed axon branch removal, stabilization of the microtubule cytoskeleton increased myelination of axons that dually innervated NMJs. Thus, the brake that synaptic competition normally places on terminal branch myelination can be overcome by increasing the mass and maturity of the microtubule cytoskeleton.How does axonal microtubule stability influence terminal axon myelination? Microtubules participate in the anterograde and retrograde transport of diverse cargoes including mitochondria and growth factors. To determine if anterograde axonal transport promotes myelination of axons that singly innervate NMJs, Wang et al. used a dominant-negative mutant of kinesin-1 heavy chain which binds cargo, but lacks the protein’s motor domain, thereby impairing transport. After confirming transport inhibition by tracking impaired movement of the β1 subunit of voltage gated sodium channels, they found that myelination and node of Ranvier formation were significantly delayed in singly innervated NMJs expressing the dominant negative kinesin. Taken together, these results suggest that synapse elimination promotes maturation of the microtubule cytoskeleton which allows more efficient delivery of promyelinating signals to the terminal branch.What could these promyelinating signals be? One obvious candidate is Neuregulin 1 type III (Nrg1-III), which has long been known to promote myelination of peripheral nervous system axons (5). Consistent with this idea, conditional deletion of Nrg1-III dramatically reduced the number of myelinated axon terminals that singly innervate NMJs but did not alter the number of dually innervated NMJs. In contrast, overexpression of Nrg1-III in a transgenic mouse removed the competition-dependent block on myelination resulting in more myelination of both dually and singly innervating axon terminals. In these same transgenic Nrg1-III mice, among those NMJs that were singly innervated, their corresponding axons had higher levels of Nrg1-III. Remarkably, even in these same transgenic overexpressers, inhibition of muscle activity reduced the amount of Nrg1-III found on singly innervated axons, consistent with the observed impairment of the microtubule-based cytoskeleton after α-Btx treatment. ERK1/2 and AKT are downstream effectors of Nrg1-III in Schwann cells and implicated in the myelination pathway. Immunostaining of Schwann cells ensheathing singly innervating axon terminals revealed higher levels of pERK and pAKT.Taken together, the experiments performed by Wang et al. (2) suggest that as multiple axons actively compete for synaptic dominance at the NMJ, the myelination of their terminal branches is delayed. Upon synapse elimination, neuromuscular activity promotes a retrograde signal that increases maturity of the microtubule cytoskeleton. Maturation of the microtubule-based cytoskeleton facilitates the transport of promyelinating signals like Nrg1-III which, when presented to Schwann cells, results in myelination of the “winner” terminal axon branch of a singly innervated NMJ (Fig. 1).Open in a separate windowFigure 1.Synapse elimination promotes myelination of terminal motor axon branches. During early development, NMJs are innervated by multiple axons that compete for endplate territory. During this time, the terminal branches of the axons are not myelinated, and the tubulin cytoskeletal network remains immature. Synaptic activity induces elimination of redundant connections, which leads the winner axon’s microtubule-based cytoskeleton to mature and increase, while the microtubule cytoskeleton is degraded in the retracting axon. The maturity of the cytoskeleton allows for kinesin dependent anterograde transport of Neuregulin 1-III, which then initiates a promyelination signaling cascade via AKT and ERK activation.To the best of our knowledge, this is the first demonstration of plasticity of myelination downstream of activity and synapse refinement in the peripheral motor nervous system. Many studies in the CNS demonstrate that de novo myelination occurs in response to neuronal activity and learning paradigms (6, 7), although the mechanisms responsible remain unknown. Thus, synapse refinement and elimination-dependent myelination may be a paradigm to uncover mechanisms of learning- and activity-dependent myelination in the CNS. Functionally, the addition of myelin to the terminal motor axon branch promotes efficient neurotransmitter release through faster action potential propagation, improved metabolic support of the axon, and more efficient depolarization of the presynaptic terminal by clustered Na⁺ channels at the terminal heminode (8). Whether any or all of these benefits also exist in the CNS remains unknown.This is also the first demonstration of postsynaptic activity driving myelination of a presynaptic axon. Although it is clear that a retrograde signal from the muscle promotes the further maturation and subsequent myelination of the terminal axon, the identity of this cue is unknown. One interesting candidate for a muscle-derived competition and axonal maturation cue is the neurotrophin brain-derived neurotrophic factor (BDNF), which is released during muscle activity (9). Consistent with this idea, BDNF promotes axon maturation by stimulating both actin polymerization and microtubule assembly (10). It will be interesting to test the role of trophic factors in activity-dependent synapse elimination and subsequent myelination in both the CNS and PNS.In conclusion, Wang et al. (2) is an excellent addition to a growing body of research that demonstrates how neuronal activity promotes and modulates myelination. Furthermore, it stands as another example of how using simple model systems, such as the NMJ, may provide insights and have important implications for much more complicated biological systems.     

Miro1‐dependent mitochondrial positioning drives the rescaling of presynaptic Ca2+ signals during homeostatic plasticity

Mitochondrial trafficking is influenced by neuronal activity, but it remains unclear how mitochondrial positioning influences neuronal transmission and plasticity. Here, we use live cell imaging with the genetically encoded presynaptically targeted Ca²⁺ indicator, SyGCaMP5, to address whether presynaptic Ca²⁺ responses are altered by mitochondria in synaptic terminals. We find that presynaptic Ca²⁺ signals, as well as neurotransmitter release, are significantly decreased in terminals containing mitochondria. Moreover, the localisation of mitochondria at presynaptic sites can be altered during long‐term activity changes, dependent on the Ca²⁺‐sensing function of the mitochondrial trafficking protein, Miro1. In addition, we find that Miro1‐mediated activity‐dependent synaptic repositioning of mitochondria allows neurons to homeostatically alter the strength of presynaptic Ca²⁺ signals in response to prolonged changes in neuronal activity. Our results support a model in which mitochondria are recruited to presynaptic terminals during periods of raised neuronal activity and are involved in rescaling synaptic signals during homeostatic plasticity.     

ELECTRON MICROSCOPE HISTOCHEMICAL EVIDENCE FOR A PARTIAL OR TOTAL BLOCK OF THE TRICARBOXYLIC ACID CYCLE IN THE MITOCHONDRIA OF PRESYNAPTIC AXON TERMINALS

Respiration-linked, massive accumulation of Sr²⁺ is used to reveal the coupled oxidation of pyruvate, α-oxoglutarate, succinate, and malate by in situ mitochondria. All of these substrates were actively oxidized in the dendritic and perikaryal mitochondria, but no α-oxoglutarate or succinate utilization could be demonstrated in the mitochondria of the presynaptic axon terminals. A block at an early step of α-oxoglutarate and succinate oxidation is proposed to account for the negative histochemical results, since the positive reaction with pyruvate and malate proves that these mitochondria possess an intact respiratory chain and energy-coupling mechanism essential for Sr²⁺ accumulation. This indicates that the mitochondria in the axon terminals would be able to generate energy for synaptic function with at least some of the respiratory substrates. With regard to the block in the tricarboxylic acid cycle, the oxaloacetate necessary for citrate formation is suggested to be provided by fixation of CO₂ into some of the pyruvate.     

Identified motor terminals in Drosophila larvae show distinct differences in morphology and physiology

In Drosophila, the type I motor terminals innervating the larval ventral longitudinal muscle fibers 6 and 7 have been the most popular preparation for combining synaptic studies with genetics. We have further characterized the normal morphological and physiological properties of these motor terminals and the influence of muscle size on terminal morphology. Using dye-injection and physiological techniques, we show that the two axons supplying these terminals have different innervation patterns: axon 1 innervates only muscle fibers 6 and 7, whereas axon 2 innervates all of the ventral longitudinal muscle fibers. This difference in innervation pattern allows the two axons to be reliably identified. The terminals formed by axons 1 and 2 on muscle fibers 6 and 7 have the same number of branches; however, axon 2 terminals are approximately 30% longer than axon 1 terminals, resulting in a corresponding greater number of boutons for axon 2. The axon 1 boutons are approximately 30% wider than the axon 2 boutons. The excitatory postsynaptic potential (EPSP) produced by axon 1 is generally smaller than that produced by axon 2, although the size distributions show considerable overlap. Consistent with vertebrate studies, there is a correlation between muscle fiber size and terminal size. For a single axon, terminal area and length, the number of terminal branches, and the number of boutons are all correlated with muscle fiber size, but bouton size is not. During prolonged repetitive stimulation, axon 2 motor terminals show synaptic depression, whereas axon 1 EPSPs facilitate. The response to repetitive stimulation appears to be similar at all motor terminals of an axon.     

SMN requirement for synaptic vesicle, active zone and microtubule postnatal organization in motor nerve terminals

Low levels of the Survival Motor Neuron (SMN) protein produce Spinal Muscular Atrophy (SMA), a severe monogenetic disease in infants characterized by muscle weakness and impaired synaptic transmission. We report here severe structural and functional alterations in the organization of the organelles and the cytoskeleton of motor nerve terminals in a mouse model of SMA. The decrease in SMN levels resulted in the clustering of synaptic vesicles (SVs) and Active Zones (AZs), reduction in the size of the readily releasable pool (RRP), and the recycling pool (RP) of synaptic vesicles, a decrease in active mitochondria and limiting of neurofilament and microtubule maturation. We propose that SMN is essential for the normal postnatal maturation of motor nerve terminals and that SMN deficiency disrupts the presynaptic organization leading to neurodegeneration.     

The colocalization of neurotransmitters in the presynaptic boutons of inhibitory synapses in the lamprey spinal cord]

Using the electron microscopy immunocytochemistry, the GABA and glycine immunoreactivity was studied in presynaptic axon terminals of the spinal cord central gray in the lamprey Lampetra fluviatilis. All immunopositive presynaptic terminals contacting motoneurones or non-identified post-synaptic profiles were divided into only GABA- (44%), only glycine-immunopositive terminals (26%), and both GABA- and glycine-containing terminals (30%). The glycine-immunopositive axon terminals contained flattened synaptic vesicles. Large dense core vesicles were co-localised with conventional synaptic vesicles in 74% of GABA-containing presynaptic terminals.     

SYNAPSES IN THE CENTRAL NERVOUS SYSTEM

A number of different synapses have been described in the medulla, cerebellar cortex, and cerebral cortex of the rat. All of these possess the same fundamental fine structure as follows: 1. Close apposition of the limiting membranes of presynaptic and postsynaptic cells without any protoplasmic continuity across the synapse. The two apposed membranes are separated by a cleft about 200 A wide, and display localized regions of thickening and increased density. 2. The presynaptic expansion of the axon, the end-foot or bouton terminal, contains a collection of mitochondria and clusters of small vesicles about 200 to 650 A in diameter. Although the significance of these structures in the physiology of the synapse is still unknown, two suggestions are made: that the mitochondria, by means of the relation between their enzymatic activity and ion transport, participate in the electrical phenomena about the synapse; and that the small synaptic vesicles provide the morphological representation of the prejunctional, subcellular units of neurohumoral discharge at the synapse demanded by physiological evidence.     

Programmed cell death: Cytochemical and X-ray microanalytical characterization of calcium compartments in neuromuscular junctions during the normal breakdown of the intersegmental muscles in the giant silkmothAntheraea polyphemus

Summary Calcium stores were cytochemically demonstrated using a combined oxalate—pyroantimonate method in the neuromuscular junctions of the degenerating intersegmental muscles in the giant silkmothAntheraea polyphemus. The elemental composition of punctate precipitates of the reaction product was determined by electron probe X-ray microanalysis of unstained thin sections by energy-dispersive spectrometry and wavelength-dispersive spectrometry. The wavelength-dispersive spectra collected over terminal axons demonstrate a significant calcium signal and a trace of antimony.During the rapid lytic phase of spontaneous muscle degeneration, the calcium punctate deposits were detected in presynaptic terminals in the following sites: the synaptic vesicles and the mitochondria. Calcium precipitates were also found in the dense bodies and the mitochondria encountered in the glial convolutions. No calcium deposit was seen in the synaptic clefts and intercellular spaces of the subsynaptic reticulum of type I and type II. A comparison of calcium to antimony ratios between the terminal axons and the sarcoplasmic lysosomes revealed highly significant differences (P<0.001). Such a variability of the calcium to antimony ratio may be related to different conditions of precipitation or antimony diffusion in the different cell compartments. It was concluded that such synaptic terminals do not appear damaged in spite of the muscle degeneration and presumably continue to perform vital functions while the muscles are no longer contractile 20 h after adult ecdysis.     

Type I Brain Hexokinase: Axonal Transport and Membrane Associations Within Central Nervous System Presynaptic Terminals

Abstract: While studying the delivery of cytoplasmic proteins to the presynaptic terminals of CNS neurons, we discovered unique characteristics of one protein (p118) conveyed in slow component b (SCb) of axonal transport, the large group of proteins representing the cytoplasmic matrix. Alone among the SCb group, p118 coisolated with the synaptic junctional complex on biochemical fractionation of the radiolabeled synaptic regions. Purification and amino acid sequencing of this protein revealed it is most likely the guinea pig form of type I (brain) hexokinase (ATP: d -hexose 6-phosphotransferase, EC 2.7.1.1). Further biochemical treatments were consistent with this identity. The majority of type I brain hexokinase has been thought to be associated primarily with membranes, in particular the mitochondrial outer membrane. We found that the majority of type I hexokinase is transported toward the terminals at a rate at least 10 times slower than that exhibited by the maximal or average rate of mitochondria. This suggests that, in the axon, the enzyme exhibits transient or dynamic interactions with mitochondria that are moving more rapidly. It is not clear whether hexokinase binds exclusively to mitochondria, or also exhibits association with nonmitochondrial membranes. The unexpected enrichment of hexokinase during synaptic junctional complex purification may result from its strong association with the presynaptic membrane portion of the synapse.