Synaptogenesis in the neural ganglia of the heart in the human embryo

Electron microscopy was used to study synapse development in the cardiac ganglia of human fetuses ranging from 8 to 27 weeks of ovulation time. Staining with ethanolic phosphotungstic acid was used for analysis of synaptic active zones. Specialization of interneuronal links begins with the appearance of electron dense material on plasmalemmas of nerve cells in the places of simple contacts. First synapses with single synaptic vesicles and short osmiophilic zones were found in cardiac ganglia in 8-week-old fetuses. Large granular vesicles and mitochondria vesicles are formed from cisternae of agranular endoplasmic reticulum in the preterminal parts of axons and moved by axoplasmic transport to the osmiophilic zones of future synapses. Axodendritic synapses appeared earlier in the cardiac ganglia than axosomatic ones, the latter were observed from the middle of gestation. Transient neuroglial synapse-like contacts were found in the cardiac ganglia. Staining with phosphotungstic acid made it possible to distinguish the degree of synapse maturation according to active synaptic zones. The peculiarities of synaptic development in cardiac ganglia in comparison with that in the central nervous system may be accounted for by different origins of the neural tube and of neural crest and by the level of their phylogenic development.     

Colocalization of synapsin and actin during synaptic vesicle recycling

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.     

Astrocytes optimize the synaptic transmission of information

Chemical synapses transmit information via the release of neurotransmitter-filled vesicles from the presynaptic terminal. Using computational modeling, we predict that the limited availability of neurotransmitter resources in combination with the spontaneous release of vesicles limits the maximum degree of enhancement of synaptic transmission. This gives rise to an optimal tuning that depends on the number of active zones. There is strong experimental evidence that astrocytes that enwrap synapses can modulate the probabilities of vesicle release through bidirectional signaling and hence regulate synaptic transmission. For low-fidelity hippocampal synapses, which typically have only one or two active zones, the predicted optimal values lie close to those determined by experimentally measured astrocytic feedback, suggesting that astrocytes optimize synaptic transmission of information.     

Localization of smg p25A/rab3A p25, a small GTP-binding protein, at the active zone of the rat neuromuscular junction.

smg p25A is a small G protein which has been suggested to regulate neurotransmitter release from the synapses. We investigated here the ultrastructural localization of this small G protein in the rat neuromuscular junction by an immunoperoxidase method. The results showed that smg p25A was distributed non-uniformly on the presynaptic plasma membrane and among the synaptic vesicles with the focal accumulation on the discrete presynaptic sites which corresponded to the active zones, the regions of the presynaptic plasma membrane specialized for the exocytosis of the synaptic vesicles. This unique distribution of smg p25A suggests that it plays an important role in the attachment and fusion of the synaptic vesicles with the active zones.     

The actin cytoskeleton and neurotransmitter release: an overview

Here we review evidence that actin and its binding partners are involved in the release of neurotransmitters at synapses. The spatial and temporal characteristics of neurotransmitter release are determined by the distribution of synaptic vesicles at the active zones, presynaptic sites of secretion. Synaptic vesicles accumulate near active zones in a readily releasable pool that is docked at the plasma membrane and ready to fuse in response to calcium entry and a secondary, reserve pool that is in the interior of the presynaptic terminal. A network of actin filaments associated with synaptic vesicles might play an important role in maintaining synaptic vesicles within the reserve pool. Actin and myosin also have been implicated in the translocation of vesicles from the reserve pool to the presynaptic plasma membrane. Refilling of the readily releasable vesicle pool during intense stimulation of neurotransmitter release also implicates synapsins as reversible links between synaptic vesicles and actin filaments. The diversity of actin binding partners in nerve terminals suggests that actin might have presynaptic functions beyond synaptic vesicle tethering or movement. Because most of these actin-binding proteins are regulated by calcium, actin might be a pivotal participant in calcium signaling inside presynaptic nerve terminals. However, there is no evidence that actin participates in fusion of synaptic vesicles.     

Rapid Ultrastructural Changes in the CA3 Field of Hippocampus of Krushinskii–Molodkina Rats after Acoustic Stress

Morphometric analysis of electron microphotographs of hippocampus of Krushinskii–Molodkina rats with hereditary predisposition to audiogenic epilepsy revealed rapid reversible changes in synapses between terminals of mossy fibers and dendritic spines of pyramidal neurons in the field CA3 after convulsions induced by stressing auditory effects. It was established that an initial increase of the number, size, and perforation of active zones, which indicates activation of synaptic transmission, was subsequently replaced by a decrease of these parameters as well as of spine invaginations and by an increase of the number of dense-core synaptic vesicles in active zones of synapses. Previous administration of melatonin was observed to lead to a significant decrease of intensity of manifestation of the convulsion symptoms and of the degree of the above ultrastructural changes, which might be considered an evidence for melatonin sedative and stress-limiting, as well as anti-epileptic and neuroprotective properties.     

Actin study of the synapses of surviving hippocampal slices during long-term potentiation

A study was made of the synaptic actin ultrastructural localization in the hippocampal slices at long-lasting potentiation of area CA, using myosin subfragment-1 labeling. A specific qualitative ultrastructural sign of the potentiated hippocampal synapses was revealed for the first time - the formation in spines of rodlike bundles of actin filaments resembling the cilia. They penetrate the spine stalks to pass through the spine core towards the postsynaptic densities of active zones. The thinner bridges link the filament bundles with the actin cytoskeleton meshwork, with spine apparatus cisterns and with postsynaptic membranes of the active zones. Besides the increasing density of the presynaptic actin meshwork was shown. The changes in the actin cytoskeleton being taken into consideration, its contractile properties account for some morphofunctional features of the potentiated synapses known before and predict previously unknown ones.     

Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon

Mutant mice lacking the central region of the presynaptic active zone protein Bassoon were generated to establish the role of this protein in the assembly and function of active zones as sites of synaptic vesicle docking and fusion. Our data show that the loss of Bassoon causes a reduction in normal synaptic transmission, which can be attributed to the inactivation of a significant fraction of glutamatergic synapses. At these synapses, vesicles are clustered and docked in normal numbers but are unable to fuse. Phenotypically, the loss of Bassoon causes spontaneous epileptic seizures. These data show that Bassoon is not essential for synapse formation but plays an essential role in the regulated neurotransmitter release from a subset of glutamatergic synapses.     

The diverse roles of ribbon synapses in sensory neurotransmission

Sensory synapses of the visual and auditory systems must faithfully encode a wide dynamic range of graded signals, and must be capable of sustained transmitter release over long periods of time. Functionally and morphologically, these sensory synapses are unique: their active zones are specialized in several ways for sustained, rapid vesicle exocytosis, but their most striking feature is an organelle called the synaptic ribbon, which is a proteinaceous structure that extends into the cytoplasm at the active zone and tethers a large pool of releasable vesicles. But precisely how does the ribbon function to support tonic release at these synapses? Recent genetic and biophysical advances have begun to open the 'black box' of the synaptic ribbon with some surprising findings and promise to resolve its function in vision and hearing.     

Neurotransmitter release at ribbon synapses in the retina

The synapses of photoreceptors and bipolar cells in the retina are easily identified ultrastructurally by the presence of synaptic ribbons, electron-dense bars perpendicular to the plasma membrane at the active zones, extending about 0.5 microm into the cytoplasm. The neurotransmitter, glutamate, is released continuously (tonically) from these 'ribbon synapses' and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven bursts of release at conventional synapses. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but a few differences have been identified that may be important determinants of tonic transmitter release. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.     

Drosophila ankyrin 2 is required for synaptic stability

Synaptic connections are stabilized through transsynaptic adhesion complexes that are anchored in the underlying cytoskeleton. The Drosophila neuromuscular junction (NMJs) serves as a model system to unravel genes required for the structural remodeling of synapses. In a mutagenesis screen for regulators of synaptic stability, we recovered mutations in Drosophila ankyrin 2 (ank2) affecting two giant Ank2 isoforms that are specifically expressed in the nervous system and associate with the presynaptic membrane cytoskeleton. ank2 mutant larvae show severe deficits in the stability of NMJs, resulting in a reduction in overall terminal size, withdrawal of synaptic boutons, and disassembly of presynaptic active zones. In addition, lack of Ank2 leads to disintegration of the synaptic microtubule cytoskeleton. Microtubules and microtubule-associated proteins fail to extend into distant boutons. Interestingly, Ank2 functions downstream of spectrin in the anchorage of synaptic microtubules, providing the cytoskeletal scaffold that is essential for synaptic stability.     

Optimizing synaptic architecture and efficiency for high-frequency transmission

Bursts of neuronal activity are transmitted more effectively as synapses mature. However, the mechanisms that control synaptic efficiency during development are poorly understood. Here, we study postnatal changes in synaptic ultrastructure and exocytosis in a calyx-type nerve terminal. Vesicle pool size, exocytotic efficiency (amount of exocytosis per Ca influx), Ca current facilitation, and the number of active zones (AZs) increased with age, whereas AZ area, number of docked vesicles per AZ, and release probability decreased with age. These changes led to AZs that are less prone to multivesicular release, resulting in reduced AMPA receptor saturation and desensitization. A greater multiplicity of small AZs with few docked vesicles, a larger pool of releasable vesicles, and a higher efficiency of release thus promote prolonged high-frequency firing in mature synapses.     

Altered synaptic development and active zone spacing in endocytosis mutants

Many types of synapses have highly characteristic shapes and tightly regulated distributions of active zones, parameters that are important to the function of neuronal circuits. The development of terminal arborizations must therefore include mechanisms to regulate the spacing of terminals, the frequency of branching, and the distribution and density of release sites. At present, however, the mechanisms that control these features remain obscure. Here, we report the development of supernumerary or "satellite" boutons in a variety of endocytic mutants at the Drosophila neuromuscular junction. Mutants in endophilin, synaptojanin, dynamin, AP180, and synaptotagmin all show increases in supernumerary bouton structures. These satellite boutons contain releasable vesicles and normal complements of synaptic proteins that are correctly localized within terminals. Interestingly, however, synaptojanin terminals have more active zones per unit of surface area and more dense bodies (T-bars) within these active zones, which may in part compensate for reduced transmission per active zone. The altered structural development of the synapse is selectively encountered in endocytosis mutants and is not observed when synaptic transmission is reduced by mutations in glutamate receptors or when synaptic transmission is blocked by tetanus toxin. We propose that endocytosis plays a critical role in sculpting the structure of synapses, perhaps through the endocytosis of unknown regulatory signals that organize morphogenesis at synaptic terminals.     

Involvement of neuropeptide mechanisms in the integration of heterotopic dentate fascia grafts and recipient brain

Embryonic dentate fascia was grafted into the somatosensory neocortex of adult rats. Nine months post-grafting, the ultrastructural and morphometric analysis of the giant synapses established between the grafted granular neurons and inappropriate targets in the recipient brain was performed. As compared to the intact synaptic endings in the control hippocampus, differences were found in both the number and distribution of large dense-core synaptic vesicles, which store the neuropeptide co-transmitters. The peptidergic vesicle proportion (of total vesicle pool) within the ectopic giant synapses was 5.8 +/- 0.6% (versus 3.3 +/- 0.6% in the control). Clusters of large dense-core vesicles near the active zones of aberrant connections were observed almost 7.9 times more frequently than that of normal contacts. These data provide evidence that neuropeptide transmitters are critical for the maintenance of synaptic connections between the heterotopic dentate grafts and host brain.     

Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).     

视皮层LTP维持阶段的突触形态计量学研究

本实验使用１８～２０ｄ的幼年大鼠视皮层脑片标本,在ＬＴＰ出现后３ｈ取局部微脑片固定进行ＬＴＰ维持阶段超微结构的研究。分别与孵育相同时间而未予任何刺激的脑片和仅给予测试刺激的脑片作比较。运用图像分析仪分别对三组电镜结果进行以下参数的测量：（１）突触间隙的宽度;（２）突触后致密物（ＰＳＤ）的厚度;（３）活性区的长度;和（４）突触界面曲率。用双盲法对突触数目进行计量,并用立体计量学方法对各种突触类型进行定量,所得数据用方差分析进行统计学处理。结果显示：（１）ＬＴＰ形成后１５ｈ左右,其反应达到峰值,然后维持在最高水平一直到３ｈ仍无下降趋势;（２）突触间隙的宽度较两个对照组明显增宽;（３）ＰＳＤ的厚度也明显增厚;（４）活性区的面密度及突触界面曲率明显增加;（５）总突触数目和棘突触数目的数密度较空白对照明显增高;（６）穿孔性突触的数密度与对照组相比明显增加。结果提示：活性区面密度的增加及突触界面曲率的增大可能是ＬＴＰ维持的形态学基础。穿孔性突触的形成与ＬＴＰ的维持密切相关。     

Ultrastructure of the Mauthner neurons in goldfish fry after their adaptation to vestibular stimulation

The ultrastructure of the Mauthner cells (M-cells) of goldfish fries was investigated under four different functional states: a) intact (native fishes), b) fatigue (intact fishes subjected to a prolonged vestibular stimulation), c) adapted (intact fishes after a prolonged training session of the daily short vestibular stimuli), d) excited (adapted fishes subjected to a prolonged vestibular stimulation). It has been first found that the fatigue of the M-cells may result in destructive changes of their cytoskeleton. Besides, in the afferent synapses of both adapted and excited M-cells numerous dense-cored vesicles were revealed near the active zones. The data show the neuronal cytoskeleton to be the central target susceptible to damage upon stimulation. The training leads presumably to stabilization of the cytoskeleton ultrastructure. The dense-cored vesicles were suggested to play an active role in the process.     

The presynaptic active zone protein bassoon is essential for photoreceptor ribbon synapse formation in the retina

The photoreceptor ribbon synapse is a highly specialized glutamatergic synapse designed for the continuous flow of synaptic vesicles to the neurotransmitter release site. The molecular mechanisms underlying ribbon synapse formation are poorly understood. We have investigated the role of the presynaptic cytomatrix protein Bassoon, a major component of the photoreceptor ribbon, in a mouse retina deficient of functional Bassoon protein. Photoreceptor ribbons lacking Bassoon are not anchored to the presynaptic active zones. This results in an impaired photoreceptor synaptic transmission, an abnormal dendritic branching of neurons postsynaptic to photoreceptors, and the formation of ectopic synapses. These findings suggest a critical role of Bassoon in the formation and the function of photoreceptor ribbon synapses of the mammalian retina.     

Inactivity produces increases in neurotransmitter release and synapse size.

When hippocampal synapses in culture are pharmacologically silenced for several days, synaptic strength increases. The structural correlate of this change in strength is an increase in the size of the synapses, with all synaptic components--active zone, postsynaptic density, and bouton--becoming larger. Further, the number of docked vesicles and the total number of vesicles per synapse increases, although the number of docked vesicles per area of active zone is unchanged. In parallel with these anatomical changes, the physiologically measured size of the readily releasable pool (RRP) and the release probability are increased. Ultrastructural analysis of individual synapses in which the RRP was previously measured reveals that, within measurement error, the same number of vesicles are docked as are estimated to be in the RRP.