Estradiol regulates large dense core vesicles in the hippocampus of adult female rats

Previous work has shown that the steroid hormone estradiol facilitates the release of anticonvulsant neuropeptides from inhibitory neurons in the hippocampus to suppress seizures. Because neuropeptides are packaged in large dense core vesicles, estradiol may facilitate neuropeptide release through regulation of dense core vesicles. In the current study, we used serial section electron microscopy in the hippocampal CA1 region of adult female rats to test three hypotheses about estradiol regulation of dense core vesicles: (1) Estradiol increases the number of dense core vesicles in axonal boutons, (2) Estradiol increases the size of dense core vesicles in axonal boutons, (3) Estradiol shifts the location of dense core vesicles toward the periphery of axonal boutons, potentially lowering the threshold for neuropeptide release during seizures. We found that estradiol increases the number and size of dense core vesicles in inhibitory axonal boutons, consistent with increased neuropeptide content, but does not shift the location of dense core vesicles closer to the bouton periphery. These effects were specific to large dense core vesicles (>80 nm) in inhibitory boutons. Estradiol had no effects on small dense core vesicles or dense core vesicles in excitatory boutons. Our results indicate that estradiol suppresses seizures at least in part by increasing the potentially releasable pool of neuropeptides in the hippocampus, and that estradiol facilitation of neuropeptide release involves a mechanism other than mobilization of dense core vesicles toward sites of release.     

Ultrastructural localization of active zone and synaptic vesicle proteins in a preassembled multi-vesicle transport aggregate

Although it has been suggested that presynaptic active zone (AZ) may be preassembled, it is still unclear which entities carry the various proteins to the AZ during synaptogenesis. Here, I propose that aggregates of dense core vesicles (DCV) and small clear vesicles in the axons of young rat hippocampal cultures are carriers containing preformed AZ and synaptic vesicle (SV) components on their way to developing synapses. The aggregates were positively labeled with antibodies against Bassoon and Piccolo (two AZ cytomatrix proteins), VAMP, SV2, synaptotagmin (three SV membrane proteins), and synapsin I (a SV-associated protein). Bassoon and Piccolo labeling were localized at dense material both in the aggregates and at the AZ. In addition to the SV at the synapses, the SV membrane proteins labeled the clear vesicles in the aggregate as well as many other SV-like and pleiomorphic vesicular structures in the axons, and synapsin I labeling was associated with the vesicles in the aggregates. In single sections, these axonal vesicle aggregates were approximately 0.22 by 0.13 microm in average dimensions and contain one to two DCV and five to six small clear vesicles. Serial sections confirmed that the aggregates were not synaptic junctions sectioned en face. Labeling intensities of Bassoon and Piccolo measured from serially sectioned transport aggregates and AZ were within range of each other, suggesting that one or a few aggregates, but not individual DCV, can carry sufficient Bassoon and Piccolo to form an AZ. The present findings provide the first ultrastructural evidence localizing various AZ and SV proteins in a preassembled multi-vesicle transport aggregate that has the potential to quickly form a functional active zone.     

Assembly of presynaptic active zones from cytoplasmic transport packets

Little is known about presynaptic assembly during central nervous system synaptogenesis. Here we used time-lapse fluorescence imaging, immunocytochemistry and electron microscopy to study hippocampal neuronal cultures transfected with a fusion construct of the presynaptic vesicle protein VAMP and green fluorescent protein. Our results suggest that major cytoplasmic and membrane-associated protein precursors of the presynaptic active zone are transported along developing axons together as discrete packets. Retrospective electron microscopy demonstrated varied vesicular and tubulovesicular membrane structures. Packets containing these heterogeneous structures were stabilized specifically at new sites of dendrite- and axon-initiated cell-cell contact; within less than one hour, evoked vesicle recycling was observed at these putative nascent synapses. These observations suggest that substantial membrane remodeling may be necessary to produce the uniform vesicles typical of the mature active zone, and that many presynaptic proteins may be united early in their biogenesis and sorting pathways.     

Development of synaptic structure and function in organotypic cultures of the rat hippocampus

The sequence of development of synapses, as well as the ultrastructure of axonal growth cones, has been investigated electron microscopically in tissue cultures of the newborn rat hippocampus. During differentiation of the tissue cultures, the formation of synapses is preceded by identifiable growth cones. A characteristic feature of axonal growth cones is the presence of numerous large clear vesicles which vary in diameter from ~100 to 150 nm. The first immature synapses were formed on the 5th, 6th or 7th day in vitro on the growth cones of differentiating neuronal processes. Axonal growth cones are occasionally found to be presynaptic to a dendrite. At first axo-dendritic synapses, most of them being en passant, arise, whereas axo-somatic and axo-spinous-dendritic synapses of different complex structures appear later.It is suggested that the earliest signs of synaptogenesis are vesicular structures (‘growth’ vesicles and few synaptic vesicles), which occur in growth cones, axons and presynaptic boutons of immature synaptic contacts even before formation of the specialized pre- and postsynaptic membranes.     

Synaptic vesicle dynamics in the mossy fiber-CA3 presynaptic terminals of mouse hippocampus

The mossy fiber (MF)-CA3 synapse in the hippocampus is unique in the CNS because of its wide dynamic range of transmitter release during short- and long-term plasticity. The presynaptic mechanisms underlying the fidelity of transmission were investigated for the MF-CA3 synapses. The relative size of readily releasable pool (RRP) of vesicles was estimated by counting the number of docked vesicles at an active zone (AZ) on the transmission electron microscopy (TEM) image. The size of the releasable pool and the exo-endocytosis kinetics were directly measured from individual large MF boutons in hippocampal slices of transgenic mice that selectively express synaptopHluorin (SpH), a pH-sensitive GFP fused to the lumenal aspect of one of the vesicular membrane proteins, VAMP-2, in these boutons. Here we found (1) there are distinct two vesicle pools, the resting pool which is resistant to exocytosis, and the releasable pool, (2) the initially docked vesicles are easily depleted and the RRP is maintained by refilling from the reserve subpopulation of releasable pool ("reserve" releasable pool), and (3) the contribution of rapid reuse of recycled vesicles is relatively small. Therefore, the fidelity of transmission is suggested to be ensured by the rapid refilling rate of RRP.     

Timing and efficacy of transmitter release at mossy fiber synapses in the hippocampal network

It is widely accepted that the hippocampus plays a major role in learning and memory. The mossy fiber synapse between granule cells in the dentate gyrus and pyramidal neurons in the CA3 region is a key component of the hippocampal trisynaptic circuit. Recent work, partially based on direct presynaptic patch-clamp recordings from hippocampal mossy fiber boutons, sheds light on the mechanisms of synaptic transmission and plasticity at mossy fiber synapses. A high Na⁺ channel density in mossy fiber boutons leads to a large amplitude of the presynaptic action potential. Together with the fast gating of presynaptic Ca²⁺ channels, this generates a large and brief presynaptic Ca²⁺ influx, which can trigger transmitter release with high efficiency and temporal precision. The large number of release sites, the large size of the releasable pool of vesicles, and the huge extent of presynaptic plasticity confer unique strength to this synapse, suggesting a large impact onto the CA3 pyramidal cell network under specific behavioral conditions. The characteristic properties of the hippocampal mossy fiber synapse may be important for pattern separation and information storage in the dentate gyrus-CA3 cell network.     

Visualization of changes in presynaptic function during long-term synaptic plasticity.

Controversy exists regarding the site of modification of synaptic transmission during long-term plasticity in the mammalian hippocampus. Here we used a fluorescent marker of presynaptic activity, FM 1-43, to directly image changes in presynaptic function during both short-term and long-term forms of plasticity at presynaptic boutons of CA3-CA1 excitatory synapses in acute hippocampal slices. We demonstrated enhanced presynaptic function during long-term potentiation (LTP) induced either chemically (with tetraethylammonium), or by high-frequency (200-Hz) electrical stimulation. Both of these forms of LTP required activation of L-type voltage-gated calcium channels and NMDA receptors in the postsynaptic CA1 neuron. These results thus implied that a long-lasting increase in the efficacy of synaptic transmission is likely to depend, at least in part, on enhanced transmitter release from the presynaptic neuron.     

大鼠海马结构在空间辨别性学习记忆时的突触形态学观察

目的：探讨学习记忆活动是否可以引起大鼠海马结构发生突触可塑变化。方法：电子显微镜下对模型大鼠和对照大鼠的海马结构内突触复合体的形态进行了对比观察。结果：(1)模型大鼠海马CJ43区多形层内多为神经纤维，其中无髓纤维占多数，有髓纤维很少。在无髓纤维间见到形状不规则的轴突终末与周围的树突和树突棘分别形成轴—树突触和轴棘突触，后者多见。突触前膨大内充满圆形清亮、无核心的突触小泡和线粒体。突触后成分多为树突棘并在多处与突触前膜形成活性区，树突棘内见到棘器。(2)对照大鼠海马CA3区多形层内含有大量无髓纤维并见到较少的轴—树突触。该突触形状规则且活性带面积较小，突触后成分多为一个，突触小泡清亮、圆形无核心，偶见线粒体。(3)经Timm染色的两组大鼠海马CA3区多形层的超微结构特点是银颗粒仅沉积在苔藓纤维的髓鞘内和苔藓纤维的大终扣内，线粒体、树突干和树突棘内均无银颗粒。结论：正常生理活动和空间辨别性学习记忆活动均可引致大鼠海马CA3区多形层突触可塑性变化，但两者在形态上有所不同。     

Synaptic vesicle dynamics in the mossy fiber-CA3 presynaptic terminals of mouse hippocampus

The mossy fiber (MF)-CA3 synapse in the hippocampus is unique in the CNS because of its wide dynamic range of transmitter release during short- and long-term plasticity. The presynaptic mechanisms underlying the fidelity of transmission were investigated for the MF-CA3 synapses. The relative size of readily releasable pool (RRP) of vesicles was estimated by counting the number of docked vesicles at an active zone (AZ) on the transmission electron microscopy (TEM) image. The size of the releasable pool and the exo-endocytosis kinetics were directly measured from individual large MF boutons in hippocampal slices of transgenic mice that selectively express synaptopHluorin (SpH), a pH-sensitive GFP fused to the lumenal aspect of one of the vesicular membrane proteins, VAMP-2, in these boutons. Here we found (1) there are distinct two vesicle pools, the resting pool which is resistant to exocytosis, and the releasable pool, (2) the initially docked vesicles are easily depleted and the RRP is maintained by refilling from the reserve subpopulation of releasable pool (“reserve” releasable pool), and (3) the contribution of rapid reuse of recycled vesicles is relatively small. Therefore, the fidelity of transmission is suggested to be ensured by the rapid refilling rate of RRP.     

Constitutive sharing of recycling synaptic vesicles between presynaptic boutons

The synaptic vesicle cycle is vital for sustained neurotransmitter release. It has been assumed that functional synaptic vesicles are replenished autonomously at individual presynaptic terminals. Here we tested this assumption by using FM dyes in combination with fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured rat hippocampal neurons. After photobleaching, synapses acquired recently recycled FM dye-labeled vesicles originating from nonphotobleached synapses by a process requiring dynamic actin turnover. The imported vesicles entered the functional pool at their host synapses, as revealed by the exocytic release of the dye upon stimulation. FM1-43 photoconversion and ultrastructural analysis confirmed the incorporation of imported vesicles into the presynaptic terminal, where they mixed with the native vesicle pools. Our results demonstrate that synaptic vesicle recycling is not confined to individual presynaptic terminals as is widely believed; rather, a substantial proportion of recycling vesicles are shared constitutively between boutons.     

A quantitative analysis of the laminar distribution of synaptic boutons in field CA3 of the rat hippocampus

We analyzed the laminar distribution of synaptic boutons in field CA3 of the rat hippocampus using a large montage electron micrograph. The size of boutons and synaptic vesicles was measured using a computer-assisted digitizing system. In all, 3353 synaptic boutons were observed in a 15 microm x 100 microm strip. Of these, 86.3% contained spherical vesicles (S-boutons), 12% contained flat vesicles (F-boutons), and 1.7% were mossy terminals (M-boutons). S-boutons were distributed widely in the strata moleculare (st. Mol), radiatum (st. Rad), and oriens (st. Ori), but there were only a few in the strata lucidum (st. Luc) and pyramidale (st. Pyr). The upper portions of both the st. Rad and Ori contained slightly fewer boutons. In terms of the location of synaptic contacts, 83% of all S-boutons were found on the dendritic spines and the rest were on the dendritic shafts. S-boutons on the dendritic shafts were observed more frequently in the st. Mol than in the other strata. According to the morphometry of the size of synaptic vesicles, S-boutons with small vesicles (mean vesicle area <1109 nm(2)) were located exclusively in the st. Mol, S-boutons with medium-sized vesicles (mean vesicle area 1109-1482 nm(2)) were observed in all strata, and S-boutons with large vesicles (mean vesicle area >1482 nm(2)) were distributed in the st. Luc and Ori, but not in the st. Mol. F-boutons were predominantly distributed in the upper half of the st. Mol and in the area around the st. Pyr, although they were observed in all strata. In the st. Mol, all the F-boutons were in contact with dendritic shafts, while near the st. Pyr, F-boutons were found exclusively on somata, the proximal parts of the dendritic shafts, and the initial segments of axons. The average F-bouton was smaller in the st. Mol (0.23 microm(2)) than near the st. Pyr (0.39 microm(2)). In this synapto-architectural study of the hippocampal CA3 region using large montage electron micrographs, we observed (1) an intimate relationship between synapse distribution and the dendritic structure of pyramidal neurons, (2) the distribution of different types of boutons containing vesicles of various size, and (3) two different plausible foci of postsynaptic inhibition where F-boutons were distributed densely, and (4) estimated the input ratios of pyramidal neurons.     

Loss of synaptotagmin IV results in a reduction in synaptic vesicles and a distortion of the Golgi structure in cultured hippocampal neurons

Fusion of synaptic vesicles with the plasma membrane is mediated by the SNARE (soluble NSF attachment receptor) proteins and is regulated by synaptotagmin (syt). There are at least 17 syt isoforms that have the potential to act as modulators of membrane fusion events. Synaptotagmin IV (syt IV) is particularly interesting; it is an immediate early gene that is regulated by seizures and certain classes of drugs, and, in humans, syt IV maps to a region of chromosome 18 associated with schizophrenia and bipolar disease. Syt IV has recently been found to localize to dense core vesicles in hippocampal neurons, where it regulates neurotrophin release. Here we have examined the ultrastructure of cultured hippocampal neurons from wild-type and syt IV −/− mice using electron tomography. Perhaps surprisingly, we observed a potential synaptic vesicle transport defect in syt IV −/− neurons, with the accumulation of large numbers of small clear vesicles (putative axonal transport vesicles) near the trans-Golgi network. We also found an interaction between syt IV and KIF1A, a kinesin known to be involved in vesicle trafficking to the synapse. Finally, we found that syt IV −/− synapses exhibited reduced numbers of synaptic vesicles and a twofold reduction in the proportion of docked vesicles compared to wild-type. The proportion of docked vesicles in syt IV −/− boutons was further reduced, 5-fold, following depolarization.     

大鼠皮质大颗粒泡的超微结构

用电镜在4只猫听区皮质和3只大白鼠额叶皮质中观察了大颗粒泡的超微结构。在2031个突触面上,共见到大颗粒泡282个。1.大颗粒泡的位置:在104个突触前囊中含有大颗粒泡135个,它们多位于突触前囊的后部(64/135),位于前囊的中部或接近突触前膜者较少。有的大颗粒泡不在突触前囊内,而位于轴突、树突或个别例在树突侧棘中。2.大颗粒泡数目:大脑皮质中的大颗粒泡数目较少。在各种成分中(包括突触前囊、轴突、树突以及其他成分)计见282个大颗粒泡,在一个成分中含1个大颗粒泡的占多数(78.80％)。个别例有含有6个者。多数突触前囊无大颗粒泡。3.大颗粒泡的形态:多为圆形或卵圆形,亚铃形或不整形者也有之。致密核心多数致密,有的并不致密而显得灰淡,有的致密核心呈网状,或在一个包膜内有两个核心。有的膜下环并不明显。4.大颗粒泡的大小不一。本文对大颗粒泡和某些递质的可能关系进行了讨论。     

Activity-dependent liberation of synaptic neuropeptide vesicles

Despite the importance of neuropeptide release, which is evoked by long bouts of action potential activity and which regulates behavior, peptidergic vesicle movement has not been examined in living nerve terminals. Previous in vitro studies have found that secretory vesicle motion at many sites of release is constitutive: Ca(2+) does not affect the movement of small synaptic vesicles in nerve terminals or the movement of large dense core vesicles in growth cones and endocrine cells. However, in vivo imaging of a neuropeptide, atrial natriuretic factor, tagged with green fluorescent protein in larval Drosophila melanogaster neuromuscular junctions shows that peptidergic vesicle behavior in nerve terminals is sensitive to activity-induced Ca(2+) influx. Specifically, peptidergic vesicles are immobile in resting synaptic boutons but become mobile after seconds of stimulation. Vesicle movement is undirected, occurs without the use of axonal transport motors or F-actin, and aids in the depletion of undocked neuropeptide vesicles. Peptidergic vesicle mobilization and post-tetanic potentiation of neuropeptide release are sustained for minutes.     

Light microscopic and ultrastructural localization of immunoreactive substance P in the dorsal horn of monkey spinal cord

Light- and electron-microscopic localization of substance P in the monkey spinal cord was studied by the peroxidase anti-peroxidase technique with the particular aim of examining types of interactions made by substance P-positive boutons with other neuronal elements in the dorsal horn. By light-microscopy dense labeling for immunoreactive substance P was found in laminae I, II (outer zone) and V (lateral region), consistent with findings in other mammalian species. By electron-microscopy, substance P-positive staining was mostly in unmyelinated and in some thinly myelinated small diameter fibers. Substance P-positive terminals contained both large granular vesicles (80-120 nm diameter), which were filled with reaction product, and clear round vesicles (40-60 nm). Substance P-positive large granular vesicles were sometimes observed near presynaptic sites and in contact with dense projection there. Immunoreactive substance P boutons were small to large in size (1-4 micron), formed synapses with somata and large dendrites and were the central axons of synaptic glomeruli where they were in synaptic contact with numerous small dendrites and spines. Substance P-labeled axons frequently formed synapses with dorsal horn neurons which were also postsynaptic to other types of axons. Substance P-positive profiles participated in numerous puncta adhaerentia with unlabeled cell bodies, dendrites and axons. Only rarely, some suggestive evidence was obtained indicating that axons might synapse onto substance P-containing boutons. Biochemical analysis of monkey spinal cord tissue extracts, undertaken to characterize more precisely the immunoreactive substances, indicated that only substance P and its oxide derivative were detected with the antiserum used in the immunocytochemistry. These morphological findings show that substance P is contained within a class of axon terminals, many of which have been shown previously in the monkey to originate from the dorsal root. The results suggest that modulation of substance P primary afferents terminating in the outer dorsal laminae of the monkey spinal cord occurs in part via axonal inputs onto dorsal horn neurons postsynaptic to the primary afferent.     

Direct and indirect effects of estrogen on rat hippocampus

Estrogen-induced synaptic plasticity was frequently shown by an increase of spines at apical dendrites of CA1 pyramidal neurons after systemic application of estradiol to ovariectomized rats. Recent findings question this direct endocrine regulation of synaptogenesis by estradiol. We have shown, for the first time, that estrogens are synthesized de novo in rat hippocampal neurons. By using letrozole, an inhibitor of aromatase, estradiol levels in hippocampal dispersion cultures as well as in hippocampal slice cultures were significantly suppressed. Letrozole treatment resulted in a significant decrease in the density of spines and spine synapses and in the number of presynaptic boutons. Quantitative immunohistochemistry revealed a dose-dependent downregulation of spinophilin, a spine marker, and of synaptophysin, a presynaptic marker, in the hippocampus. Surprisingly, exogenous application of estradiol to the cultures had no effect. Indirect effects of estrogens, mediated via subcortical nuclei, may help to explain this phenomenon. Implantation of estrogen-filled cannulae into the median raphe, which projects to the hippocampus, resulted in a significant increase in spine density in the hippocampus after seven days of treatment. This increase was paralleled by a decrease in the density of serotonergic innervation of the strata lacunosum moleculare and radiatum of the CA1 region. Apart from direct endocrine mechanisms our findings suggest that estradiol-induced spinogenesis in the hippocampus is also mediated by indirect mechanisms and is furthermore regulated endogenously, in a paracrine manner.     

Compensation in the number of presynaptic dense projections and synaptic vesicles in remaining parallel fibres following cerebellar lesions

Summary Our previous investigations demonstrated an increase in the size of remaining synaptic sites as an intermediate or possible alternative to sprouting plasticity. The total amount of postsynaptic contact area remained relatively constant for each target neuron even though there was a marked decrease in the number of sites on these neurons. In addition, enlarged boutons containing numerous synaptic vesicles were positioned adjacent to enlarged postsynaptic sites.The question posed by this study was to determine whether dense projections, parts of the presynaptic grids of the remaining parallel fibres, spread to cover the enlarged postsynaptic sites, or if the number of these densities increased on each site to maintain the structural organization of the presynaptic grid. In addition, the number of synaptic vesicles per bouton was quantitated to determine whether they compensated by increasing their number in relationship to the increased area of the presynaptic grid.The number of parallel fibre synapses on Purkinje cells was reduced by transection of a narrow bundle of parallel fibres accompanied by a small lesion undercutting the molecular layer to destroy granule cells contributing to this bundle. The number of presynaptic dense projections was quantitated in control and lesioned preparations (using ethanolic phosphotungstic acid staining) in order to determine their correlation to the area of each site. In addition, the average number of synaptic vesicles in boutons was compared to the average size of boutons and the average contact area of the synaptic sites. At 3 to 7 days following partial deafferentation of Purkinje cells in adult rats, the density of dense projections of parallel fibre synapses on Purkinje cell spines remained uniform. This occurred throughout a range of reduction in the number of synapses in conjunction with a reciprocal increase in the size of sites. The finding of a uniform density of these projections and an increase in the size of sites implies that each granule cell axon must gain dense projections. In addition, the remaining presynaptic boutons had a uniform density of synaptic vesicles even though the volume of the boutons and the area of the synaptic contact doubled. Thus, the number of synaptic vesicles gained in proportion to the total enlargement of the contact site and the bouton size.These results strongly suggest that deficits or losses in synaptic connections of parallel fibre on Purkinje cell spines produces a compensation in the total number of synaptic vesicles and presynaptic dense projections of the remaining boutons. An enlargement of the presynaptic grid occurs in concert with redistribution of the constant total area of membrane occupied by macromolecules (or insertion of new ones) on remaining postsynaptic sites. These compensations could be facilitating efficacy of neuronal connections after lesions or neuronal attrition by re-establishing available transmitter and release sites in proportion to the constant amount of receptor area.     

A readily retrievable pool of synaptic vesicles

Although clathrin-mediated endocytosis is thought to be the predominant mechanism of synaptic vesicle recycling, it seems to be too slow for fast recycling. Therefore, it was suggested that a presorted and preassembled pool of synaptic vesicle proteins on the presynaptic membrane might support a first wave of fast clathrin-mediated endocytosis. In this study we monitored the temporal dynamics of such a 'readily retrievable pool' of synaptic vesicle proteins in rat hippocampal neurons using a new type of probe. By applying cypHer5E, a new cyanine dye-based pH-sensitive exogenous marker, coupled to antibodies to luminal domains of synaptic vesicle proteins, we could reliably monitor synaptic vesicle recycling and demonstrate the preferential recruitment of a surface pool of synaptic vesicle proteins upon stimulated endocytosis. By using fluorescence nanoscopy of surface-labeled synaptotagmin 1, we could resolve the spatial distribution of the surface pool at the periactive zone in hippocampal boutons, which represent putative sites of endocytosis.     

Immunoelectron microscopic localization of synaptophysin in a Golgi subcompartment of developing hypothalamic neurons

Synaptophysin, previously identified as an integral membrane glycoprotein (mol. wt 38,000) characteristic of presynaptic vesicles of mature neurons, provides a molecular marker to study the origin, formation and traffic of synaptic vesicles. Using the monoclonal antibody SY38 against this polypeptide we have localized synaptophysin by immunofluorescence and electron microscope immunoperoxidase methods in cultured mouse hypothalamic neurons taken from 16-day-old fetuses which achieve synaptogenesis after 10-12 days in vitro. We have compared the localization of synaptophysin in perikarya and nerve endings as a function of age (2-19 days in vitro) and of treatment of mature neurons with nocodazole. Using immunofluorescence microscopy, synaptophysin was already detected in neuronal soma at 2 days in vitro, where the initiation of neurite development is observed. At the electron microscope level, virtually all mature synaptic boutons and varicosities showed an extensive synaptophysin labeling of synaptic vesicles at 12-13 days in culture whereas neurites showed only very few labeled vesicles. In neuronal soma taken before synapse formation (6 days in vitro), synaptophysin was selectively localized in membranes of the innermost cisternae of the Golgi zone and in vesicles of variable size and shape in the core of the Golgi zone. In contrast, after synapse formation, synaptophysin labeling was barely detected in the Golgi zone of neurons but a very strong labeling of synaptic vesicles in synaptic boutons was observed. Treatment of mature neurons (12 days in vitro) with nocodazole (10(-5) M) resulted in a conspicuous synaptophysin staining of the innermost trans-Golgi cisternae and numerous vesicles in the cytoplasm. Furthermore, an accumulation of labeled synaptic vesicles on the presynaptic membrane of nerve terminals was found. The data suggest that synaptophysin is released from the Golgi apparatus in a vesicular form, after glycosylation, and is then transported to nerve endings by a mechanism which requires integrity of microtubules.     

Synapsin-dependent development of glutamatergic synaptic vesicles and presynaptic plasticity in postnatal mouse brain

Inactivation of the genes encoding the neuronal, synaptic vesicle-associated proteins synapsin I and II leads to severe reductions in the number of synaptic vesicles in the CNS. We here define the postnatal developmental period during which the synapsin I and/or II proteins modulate synaptic vesicle number and function in excitatory glutamatergic synapses in mouse brain. In wild-type mice, brain levels of both synapsin I and synapsin IIb showed developmental increases during synaptogenesis from postnatal days 5-20, while synapsin IIa showed a protracted increase during postnatal days 20-30. The vesicular glutamate transporters (VGLUT) 1 and VGLUT2 showed synapsin-independent development during postnatal days 5-10, following which significant reductions were seen when synapsin-deficient brains were compared with wild-type brains following postnatal day 20. A similar, synapsin-dependent developmental profile of vesicular glutamate uptake occurred during the same age periods. Physiological analysis of the development of excitatory glutamatergic synapses, performed in the CA1 stratum radiatum of the hippocampus from the two genotypes, showed that both the synapsin-dependent part of the frequency facilitation and the synapsin-dependent delayed response enhancement were restricted to the period after postnatal day 10. Our data demonstrate that while both synaptic vesicle number and presynaptic short-term plasticity are essentially independent of synapsin I and II prior to postnatal day 10, maturation and function of excitatory synapses appear to be strongly dependent on synapsin I and II from postnatal day 20.