突触前膜Ca~(2 )通道作为结构元件参与突触囊泡的停靠与融合

新近提供的证据表明 ,在神经递质释放中具关键作用的突触前膜Ca2 通道 ,除为Ca2 进入胞内提供路径外 ,还通过位于其胞内环 (LⅡ ～LⅢ)中的一段称为synprint位点的肽段与突触蛋白syntaxin和SNAP 2 5等相互作用 ,参与突触囊泡停靠 /融合。本文综述了相关资料     

Disentangling the Roles of RIM and Munc13 in Synaptic Vesicle Localization and Neurotransmission

Efficient neurotransmitter release at the presynaptic terminal requires docking of synaptic vesicles to the active zone membrane and formation of fusion-competent synaptic vesicles near voltage-gated Ca²⁺ channels. Rab3-interacting molecule (RIM) is a critical active zone organizer, as it recruits Ca²⁺ channels and activates synaptic vesicle docking and priming via Munc13-1. However, our knowledge about Munc13-independent contributions of RIM to active zone functions is limited. To identify the functions that are solely mediated by RIM, we used genetic manipulations to control RIM and Munc13-1 activity in cultured hippocampal neurons from mice of either sex and compared synaptic ultrastructure and neurotransmission. We found that RIM modulates synaptic vesicle localization in the proximity of the active zone membrane independent of Munc13-1. In another step, both RIM and Munc13 mediate synaptic vesicle docking and priming. In addition, while the activity of both RIM and Munc13-1 is required for Ca²⁺-evoked release, RIM uniquely controls neurotransmitter release efficiency. However, activity-dependent augmentation of synaptic vesicle pool size relies exclusively on the action of Munc13s. Based on our results, we extend previous findings and propose a refined model in which RIM and Munc13-1 act in overlapping and independent stages of synaptic vesicle localization and release.SIGNIFICANCE STATEMENT The presynaptic active zone is composed of scaffolding proteins that functionally interact to localize synaptic vesicles to release sites, ensuring neurotransmission. Our current knowledge of the presynaptic active zone function relies on structure-function analysis, which has provided detailed information on the network of interactions and the impact of active zone proteins. Yet, the hierarchical, redundant, or independent cooperation of each active zone protein to synapse functions is not fully understood. Rab3-interacting molecule and Munc13 are the two key functionally interacting active zone proteins. Here, we dissected the distinct actions of Rab3-interacting molecule and Munc13-1 from both ultrastructural and physiological aspects. Our findings provide a more detailed view of how these two presynaptic proteins orchestrate their functions to achieve synaptic transmission.     

Presynaptic protein distribution and odour mapping in glomeruli of the olfactory bulb of Xenopus laevis tadpoles

The sensory input layer in the olfactory bulb (OB) is typically organized into spheroidal aggregates of dense neuropil called glomeruli. This characteristic compartmentalization of the synaptic neuropil is a typical feature of primary olfactory centres in vertebrates and most advanced invertebrates. In the present work we mapped the location of presynaptic sites in glomeruli across the OB using antibodies to presynaptic vesicle proteins and presynaptic membrane proteins in combination with confocal microscopy. In addition the responses of glomeruli upon mucosal application of amino acid-odorants and forskolin were monitored using functional calcium imaging. We first describe the spatial distribution of glomeruli across the main olfactory bulb (MOB) in premetamorphic Xenopus laevis. Second, we show that the heterogeneous organization of glomeruli along the dorsoventral and mediolateral axes of the MOB is associated with a differential distribution of synaptic vesicle proteins. While antibodies to synaptophysin, syntaxin and SNAP-25 uniformly labelled glomeruli in the whole MOB, intense synaptotagmin staining was present only in glomeruli in the lateral, and to a lesser extent in the intermediate, part of the OB. Interestingly, amino acid-responsive glomeruli were always located in the lateral part of the OB, and glomeruli activated by mucosal forskolin application were exclusively located in the medial part of the OB. This correlation between odour mapping and presynaptic protein distribution is an additional hint on the existence of different subsystems within the main olfactory system in larval Xenopus laevis.     

Synapsin-like Molecules in Aplysia punctata and Helix pomatia: Identification and Distribution in the Nervous System and During the Formation of Synaptic Contacts In Vitro

The distribution and biochemical features of the synapsin-like peptides recognized in Aplysia and Helix by various antibodies directed against mammalian synapsins were studied. The peptides can be extracted at low pH and are digested by collagenase; further, they can be phosphorylated by both protein kinase A and Ca²⁺/calmodulin-dependent protein kinase II. In the ganglia of both snails, they are associated with the soma of most neurons and with the neuropil; punctate immunostaining is present along the neurites. Using cocultures of a Helix serotoninergic neuron and of its target cell, we analysed the redistribution of the synapsin-like peptides during the formation of active synaptic contacts. When the presynaptic neuron is plated in isolation, both synapsin and serotonin immunoreactivities are restricted to the distal axonal segments and to the growth cones; in the presence of the target, the formation of a chemical connection is accompanied by redistribution of the synapsin and serotonin immunoreactivities that concentrate in highly fluorescent round spots scattered along the newly grown neurites located close to the target cell. Almost every spot that is stained for serotonin is also positive for synapsin. In the presynaptic cell plated alone, the number of these varicosity-like structures is substantially stable throughout the whole period; by contrast, when the presynaptic cell synapses the target, their number increases progressively parallel to the increase in the mean amplitude of cumulative excitatory postsynaptic potentials recorded at the same times. The data indicate that mollusc synapsin-like peptides to some extent resemble their mammalian homologues, although they are not exclusively localized in nerve terminals and their expression strongly correlates with the formation of active synaptic contacts.     

Synaptic Vesicle Depletion in Reticulospinal Axons is Reduced by 5-hydroxytryptamine: Direct Evidence for Presynaptic Modulation of Glutamatergic Transmission

5-hydroxytryptamine (5-HT; serotonin) is known to depress glutamatergic synaptic transmission in the spinal cord of vertebrates. To test directly whether 5-HT inhibits synaptic glutamate release, we examined its effect on the ultrastructure of synaptic vesicle clusters in giant reticulospinal axons in a lower vertebrate (lamprey; Lampetra fluviatilis ). The size of these axons makes it possible to selectively expose only a part of the presynaptic element to 5-HT, while another part of the same axon is maintained in control solution. Action potential stimulation at 20 Hz for 20 min caused a marked reduction in the number of synaptic vesicles in active zones maintained in control solution, while in the part exposed to 5-HT (20 μM) the number of synaptic vesicles per active zone was -3-fold higher. In contrast, 5-HT had no effect on the number of vesicles in resting axons. To examine whether 5-HT acts by reducing presynaptic Ca²⁺ influx, intra-axonal recordings of Ba²⁺ potentials were performed. No reduction of the axonal Ba²⁺ potential could be detected after application of 20 or 200 pM 5-HT. The present results show that 5-HT reduces the rate of synaptic exocytosis in reticulospinal axons. The effect appears to be mediated by a mechanism distinct from the presynaptic Ca²⁺ channels.     

Presynaptic size of associational/commissural CA3 synapses is controlled by fibroblast growth factor 22 in adult mice

Associational/commissural CA3–CA3 synapses define the recurrent CA3 network that generates the input to CA1 pyramidal neurons. We quantified the fine structure of excitatory synapses in the stratum radiatum of the CA3d area in adult wild type (WT) and fibroblast growth factor 22 knock‐out (FGF22KO) mice by using serial 3D electron microscopy. WT excitatory CA3 synapses are rather small yet range 10 fold in size. Spine size, however, was small and uniform and did not correlate with the size of the synaptic junction. To reveal mechanisms that regulate presynaptic structure, we investigated the role of FGF22, a target‐derived signal specific for the distal part of area CA3 (CA3d). In adult FGF22KO mice, postsynaptic properties of associational CA3 synapses were unaltered. Presynaptically, the number of synaptic vesicles (SVs), the bouton volume, and the number of vesicles in axonal regions (the super pool) were reduced. This concurrent decrease suggests concerted control by FGF22 of presynaptic size. This hypothesis is supported by the finding that WT presynapses in the proximal part of area CA3 (CA3p) that do not receive FGF22 signaling in WT mice were smaller than presynapses in CA3d in WT but of comparable size in CA3d of FGF22KO mice. Docked SV density was decreased in CA1, CA3d, and CA3p in FGF22KO mice. Because CA1 and CA3p are not directly affected by the loss of FGF22, the smaller docked SV density may be an adaptation to activity changes in the CA3 network. Thus, docked SV density potentially is a long‐term regulator for the synaptic release probability and/or the strength of short‐term depression in vivo. © 2015 Wiley Periodicals, Inc.     

Presynaptic inhibition of excitatory neurotransmission by lamotrigine in the rat amygdalar neurons

Lamotrigine (LAG) is a new antiepileptic drug which is licensed as adjunctive therapy for partial and secondary generalized seizures. In the present study, the mechanisms responsible for its antiepileptic effect were studied in rat amygdaloid slices using intracellular recording and whole-cell patch clamp techniques. Bath application of LAG (50 μM) reversibly suppressed the excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) evoked by stimulating ventral endopyriform nucleus. Synaptic response mediated by the N-methyl-D-aspartate (NMDA) receptor (EPSP _NMDA) was isolated pharmacologically by application of a solution containing non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX,10 μM) and γ-aminobutyric acid_A receptor antagonist bicuculline (20 μM). LAG produced a parallel inhibition of EPSP _NMDA. Postsynaptic depolarization induced by α-amino-5-methyl-4-isoxazole propionate (AMPA) was not altered by LAG. In addition, LAG increased the ratio of the second pulse response to the first pulse response (P₂/P₁), which is consistent with a presynaptic mode of action. The L-type Ca⁺⁺ channel blocker nifedipine (20 μM) had no effect on LAG-induced presynaptic inhibition. However, the depressant effect of LAG was markedly reduced in slices pretreated with N-type Ca⁺⁺ channel blocker ωconotoxin-GVIA (ω-CgTX-GVIA, 1 μM) or a broad spectrum Ca⁺⁺ channel blocker ω-conotoxin-MVIIC (ω-CgTX-MVIIC, 1 μM). It is concluded that a reduction in ω-CgTX-GVIA-sensitive Ca⁺⁺ currents largely contributes to LAG-induced presynaptic inhibition. © 1996 Wiley-Liss, Inc.     

Light and electron microscopic analysis of synaptic development in Macaca monkey retina as detected by immunocytochemical labeling for the synaptic vesicle protein,SV2

The development of synapses has been followed in Macaca monkey fetal and infant retina using immunocytochemical labeling for the transmembrane synaptic vesicle glycoprotein, SV2. Electron microscopy (EM) was used to verify the presence of morphological synapses at selected ages. EM immunocytochemical labeling in adult retina showed that all synaptic types contained SV2 in inner (IPL) and outer (OPL) plexiform layers. In fetal retina, SV2 expression and the appearance of morphological synapses were closely related in time, demonstrating that SV2 is a reliable marker for synaptogenesis. SV2 expression appears along a foveal to peripheral gradient. Both SV2 and synapses appear in the foveal IPL at Fd50–55, and reach the retinal edge by Fd90–103. Cone ribbon synapses and SV2 labeling are not present in the foveal OPL until Fd60. Photoreceptors in the far periphery contain SV2 by Fd119–125. This pattern demonstrates an “inner to outer” direction of synaptogenesis. Cones show SV2 labeling before rods at the same retinal eccentricity. In the cone-dominated fovea, SV2 labeling and bipolar cell ribbon-containing terminals are present at Fd55 when amacrine cell conventional terminals are very scarce, indicating that bipolar synapses precede amacrine synapses in monkey foveal IPL. SV2 labeling and bipolar terminals appear first in the outer IPL which contains “OFF” ganglion and bipolar processes in the adult, suggesting that “OFF” midget bipolar cells may form the first synapses. Both SV2 immunocytochemical labeling and EM morphology find that monkey retina follows a generalized inner before outer, and cone before rod synaptic developmental pattern, similar to that in other mammals. The cone-dominated fovea initiates synaptogenesis, and shows a different synaptic sequence from rod-dominated peripheral retina. © 1994 Wiley-Liss, Inc.     

Synaptic proteins in frontal and control brain regions of rats after exposure to spatial problems

Synaptic proteins D1, D2, D3, synaptin and 14-3-2, as well as the glial protein glutamine synthetase, were measured by crossed immunoelectrophoresis in the anteromedial (prefrontal) cortex, occipital (visual) cortex and the anterior and posterior parts of the neostriatum of rats. The 3 experimental groups consisted of rats trained to criterion in a spatial delayed alternation, those run as yoked controls and, finally, rats kept in individual cages and not subjected to any training. Statistical analysis showed that two variables: behavioral procedures and brain regions, had a significant effect. Their interaction was also significant. Further analysis revealed that only in the prefrontal cortex of the yoked control animals was there a significant decrease of the synaptic membrane proteins D1, D2 and D3. Thus, particular behavioral treatment seems capable of affecting synapses in a specific ‘association’ cortical area. The change is more easily related to the amount of ‘work’ than to formation of ‘memory trace’ within the critical area.     

Voltage-independent autocrine modulation of L-type channels mediated by ATP, opioids and catecholamines in rat chromaffin cells

The inhibition of L-type channels induced by either bath application of ATP, opioids and catecholamines or by endogenously released neurotransmitters was investigated in rat chromaffin cells with whole-cell recordings (5 mM Ba2+). In both cases, the L-type current, isolated pharmacologically using omega-toxin peptides and potentiated by Bay K 8644, was inhibited by approximately 50% with nearly no changes to the activation-inactivation kinetics. Inhibition was voltage independent at a wide range of potentials (-20 to +50 mV) and insensitive to depolarizing prepulses (+100 mV, 50 ms). Onset and offset of the inhibition were fast (time constants: tau(on) approximately 0.9 s, tau(off) approximately 3.6 s), indicating a rapid mechanism of channel modulation. Whether induced exogenously or from the released granules content in conditions of stopped cell superfusion, the neurotransmitter action was reversible and largely prevented by either intracellular GDP-beta-S, cell treatment with pertussis toxin or simultaneous application of P2y,2x delta/mu-opioidergic and alpha/beta-adrenergic antagonists. This suggests the existence of converging modulatory pathways by which autoreceptors-activated G-proteins reduce the activity of L-type channels through fast interactions. The autocrine inhibition of L-type currents, which was absent in superfused isolated cells, was effective on cell clusters, suggesting that L-type channels may be potently inhibited by cell exocytosis under physiological conditions resembling the intact adrenal glands.     

Unexpected inhibitory regulation of glutamate release from rat cerebrocortical nerve terminals by presynaptic 5-hydroxytryptamine-2A receptors

Presynaptic 5-HT(2A) receptor modulation of glutamate release from rat cerebrocortical nerve terminals (synaptosomes) was investigated by using the 5-HT(2A/2C) receptor agonist (+/-)-1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI). DOI potently inhibited 4-aminopyridine (4AP)-evoked glutamate release. Involvement of presynaptic 5-HT(2A) receptors in this modulation of 4AP-evoked release was confirmed by blockade of the DOI-mediated inhibition by the 5-HT(2A) receptor antagonist ketanserin but not by the 5-HT(2C) receptor antagonist RS102221. Inhibition of glutamate release by DOI was associated with a reduction of 4AP-evoked depolarization and downstream elevation of cytoplasmic free calcium concentration ([Ca(2+)](C)) mediated via P/Q- and N-type voltage-dependent Ca(2+) channels (VDCCs). In contrast to the DOI effect on 4AP-evoked release, the agonist had no effect on high external [K(+)] (30 mM)-induced (KCl) stimulation of VDCCs or glutamate release. Likewise, release mediated by direct Ca(2+) entry with Ca(2+) ionophore (ionomycin) or by hypertonic sucrose was unaffected by DOI. Mechanistically, DOI modulation of 4AP-evoked glutamate release appeared to involve a phospholipase C/protein kinase C signaling cascade, insofar as pretreatment of synaptosomes with the phospholipase C inhibitor U73122 or protein kinase C inhibitors Ro320432 or GF109203X all effectively occluded the inhibitory effect of the agonist. Together, these results suggest that presynaptic 5-HT(2A) receptors present on glutamatergic terminals effect an unexpected depression of glutamate release by negatively modulating nerve terminal excitability and downstream VDCC activation through a signaling cascade involving phospholipase C/protein kinase C. These observations invoke presynaptic inhibitory 5-HT(2A) receptor function as a potential target for drugs to mitigate the effects of excessive glutamatergic transmission.