Expression of olfactory receptors in different life stages and life histories of wild Atlantic salmon (Salmo salar)

It has been hypothesized that salmonids use olfactory cues to return to their natal rivers and streams. However, the key components of the molecular pathway involved in imprinting and homing are still unknown. If odorants are involved in salmon homing migration, then olfactory receptors should play a critical role in the dissipation of information from the environment to the fish. Therefore, we examined the expression profiles of a suite of genes encoding olfactory receptors and other olfactory-related genes in the olfactory rosettes of different life stages in two anadromous and one non-anadromous wild Atlantic salmon populations from Newfoundland, Canada. We identified seven differentially expressed OlfC genes in juvenile anadromous salmon compared to returning adults in both populations of anadromous Atlantic salmon. The salmon from the Campbellton River had an additional 10 genes that were differentially expressed in juveniles compared to returning adults. There was no statistically significant difference in gene expression of any of the genes in the non-anadromous population (P < 0.01). The function of the OlfC gene products is not clear, but they are predicted to be amino acid receptors. Other studies have suggested that salmon use amino acids for imprinting and homing. This study, the first to examine the expression of olfactory-related genes in wild North American Atlantic salmon, has identified seven OlfC genes that may be involved in the imprinting and homeward migration of anadromous Atlantic salmon.     

Visualizing recycling of synaptic vesicle proteins

The use of modern techniques (in particular, novel fluorescence markers of a few molecular participants of the exo-and endocytotic processes, including pH-sensitive agents, immuno-electron and laser-scanning microscopy) allows experimenters to visualize different stages of recycling of synaptic vesicle proteins. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 350–351, July–October, 2007.     

Trafficking of the vesicular acetylcholine transporter in SN56 cells: a dynamin-sensitive step and interaction with the AP-2 adaptor complex

The pathways by which synaptic vesicle proteins reach their destination are not completely defined. Here we investigated the traffic of a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) in cholinergic SN56 cells, a model system for neuronal processing of this cargo. GFP-VAChT accumulates in small vesicular compartments in varicosities, but perturbation of endocytosis with a dominant negative mutant of dynamin I-K44A impaired GFP-VAChT trafficking to these processes. The protein in this condition accumulated in the cell body plasma membrane and in large vesicular patches therein. A VAChT endocytic mutant (L485A/L486A) was also located at the plasma membrane, however, the protein was not sorted to dynamin I-K44A generated vesicles. A fusion protein containing the VAChT C-terminal tail precipitated the AP-2 adaptor protein complex from rat brain, suggesting that VAChT directly interacts with the endocytic complex. In addition, yeast two hybrid experiments indicated that the C-terminal tail of VAChT interacts with the micro subunit of AP-2 in a di-leucine (L485A/L486A) dependent fashion. These observations suggest that the di-leucine motif regulates sorting of VAChT from the soma plasma membrane through a clathrin dependent mechanism prior to the targeting of the transporter to varicosities.     

槲皮素抑制中枢神经突触前兴奋性依赖的胞吞动力学

目的 槲皮素是一种广泛分布于药用植物中的黄酮类化合物,传统被认为具有神经保护作用。本研究利用位于大鼠脑干花萼状突触的突触前神经末梢进行膜片钳记录,研究槲皮素调控突触传递和可塑性的突触前机制。方法 利用全细胞膜片钳结合膜电容记录,在突触后记录微小兴奋性突触后电流（m EPSC）,在突触前神经末梢记录钙內流和神经囊泡的释放、回收以及可立即释放库（RRP）的恢复动力学。并且利用纤维刺激在轴突给予5～200 Hz的刺激,诱发突触后EPSC,记录突触后短时程抑制（STD）。结果 100μmol/L槲皮素不影响突触后m EPSC的振幅、频率以及AMPA受体的动力学特征。在突触前神经末梢,槲皮素不改变钙内流或囊泡的释放,但显著抑制胞吐后网格蛋白依赖的慢速胞吞。抑制胞吞会导致突触前囊泡动员的减慢,降低RRP的补充速率,并且增强高频刺激下的短时程可塑性STD。结论 本研究为槲皮素调控中枢神经突触传递提供全新的突触前神经机制,槲皮素有助于抑制中枢神经过度兴奋,进而发挥神经保护作用。     

槲皮素通过抑制胞吞增强短时程抑制效应

目的 槲皮素是一种广泛分布于药用植物中的黄酮类化合物,传统被认为具有神经保护作用。在本研究中,我们利用位于大鼠脑干的花萼状突触的突触前神经末梢的进行膜片钳记录,研究槲皮素调控突触传递和可塑性的突触前机制。方法 利用全细胞膜片钳结合膜电容记录,在突触后记录微小兴奋性突触后电流（mEPSC）,在突触前神经末梢记录钙內流和神经囊泡的释放、回收以及可立即释放库（RRP）的恢复动力学。并且利用纤维刺激在轴突给予5~200 Hz的刺激,诱发突触后EPSC,记录突触后短时程抑制（STD）。结果 100 μmol/L槲皮素不影响突触后mEPSC的振幅、频率以及AMPA受体的动力学特征。在突触前神经末梢,槲皮素不改变钙内流或囊泡的释放,但显著抑制胞吐后的网格蛋白依赖的慢速胞吞。抑制胞吞会导致突触前囊泡动员的减慢,降低RRP的补充速率,并且增强高频刺激下的短时程可塑性STD。结论 本研究为槲皮素调控中枢神经突触传递提供全新的突触前神经机制,槲皮素有助于抑制中枢神经过度兴奋,进而发挥神经保护作用。     

Complexin arrests a pool of docked vesicles for fast Ca(2+)-dependent release

Regulated exocytosis requires that the assembly of the basic membrane fusion machinery is temporarily arrested. Synchronized membrane fusion is then caused by a specific trigger-a local rise of the Ca(2+) concentration. Using reconstituted giant unilamellar vesicles (GUVs), we have analysed the role of complexin and membrane-anchored synaptotagmin 1 in arresting and synchronizing fusion by lipid-mixing and cryo-electron microscopy. We find that they mediate the formation and consumption of docked small unilamellar vesicles (SUVs) via the following sequence of events: Synaptotagmin 1 mediates v-SNARE-SUV docking to t-SNARE-GUVs in a Ca(2+)-independent manner. Complexin blocks vesicle consumption, causing accumulation of docked vesicles. Together with synaptotagmin 1, complexin synchronizes and stimulates rapid fusion of accumulated docked vesicles in response to physiological Ca(2+) concentrations. Thus, the reconstituted assay resolves both the stimulatory and inhibitory function of complexin and mimics key aspects of synaptic vesicle fusion.     

Kinesin-II is required for axonal transport of choline acetyltransferase in Drosophila.

KLP64D and KLP68D are members of the kinesin-II family of proteins in Drosophila. Immunostaining for KLP68D and ribonucleic acid in situ hybridization for KLP64D demonstrated their preferential expression in cholinergic neurons. KLP68D was also found to accumulate in cholinergic neurons in axonal obstructions caused by the loss of kinesin light chain. Mutations in the KLP64D gene cause uncoordinated sluggish movement and death, and reduce transport of choline acetyltransferase from cell bodies to the synapse. The inviability of KLP64D mutations can be rescued by expression of mammalian KIF3A. Together, these data suggest that kinesin-II is required for the axonal transport of a soluble enzyme, choline acetyltransferase, in a specific subset of neurons in Drosophila. Furthermore, the data lead to the conclusion that the cargo transport requirements of different classes of neurons may lead to upregulation of specific pathways of axonal transport.     

Emerging functions of the calpain superfamily of cysteine proteases in neuroendocrine secretory pathways

The first calpain protease was discovered over 40 years ago now, yet despite the vast amount of literature that has subsequently emerged detailing their involvement in the pathophysiology of a variety of human diseases, it is only in the last decade that calpain-mediated actions along the secretory pathway have begun to emerge. However, the number of secretory pathway substrates identified and their diversity of function continues to grow. This review summarizes our current knowledge of calpain-mediated mechanisms of action that are pertinent to synaptic vesicle assembly and budding, cytoskeletal organization, endosomal recycling, and exocytotic membrane fusion.     

Structural and Functional Analysis of the CAPS SNARE-Binding Domain Required for SNARE Complex Formation and Exocytosis

1. Download high-res image (209KB)
2. Download full-size image

     

Adenosine Triphosphate and the Late Steps in Calcium-dependent Exocytosis at a Ribbon Synapse

The ATP dependence of the kinetics of Ca²⁺-dependent exocytosis after flash photolysis of caged Ca²⁺ was studied by capacitance measurements with submillisecond resolution in single synaptic terminals of retinal bipolar neurons. After control experiments verified that this combination of techniques is valid for the study of exocytosis in synaptic terminals, a comparison was made between the Ca²⁺ dependence of the rate of exocytosis in synaptic terminals internally dialyzed with MgATP, MgATP-γ-S, or no added Mg²⁺ or nucleotide. The Ca²⁺ threshold for release, the maximum rate of release, and the overall relationship between the rate of synaptic vesicle fusion and [Ca²⁺]_i were found to be independent of MgATP. A decrease in the average rate at near-threshold [Ca²⁺]_i was observed in terminals with MgATP-γ-S, but due to the small sample size is of unclear significance. The Ca²⁺ dependence of the delay between the elevation of [Ca²⁺]_i and the beginning of the capacitance rise was also found to be independent of MgATP. In contrast, MgATP had a marked effect on the ability of terminals to respond to multiple stimuli. Terminals with MgATP typically exhibited a capacitance increase to a second stimulus that was >70% of the amplitude of the first response and to a third stimulus with a response amplitude that was >50% of the first, whereas terminals without MgATP responded to a second stimulus with a response <35% of the first and rarely responded to a third flash. These results suggest a major role for MgATP in preparing synaptic vesicles for fusion, but indicate that cytosolic MgATP may have little role in events downstream of calcium entry, provided that [Ca²⁺]_i near release sites is elevated above ≈30 μM.     

New generation QuIC assays for prion seeding activity

The ability of abnormal TSE-associated forms of PrP to seed the formation of amyloid fibrils from recombinant PrP^Sen has served as the basis for several relatively rapid and highly sensitive tests for prion diseases. These tests include rPrP-PMCA (rPMCA), standard quaking-induced conversion (S-QuIC), amyloid seeding assay (ASA), real-time QuIC (RT-QuIC) and enhanced QuIC (eQuIC). Here, we summarize recent improvements in the RT-QuIC-based assays that enhance the practicality, sensitivity and quantitative attributes of assays QuIC and promote the detection of prion seeding activity in dilute, inhibitor-laden fluids such as blood plasma.     

Syntaxin-1通过激活突触递质传递加速早期突触的形成

Syntaxin-1是一种多结构域蛋白,通过与synaptobrevin-2和SNAP-25形成SNARE复合体调节囊泡融合.然而,syntaxin-1在突触形成过程中是否发挥作用,目前尚不清楚.本研究显示syntaxin-1的表达水平与突触形成过程高度相关.Syntaxin-1的R151A和I155A突变影响其在突触形成中的促进作用,而Habc结构域或跨膜结构域在突触形成中无显著作用.结果表明,syntaxin-1通过激活突触囊泡释放来加速突触的形成.     

Kiss-and-run and full-collapse fusion as modes of exo-endocytosis in neurosecretion

Neurotransmitters and hormones are released from neurosecretory cells by exocytosis (fusion) of synaptic vesicles, large dense-core vesicles and other types of vesicles or granules. The exocytosis is terminated and followed by endocytosis (retrieval). More than fifty years of research have established full-collapse fusion and clathrin-mediated endocytosis as essential modes of exo-endocytosis. Kiss-and-run and vesicle reuse represent alternative modes, but their prevalence and importance have yet to be elucidated, especially in neurons of the mammalian CNS. Here we examine various modes of exo-endocytosis across a wide range of neurosecretory systems. Full-collapse fusion and kiss-and-run coexist in many systems and play active roles in exocytotic events. In small nerve terminals of CNS, kiss-and-run has an additional role of enabling nerve terminals to conserve scarce vesicular resources and respond to high-frequency inputs. Full-collapse fusion and kiss-and-run will each contribute to maintaining cellular communication over a wide range of frequencies.     

Cell entry strategy of clostridial neurotoxins

Tetanus neurotoxin and botulinum neurotoxins are the causative agents of tetanus and botulism. They block the release of neurotransmitters from synaptic vesicles in susceptible animals and man and act in nanogram quantities because of their ability to specifically attack motoneurons. They developed an ingenious strategy to enter neurons. This involves a concentration step via complex polysialo gangliosides at the plasma membrane and the uptake and ride in recycling synaptic vesicles initiated by binding to a specific protein receptor. Finally, the neurotoxins shut down the synaptic vesicle cycle, which they had misused before to enter their target cells, via specific cleavage of protein core components of the cellular membrane fusion machinery. The uptake of four out of seven known botulinum neurotoxins into synaptic vesicles has been demonstrated to rely on binding to intravesicular segments of the synaptic vesicle proteins synaptotagmin or synaptic vesicle protein 2. This review summarizes the present knowledge about the cell receptor molecules and the mode of toxin-receptor interaction that enables the toxins' sophisticated access to their site of action.     

Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis

The nerve terminal proteome governs neurotransmitter release as well as the structural and functional dynamics of the presynaptic compartment. In order to further define specific presynaptic subproteomes we used subcellular fractionation and a monoclonal antibody against the synaptic vesicle protein SV2 for immunoaffinity purification of two major synaptosome-derived synaptic vesicle-containing fractions: one sedimenting at lower and one sedimenting at higher sucrose density. The less dense fraction contains free synaptic vesicles, the denser fraction synaptic vesicles as well as components of the presynaptic membrane compartment. These immunoisolated fractions were analyzed using the cationic benzyldimethyl-n-hexadecylammonium chloride (BAC) polyacrylamide gel system in the first and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Protein spots were subjected to analysis by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS). We identified 72 proteins in the free vesicle fraction and 81 proteins in the plasma membrane-containing denser fraction. Synaptic vesicles contain a considerably larger number of protein constituents than previously anticipated. The plasma membrane-containing fraction contains synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery and numerous other proteins potentially involved in regulating the functional and structural dynamics of the nerve terminal.