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Synaptic vesicle protein trafficking at the glutamate synapse
Abstract
Expression of the integral and associated proteins of synaptic vesicles is subject to regulation over time, by region, and in response to activity. The process by which changes in protein levels and isoforms result in different properties of neurotransmitter release involves protein trafficking to the synaptic vesicle. How newly synthesized proteins are incorporated into synaptic vesicles at the presynaptic bouton is poorly understood. During synaptogenesis, synaptic vesicle proteins sort through the secretory pathway and are transported down the axon in precursor vesicles that undergo maturation to form synaptic vesicles. Changes in protein content of synaptic vesicles could involve the formation of new vesicles that either mix with the previous complement of vesicles or replace them, presumably by their degradation or inactivation. Alternatively, new proteins could individually incorporate into existing synaptic vesicles, changing their functional properties. Glutamatergic vesicles likely express many of the same integral membrane proteins and share certain common mechanisms of biogenesis, recycling, and degradation with other synaptic vesicles. However, glutamatergic vesicles are defined by their ability to package glutamate for release, a property conferred by the expression of a vesicular glutamate transporter (VGLUT). VGLUTs are subject to regional, developmental, and activity-dependent changes in expression. In addition, VGLUT isoforms differ in their trafficking, which may target them to different pathways during biogenesis or after recycling, which may in turn sort them to different vesicle pools. Emerging data indicate that differences in the association of VGLUTs and other synaptic vesicle proteins with endocytic adaptors may influence their trafficking. These observations indicate that independent regulation of synaptic vesicle protein trafficking has the potential to influence synaptic vesicle protein composition, the maintenance of synaptic vesicle pools, and the release of glutamate in response to changing physiological requirements.
... Several classes of vesicular transporters have been characterized, each associated with a different neurotransmitter: acetylcholine (VAChT); monoamines (VMAT2); GABA and glycine (VGAT); and glutamate (VGluT1, VGluT2, VGluT3). Activity-dependent regulation of their expression can alter the number of transporters localized to each vesicle, impacting vesicle filling, quantal size, and the amount and probability of neurotransmitter release (Blakely and Edwards 2012;Edwards 2007;Erickson et al. 2006;Fei et al. 2008;Hnasko and Edwards 2012;Omote et al. 2011;Santos et al. 2009;Takamori et al. 2006;Wilson et al. 2005;Wojcik et al. 2004). Accordingly, these transporters contribute to pre-and postsynaptic homeostatic mechanisms that regulate the balance of excitatory and inhibitory neurotransmission (Coleman et al. 2010;De Gois et al. 2005;Lazarevic et al. 2013;Rich and Wenner 2007;Turrigiano 2008;Turrigiano and Nelson 2004;Wilson et al. 2005). ...
... Although the potential significance of these structural details may not be immediately obvious, they have potentially profound implications for the nature of excita-tory neurotransmission in TC and CC circuits. VGluT2 has been associated with a higher probability of glutamate release than VGluT1 in some brain regions (Blakely and Edwards 2012;Hnasko and Edwards 2012;Santos et al. 2009;Varoqui et al. 2002). In addition, the number of transporters located on each vesicle has an impact on vesicle filling, quantal size, and the amount of neurotransmitter released (Blakely and Edwards 2012;Edwards 2007;Erickson et al. 2006;Fei et al. 2008;Hnasko and Edwards 2012;Omote et al. 2011;Santos et al. 2009;Takamori et al. 2006;Wilson et al. 2005;Wojcik et al. 2004). ...
... VGluT2 has been associated with a higher probability of glutamate release than VGluT1 in some brain regions (Blakely and Edwards 2012;Hnasko and Edwards 2012;Santos et al. 2009;Varoqui et al. 2002). In addition, the number of transporters located on each vesicle has an impact on vesicle filling, quantal size, and the amount of neurotransmitter released (Blakely and Edwards 2012;Edwards 2007;Erickson et al. 2006;Fei et al. 2008;Hnasko and Edwards 2012;Omote et al. 2011;Santos et al. 2009;Takamori et al. 2006;Wilson et al. 2005;Wojcik et al. 2004). Given these differences in the synaptic properties associated with VGluT1 and VGluT2, rather different influences could be conferred upon a variety of postsynaptic targets. ...
Vesicular transporter proteins are an essential component of the presynaptic machinery that regulates neurotransmitter storage and release. They also provide a key point of control for homeostatic signaling pathways that maintain balanced excitation and inhibition following changes in activity levels, including the onset of sensory experience. To advance understanding of their roles in the developing auditory forebrain, we tracked the expression of the vesicular transporters of glutamate (VGluT1, VGluT2) and GABA (VGAT) in primary auditory cortex (A1) and medial geniculate body (MGB) of developing mice (P7, P11, P14, P21, adult) before and after ear canal opening (~P11-P13). RNA sequencing, in situ hybridization, and immunohistochemistry were combined to track changes in transporter expression and document regional patterns of transcript and protein localization. Overall, vesicular transporter expression changed the most between P7 and P21. The expression patterns and maturational trajectories of each marker varied by brain region, cortical layer, and MGB subdivision. VGluT1 expression was highest in A1, moderate in MGB, and increased with age in both regions. VGluT2 mRNA levels were low in A1 at all ages, but high in MGB, where adult levels were reached by P14. VGluT2 immunoreactivity was prominent in both regions. VGluT1 (+) and VGluT2 (+) transcripts were co-expressed in MGB and A1 somata, but co-localization of immunoreactive puncta was not detected. In A1, VGAT mRNA levels were relatively stable from P7 to adult, while immunoreactivity increased steadily. VGAT (+) transcripts were rare in MGB neurons, whereas VGAT immunoreactivity was robust at all ages. Morphological changes in immunoreactive puncta were found in two regions after ear canal opening. In the ventral MGB, a decrease in VGluT2 puncta density was accompanied by an increase in puncta size. In A1, perisomatic VGAT and VGluT1 terminals became prominent around the neuronal somata. Overall, the observed changes in gene and protein expression, regional architecture, and morphology relate to-and to some extent may enable-the emergence of mature sound-evoked activity patterns. In that regard, the findings of this study expand our understanding of the presynaptic mechanisms that regulate critical period formation associated with experience-dependent refinement of sound processing in auditory forebrain circuits.
... period of depolarization, figure 58) (Neves & Lagnado, 1999;Rizzoli & Betz, 2005;Santos et al., 2009). ...
... The de novo production of secretory vesicles has already been described in the chapter 2 1-c-i-2. To In the case of SVP axonal transport, KIF1A or KIF1Bβ are recruited based on the subset of SPs they carry Santos et al., 2009). For example, synaptophysin is transported by KIF1A . ...
Neuronal circuits are at the basis of behaviors such as motor coordination or learning and memory. As being part of a network, neurons communicate at synapses through finely tuned molecular and cellular processes. One key mechanism regulating synapse homeostasis involves transport of vesicles within axons and dendrites which is dysregulated in many neurological disorders such as Rett syndrome, Alzheimer’s (AD) and Huntington’s diseases (HD). Thus, deciphering the regulation of vesicular transport within neurites in physiological context is crucial to understand, and potentially restore, the consequences of these dysregulations in pathological contexts.Huntingtin (HTT) protein, known for its devastating role in HD when mutated, is a key actor of axonal transport. It promotes and regulates vesicular transport in neurites by scaffolding adaptors and molecular motors. Particularly, HTT phosphorylation status on S421 regulates the directionality of BDNF, APP and VAMP-7 vesicles within neurites in cultured and transfected neurons. However, several questions remain to be elucidated regarding the mechanisms and the consequences of this HTT-dependent regulation of axonal transport such as the neuritic specificity (axons or dendrites) and the behavioral consequences of such modifications. Finally, we do not know whether transport regulation can be influenced in pathological conditions to restore disease-associated phenotypes in vivo.This thesis aims at characterizing in vivo the mechanisms and the consequences of axonal transport regulation of three different vesicles through the phosphorylation of Huntingtin at S421 and to investigate its propensity to restore disease-associated phenotypes in mouse models of human neurological disorders.In order to reproduce in vitro the in vivo networks associated to neurological disorders we used microfluidic devices. We investigated the transport of Amyloid Precursor Protein (APP) vesicles, precursors of synaptic vesicles (SVPs) or dense-core vesicles (DCVs) in neurons in which the HTT phosphorylation status was modified. These neurons came from mice in which Serine 421 has been replaced by an aspartic acid to mimic the phosphorylated form of HTT (HTTS421D) or by an alanine to mimic the unphosphorylatable form of HTT (HTTS421A).Transport of APP vesicles is impaired in AD. We investigated APP transport and accumulation at synapses within a corticocortical circuit. We found that Akt-mediated HTT phosphorylation at S421 regulates the directionality of APP containing vesicles in axons but not in dendrites: the phospho-defective form of HTT decreases axonal anterograde flux of APP and reduces its levels at presynaptic zones both in vitro and in vivo. Reducing anterograde flux of APP in familial AD mouse model restored synapse homeostasis in vivo and memory deficits (Publication 21; Bruyere*, Abada*, Vitet* et al., eLife, 2020).SVP axonal transport regulates the number of SVs at the synapse, which, within a corticostriatal synapse, is essential for motor skill learning. We found that HTT phosphorylation increases the recruitment of the molecular motor KIF1A on SVPs, thus promoting anterograde transport and the probability of release. Silencing KIF1A in the corticostriatal network of HTTS421D mice, we found that pHTTS421 increases the number of SVs at the synapse and impairs procedural memory through a specific HTT-KIF1A dependent mechanism. This study defines a pathway by which axonal transport of SVP impact the behavioral phenotype. (Publication 2; Vitet et al, in prep)Finally, it has been shown that BDNF transport within DCVs is dysregulated in the corticostriatal network of Rett syndrome’s patients. We found that endogenous HTT phosphorylation at S421 or a chemical inhibitor of calcineurin (FK506) rescue BDNF transport in the corticostriatal network, neuronal communication, and behaviors of Rett Syndrome mice (Publication 3; Ehinger et al., Embo Mol Med,2020).
... ECa 233 also showed a neuritogenic effect, promoting neurite outgrowth on neuroblastoma cells via ERK1/2 and Akt pathways 8 . Moreover, ECa 233 demonstrated neuroprotective effects by attenuating the learning and memory deficit induced by transient bilateral occlusion of common carotid arteries (T2VO) 9 or cerebral infusion of amyloid beta peptide 25-35 fragments (Aβ [25][26][27][28][29][30][31][32][33][34][35] 10 . These neuroprotective effects were proposed to act through anti-oxidant and anti-inflammatory mechanisms of ECa 233. ...
... According to the study of Yin and colleagues in 2015 30 , asiaticoside, which is the other major active constituent of ECa 233, also promoted the expression of p-Akt, GSK-3, synaptophysin (SYN) and PSD-95 in high glucose-treated neuroblastoma cells 30 . They suggested that asiaticoside could mediate the alteration of presynaptic terminals because it promoted an expression of SYN, a synaptic vesicle membrane protein related to the release of glutamate vesicles from presynaptic terminals, and also promoted downstream Trk-signaling cascades 30,31 . ...
The herb Centella asiatica has long been considered a memory tonic. A recent review found no strong evidence for improvement of cognitive function, suggesting negative results were due to limitations in dose, standardization and product variation. We used a standardized extract of C. asiatica (ECa 233) to study behavioral, cellular and molecular effects on learning and memory enhancement. ECa 233 (10, 30, and 100 mg/kg) was given orally to normal rats twice a day for 30 days. We used the Morris water maze to test spatial learning and performed acute brain slice recording to measure changes of synaptic plasticity in the hippocampus, a core brain region for memory formation. Plasticity-related protein expressions (NR2A, NR2B, PSD-95, BDNF and TrkB) in hippocampus was also measured. Rats receiving 10 and 30 mg/kg doses showed significantly enhanced memory retention, and hippocampal long-term potentiation; however, only the 30 mg/kg dose showed increased plasticity-related proteins. There was an inverted U-shaped response of ECa 233 on memory enhancement; 30 mg/kg maximally enhanced memory retention with an increase of synaptic plasticity and plasticity-related proteins in hippocampus. Our data clearly support the beneficial effect on memory retention of a standardized extract of Centella asiatica within a specific therapeutic range.
... Vesicular transporters undergo complex regulation of their trafficking, which has been well studied in the case of VAChT and vesicular monoamine transporters (VMAT1-2) (Tan et al. 1998;Krantz et al. 2000;Pothos et al. 2000;Santos et al. 2001Santos et al. , 2009Barbosa et al. 2002;Ferreira et al. 2005;Yao and Hersh 2007;Fei et al. 2008). For example, VAChT follows the clathrin-dependent endocytotic pathways before its targeting to varicosities (Tan et al. 1998;Krantz et al. 2000;Pothos et al. 2000;Santos et al. 2001;Ferreira et al. 2005;Fei et al. 2008;Santos et al. 2009; Barbosa et al. 2002). ...
... Vesicular transporters undergo complex regulation of their trafficking, which has been well studied in the case of VAChT and vesicular monoamine transporters (VMAT1-2) (Tan et al. 1998;Krantz et al. 2000;Pothos et al. 2000;Santos et al. 2001Santos et al. , 2009Barbosa et al. 2002;Ferreira et al. 2005;Yao and Hersh 2007;Fei et al. 2008). For example, VAChT follows the clathrin-dependent endocytotic pathways before its targeting to varicosities (Tan et al. 1998;Krantz et al. 2000;Pothos et al. 2000;Santos et al. 2001;Ferreira et al. 2005;Fei et al. 2008;Santos et al. 2009; Barbosa et al. 2002). ...
... We show for the first time that pulmonary vascular cells release glutamate rapidly, in a calcium-dependent manner and that ET-1 is one trigger for glutamate release from PASMC. ET-1 is known to enhance basal glutamate release from sensory neurons with subsequent NMDAR activation 41 and to trigger glutamate efflux from astrocytes 42 . Thus glutamate release and consequent NMDAR activation may mediate some of the deleterious effects of ET-1 on pulmonary arteries during PAH. ...
... présentent un pattern complémentaire dans le cerveau tandis que VGLUT3 est exprimé par des populations neuronales considérées traditionnellement comme n'étant pas glutamatergiques, tels que les neurones cholinergiques, sérotoninergiques et GABAergique où il permet la co-libération du glutamate avec les autres neurotransmetteurs39,41 . Le transport de glutamate dans ces vésicules est un transport actif et consomme donc de l'énergie : ces vésicules contiennent des pompes à protons hydrolysant des molécule d'ATP (V-type H+-ATPase). ...
L'hypertension artérielle pulmonaire (HTAP) est une maladie rare caractérisée par une augmentation de la pression artérielle pulmonaire moyenne liée à un important remodelage de la paroi vasculaire obstruant progressivement les petites artères pulmonaires. Le récepteur NMDA (NMDAR) est un récepteur au glutamate jouant un rôle crucial dans la transmission synaptique neuronale. Il est aussi présent dans des cellules périphériques, notamment les cellules vasculaires aortiques et cérébrales, et participe à leur prolifération. De plus, le NMDAR contribue à la prolifération des cellules cancéreuses. Puisque dans l'HTAP, les cellules vasculaires pulmonaires présentent un phénotype cancer-like, hyperprolifératif et résistant à l'apoptose, nous avons émis l'hypothèse selon laquelle les NMDARs vasculaires pulmonaires pourraient contribuer au remodelage vasculaire et conduire à l'HTAP. Nous avons montré que les cellules vasculaires pulmonaires expriment physiologiquement les principaux éléments d'une communication glutamatergique fonctionnelle via le NMDAR. Dans l'HTAP, le glutamate s'accumule dans les vaisseaux remodelés et l'endothéline-1, un acteur majeur du remodelage vasculaire, induit la libération du glutamate par les cellules musculaires lisses. Le NMDAR est mobilisé dans les cellules vasculaires et sa fonction pourrait être altérée en raison d'un déséquilibre dans le ratio d'expression de ses sous-unités. L'activation du NMDAR contribue à la prolifération des cellules vasculaires pulmonaires et à l'angiogenèse, éléments clés de la physiopathologie de l'HTAP. Des études réalisées sur des souris n'exprimant pas les NMDARs vasculaires ou utilisant des antagonistes du NMDAR mettent en évidence le rôle du NMDAR dans l'hypertension pulmonaire expérimentale. Ces résultats suggèrent que le NMDAR est un nouvel acteur du remodelage vasculaire et qu'il représente une nouvelle cible thérapeutique de l'HTAP. Ils apportent également de nouveaux éléments alimentant l'analogie entre le système vasculaire et le système nerveux.
... Vesicular transporters undergo complex regulation of their trafficking, which has been well studied in the case of VAChT and vesicular monoamine transporters (VMAT1-2) (Tan et al. 1998;Krantz et al. 2000;Pothos et al. 2000;Santos et al. 2001Santos et al. , 2009Barbosa et al. 2002;Ferreira et al. 2005;Yao and Hersh 2007;Fei et al. 2008). For example, VAChT follows the clathrin-dependent endocytotic pathways before its targeting to varicosities (Tan et al. 1998;Krantz et al. 2000;Pothos et al. 2000;Santos et al. 2001;Ferreira et al. 2005;Fei et al. 2008;Santos et al. 2009; Barbosa et al. 2002). ...
... Vesicular transporters undergo complex regulation of their trafficking, which has been well studied in the case of VAChT and vesicular monoamine transporters (VMAT1-2) (Tan et al. 1998;Krantz et al. 2000;Pothos et al. 2000;Santos et al. 2001Santos et al. , 2009Barbosa et al. 2002;Ferreira et al. 2005;Yao and Hersh 2007;Fei et al. 2008). For example, VAChT follows the clathrin-dependent endocytotic pathways before its targeting to varicosities (Tan et al. 1998;Krantz et al. 2000;Pothos et al. 2000;Santos et al. 2001;Ferreira et al. 2005;Fei et al. 2008;Santos et al. 2009; Barbosa et al. 2002). ...
... Because glutamate excitotoxicity is one major cause of neuronal dysfunction upon injury (Aarts & Tymianski, 2004); we also hypothesized that incubation period would positively correlate with changes in glutamate release machinery. Vesicular glutamate transporters (VGluts) are presynaptic proteins located on synaptic vesicles that translocate glutamate into the synaptic vesicle lumen, protecting it from degradation before calcium-dependent, exocytotic release into the synaptic cleft (Santos, Li, & Voglmaier, 2009). Because VGlut1 levels determine the amount of glutamate stored and released per vesicle, and variations in VGlut1 expression regulate the efficacy of glutamate synaptic transmission (Santos et al., 2009), we examined VGlu1 immunohistochemistry. ...
... Vesicular glutamate transporters (VGluts) are presynaptic proteins located on synaptic vesicles that translocate glutamate into the synaptic vesicle lumen, protecting it from degradation before calcium-dependent, exocytotic release into the synaptic cleft (Santos, Li, & Voglmaier, 2009). Because VGlut1 levels determine the amount of glutamate stored and released per vesicle, and variations in VGlut1 expression regulate the efficacy of glutamate synaptic transmission (Santos et al., 2009), we examined VGlu1 immunohistochemistry. ...
Introduction
With its preservation of cytoarchitecture and synaptic circuitry, the hippocampal slice preparation has been a critical tool for studying the electrophysiological effects of pharmacological and genetic manipulations. To analyze the maximum number of slices or readouts per dissection, long incubation times postslice preparation are commonly used. We were interested in how slice integrity is affected by incubation postslice preparation.
Methods
Hippocampal slices were prepared by three different methods: a chopper, a vibratome, and a rotary slicer. To test slice integrity, we compared glycogen levels and immunohistochemistry of selected proteins in rat hippocampal slices immediately after dissection and following 2 and 4 hr of incubation.
Results
We found that immunoreactivity of the dendritic marker microtubule‐associated protein 2 (Map2) drastically decreased during this incubation period, whereas immunoreactivity of the glutamate transporter VGlut1 did not significantly change with incubation time. Astrocytic and microglial cell numbers also did not significantly change with incubation time whereas glycogen levels markedly increased during incubation.
Conclusion
Immunoreactivity of the dendritic marker Map2 quickly decreased after dissection with all the slicing methods. This work highlights a need for caution when using long incubation periods following slice preparation.
... Vesicular transporters undergo complex regulation of their trafficking, which has been well studied in the case of VAChT and vesicular monoamine transporters (VMAT1-2) (Barbosa et al. 2002, Fei et al. 2008, Ferreira et al. 2005, Pothos et al. 2000, Santos et al. 2001, Santos et al. 2009, Tan et al. 1998, Yao & Hersh 2007. For example, VAChT follows the clathrin dependent endocytotic pathways before its targeting to varicosities (Tan et al. 1998, Pothos et al. 2000, Santos et al. 2001, Barbosa et al. 2002, Ferreira et al. 2005, Fei et al. 2008, Santos et al. 2009). ...
... Vesicular transporters undergo complex regulation of their trafficking, which has been well studied in the case of VAChT and vesicular monoamine transporters (VMAT1-2) (Barbosa et al. 2002, Fei et al. 2008, Ferreira et al. 2005, Pothos et al. 2000, Santos et al. 2001, Santos et al. 2009, Tan et al. 1998, Yao & Hersh 2007. For example, VAChT follows the clathrin dependent endocytotic pathways before its targeting to varicosities (Tan et al. 1998, Pothos et al. 2000, Santos et al. 2001, Barbosa et al. 2002, Ferreira et al. 2005, Fei et al. 2008, Santos et al. 2009). ...
Striatal cholinergic interneurons ( CIN ) are pivotal for the regulation of the striatal network. Acetylcholine ( AC h) released by CIN is centrally involved in reward behavior as well as locomotor or cognitive functions. Recently, BAC transgenic mice expressing channelrhodopsin‐2 (ChR2) protein under the control of the choline acetyltransferase (Ch AT ) promoter (Ch AT –ChR2) and displaying almost 50 extra copies of the VAC hT gene were used to dissect cholinergic circuit connectivity and function using optogenetic approaches. These mice display over‐expression of the vesicular acetylcholine transporter ( VAC hT) and increased cholinergic tone. Consequently, Ch AT –ChR2 mice are a valuable model to investigate hypercholinergic phenotypes. Previous experiments established that Ch AT –ChR2 mice display an increased sensitivity to amphetamine induced‐locomotor activity and stereotypes. In the present report, we analyzed the impact of VAC hT over‐expression in the striatum of Ch AT ‐ChR2 mice. Ch AT ‐ChR2 mice displayed increased locomotor sensitization in response to low dose of cocaine. In addition, we observed a dramatic remodeling of the morphology of CIN in Ch AT ‐ChR2 transgenic mice. VAC hT immunolabeling was markedly enhanced in the soma and terminal of CIN from Ch AT ‐ChR2 mice as previously shown (Crittenden et al . 2014). Interestingly, the number of cholinergic varicosities was markedly reduced (−87%) whereas their size was significantly increased (+177%). Moreover, VAC hT over‐expression dramatically modified its trafficking along the somatodendritic and axonal arbor. These findings demonstrate that Ch AT –ChR2 mice present major alterations of CIN neuronal morphology and increased behavioral sensitization to cocaine, supporting the notion that the increased levels of VAC hT observed in these mice make them fundamentally different from wild‐type mice.
image
... Usually two to four boutons, sometimes one bouton, attach to a dendritic shaft by multiple puncta adherentia junctions (PAJs) and wrap around a highly branched dendritic spine, known as thorny excrescences, where multiple synaptic junctions (SJs), neurotransmitter release sites, are formed (Amaral & Dent, 1981). Electron microscopically, three types of pools of the synaptic vesicles (SVs) in the mossy fiber bouton are known: the readily releasable pool in which SVs are docked to the active zones (AZs) or clustered around the AZs, the recycling pool in which SVs are recruited to the AZs by intense stimulation, and the reserve pool in which SVs are far from the AZs and tethered by cytoskeletal elements (Chamberland & T oth, 2016;Santos, Li, & Voglmaier, 2009). Postsynaptic densities (PSDs) are located at the heads of the spine branches and face toward the AZs. ...
... Before each volume was collected, a low magnification (5003) SVs are recruited to the AZs by intense stimulation, and the reserve pool in which SVs are far from the AZs and tethered by cytoskeletal elements (Chamberland & T oth, 2016;Santos et al., 2009). In the readily releasable pool, two groups of SVs, "docked SVs" and "undocked ...
A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply-branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin-deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin-deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin-1, nectin-3, and N-cadherin were localized at the control typical PAJs, whereas nectin-1 and nectin-3 were localized at the afadin-deficient atypical PAJs to extents lower than those in the control synapse and N-cadherin was localized at their non-junctional flanking regions. These results indicate that the atypical PAJs are formed by nectin-1 and nectin-3 independently of afadin and N-cadherin and that the typical PAJs are formed by afadin and N-cadherin cooperatively with nectin-1 and nectin-3. Serial block face-scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin-deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses. This article is protected by copyright. All rights reserved.
... Cytoskeleton-based trafficking mechanics have long been explored because of their essential role in neuronal function and maintenance (Westrum et al., 1983;Okada et al., 1995;Sorra et al., 2006;Perlson and Holzbaur, 2007;Tao-Cheng, 2007;Hirokawa et al., 2009;Staras and Branco, 2010;Tang et al., 2013;Wu et al., 2013;Maeder et al., 2014; Guedes-Dias Gramlich et al., 2021;Watson et al., 2023). Protein trafficking via cytoskeleton transport is essential for synaptogenesis (Perlson and Holzbaur, 2007;Santos et al., 2009;Klassen et al., 2010;Wu et al., 2013;Guedes-Dias and Holzbaur, 2019;Kurshan and Shen, 2019;Watson et al., 2023) and to replace older proteins with newer proteins for efficient function (Cohen et al., 2013;Dörrbaum et al., 2018Dörrbaum et al., , 2020Heo et al., 2018;Truckenbrodt et al., 2018;Jähne et al., 2021;Watson et al., 2023). Protein recycling involves two steps: first, newly synthesized proteins from the soma are trafficked to presynaptic sites; second, older synaptic vesicles (SVs) must return to the soma from presynaptic sites. ...
Presynapses locally recycle synaptic vesicles to efficiently communicate information. During use and recycling, proteins on the surface of synaptic vesicles break down and become less efficient. In order to maintain efficient presynaptic function and accommodate protein breakdown, new proteins are regularly produced in the soma and trafficked to presynaptic locations where they replace older protein-carrying vesicles. Maintaining a balance of new proteins and older proteins is thus essential for presynaptic maintenance and plasticity. While protein production and turnover have been extensively studied, it is still unclear how older synaptic vesicles are trafficked back to the soma for recycling in order to maintain balance. In the present study, we use a combination of fluorescence microscopy, hippocampal cell cultures, and computational analyses to determine the mechanisms that mediate older synaptic vesicle trafficking back to the soma. We show that synaptic vesicles, which have recently undergone exocytosis, can differentially utilize either the microtubule or the actin cytoskeleton networks. We show that axonally trafficked vesicles traveling with higher speeds utilize the microtubule network and are less likely to be captured by presynapses, while slower vesicles utilize the actin network and are more likely to be captured by presynapses. We also show that retrograde-driven vesicles are less likely to be captured by a neighboring presynapse than anterograde-driven vesicles. We show that the loss of synaptic vesicle with bound molecular motor myosin V is the mechanism that differentiates whether vesicles will utilize the microtubule or actin networks. Finally, we present a theoretical framework of how our experimentally observed retrograde vesicle trafficking bias maintains the balance with previously observed rates of new vesicle trafficking from the soma.
... Critically, glutamate, the most prevalent neurotransmitter in the frontal cortex, is packaged and released from presynaptic vesicles. Glutamatergic neurons are capable of many different forms of LTD and thus many forms of neuroplasticity [122] one of which, endocannabinoid-dependent LTD, is common in the forebrain to both glutamatergic and GABAergic synapses, particularly in the prefrontal cortex [123]. This form of synaptic plasticity can, among many things, modulate dopamine signaling and affect motivated behaviors including addiction. ...
Introduction:
Flame retardants (FRs) are common bodily and environmental pollutants, creating concern about their potential toxicity. We and others have found that the commercial mixture, FireMaster® 550 (FM 550) or its individual brominated (BFR) and organophosphate ester (OPFR) components are potential developmental neurotoxicants. Using Wistar rats, we previously reported developmental exposure to FM 550 or its component classes produced sex- and compound-specific effects on adult socioemotional behaviors. The underlying mechanisms driving the behavioral phenotypes are unknown.
Methods:
To further mechanistic understanding, here we conducted transcriptomics in parallel with a novel lipidomics approach using cortical tissues from newborn siblings of the rats in the published behavioral study. Inclusion of lipid composition is significant because it is rarely examined in developmental neurotoxicity studies. Pups were gestationally exposed via oral dosing to the dam to FM 550 or the BFR or OPFR components at environmentally relevant doses.
Results:
The neonatal cortex was highly sexually dimorphic in lipid and transcriptome composition, and males were more significantly impacted by FR exposure. Multiple adverse modes of action for the BFRs and OPFRs on neurodevelopment were identified, with the OPFRs more disruptive than the BFRs via multiple mechanisms including dysregulation of mitochondrial function and disruption of cholinergic and glutamatergic systems. Disrupted mitochondrial function by environmental factors has been linked to higher risk of autism spectrum disorders. Impacted lipid classes included the ceramides, sphingomyelins, and triacylglycerides. Robust ceramide upregulation in the OPFR females could suggest heightened risk of brain metabolic disease.
Conclusions:
This study reveals multiple mechanisms by which the components of a common FR mixture are developmentally neurotoxic and that the OPFRs may be the compounds of greatest concern.
... So we can conclude that the production of glutamate in the hippocampus depends on the duration of HU. VGLUTs control all steps of glutamatergic transmission, i.e., loading of glutamate into synaptic vesicular (SV), cargo SV to the presynaptic terminals, release of glutamate and recycling of SV (Santos et al. 2009;Hackett and Ueda 2015), and neurotransmitter synthesis directly correlates with vesicle loading Jin et al. 2003). Thus, our data revealed a depletion of glutamatergic system of the hippocampus caused by short-term HU. ...
... In contrast, the clathrin-mediated endocytosis and the bulk endocytosis are particularly slow (10 seconds to minutes) as they follow the full collapse of SVs into the presynaptic membrane and new reconstruction of SVs (Mueller et al., 2004). The mechanism of clathrin-mediated endocytosis has been well characterized and requires the recruitment of a clathrin coat by heterotetrameric adaptor complexes AP2 (adaptor protein 2) and AP180, the acquisition of a curvature of the membrane with formation of a coated pit, the subsequent scission and detachment of the vesicle from the plasma membrane by GTPase dynamin and a final step of uncoating (Brodin et al., 2000;Santos et al., 2009). ...
Amyloid beta (Aβ) is a peptide derived from proteolytic cleavage of amyloid precursor protein (APP) by β and γ-secretases. While multiple isoforms of this protein are present in the human brain, to date research on Aβ has focused on the pathological effect of the predominant Aβ1-42 species as the major component of senile plaques present in Alzheimer´s disease brains. More recently, the discovery of Aβ presence at low levels in normal healthy brains has prompted investigation of its physiological function. These new findings have reported a previously unknown positive regulatory effect of Aβ on long-term potentiation (LTP) and memory, suggesting its beneficial physiological role when present in low concentrations in the brain (Puzzo et al., 2008;Puzzo et al., 2011). Interestingly, the N-terminal Aβ fragment has been suggested to be particularly effective in this neuromodulation (Lawrence et al., 2014;Richter et al., 2018). Despite of several hints pointing to role of Aβ and other APP-derived fragments in the regulation of presynaptic function, a systematic investigation has not been performed yet. To close this gap, we explored the potential role of physiological concentrations of APP/Aβ-derived fragments in the modulation of presynaptic activity and the molecular and cellular mechanisms involved in this process. We identified distinct effects of N- and C-terminal part of Aβ1-42 on recycling of synaptic vesicles (SVs) in cultured primary neurons. While the N-terminal Aβ fragment (Aβ1-16) enhanced synaptic transmission via an increase in the fraction of recycling pool of SVs, the C- terminal fragment (Aβ17-42) had no effect. The observed enhancement of SV recycling driven by the N-terminal Aβ fragment required functional alpha 7 nicotinic acetylcholine receptors (α7nAchRs). It was completely blocked by α-Bungarotoxin (BgTx), a selective antagonist of these receptors, and was absent in neurons from α7nAchRs knockout mice. Our data suggested that Aβ acts as a positive allosteric modulator of α7nAchRs rather than an activator, increasing nicotine-evoked Ca2+ currents, as well as the phosphorylation levels of CREB at serine 133. Furthermore, Aβ attenuated synaptic depletion of SV recycling pool in response to multiple field stimulations, thus regulating the availability of SVs for neurotransmission. This was connected with an increase in the calcineurin activity and changes in the phosphorylation of synapsin 1 at serine 551, 603 and 9. Intriguingly, Aβ-increased SV recycling also required N-type Ca2+ channels, suggesting a functional link between α7nAChRs activation and N-type modulation. In conclusion, our findings demonstrate a unique role for the N-terminal Aβ-fragment (Aβ1-16) as an enhancer of presynaptic SV cycling through regulation of SV availability via changes in phosphorylation status of synapsin 1. Moreover, we uncovered a molecular pathway behind this process involving calcineurin/CDK5, PKA and/or CAMKI/IV, and CAMKII signaling. A failure of precise regulation of SV recycling upon disruption of normal Aβ metabolism may contribute to synaptic dysfunction occurring at the early stage of Alzheimer’s disease and the pharmacological strategies targeting this process might open up the possibilities for future therapies in this condition.
... S2, A and B). Furthermore, SV proteins, here the vesicular glutamate transporter protein (VGlut; Santos et al., 2009), were also strongly accumulating in the rab2 −/− motoneuron somata (Fig. 1,F and G;and Fig. S1,C and H). However, in contrast to RIM-BP or UNC13A, VGlut aggregates were 2.5-fold larger than BRP aggregates (Fig. S1 D) and, interestingly, localized rather adjacent to the BRP accumulations. ...
Reliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals, is prerequisite for successful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. We show here that in mutants of the small GTPase Rab2, both active zone and synaptic vesicle proteins accumulated in the neuronal cell body at the trans-Golgi and were, consequently, depleted at synaptic terminals, provoking neurotransmission deficits. Ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40 × 60 nm, segregating in subfractions either positive for active zone or synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, Rab2 acts upstream of Arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we identified a Golgi-associated assembly sequence of presynaptic precursor biogenesis dependent on a Rab2-regulated protein export and sorting step at the trans-Golgi.
... Synaptophysin, a major presynaptic marker, presents in almost all presynaptic endings (33). ...
Effective treatments for chemotherapy-induced peripheral neuropathy (CIPN) remain unavailable. Given the significance of spinal cord glutamate transporters in neuronal plasticity and central sensitization, this study investigated the role of excitatory amino acid transporter 2 (EAAT2) and vesicular-glutamate transporter 2 (VGLUT2) in the development of paclitaxel-induced painful neuropathy. Paclitaxel (2 mg/kg, i.p., cumulative dose 8mg/kg) induced long-lasting mechanical allodynia (> 28days) with increased glutamate concentration and decreased EAAT2 expression with no changes in GABA/glycine or VGAT (vesicular GABA transporter) in rat spinal dorsal horn. VGLUT2 expression was upregulated and co-expressed with enhanced synaptophysin, characterizing nociceptive afferent sprouting and new synapse formation of glutamatergic neurons in the spinal cord dorsal horn. HDAC2 and transcription factor YY1 were also upregulated, and their interaction and co-localization were confirmed following paclitaxel treatment using co-immunoprecipitation (Co-IP). Inhibition or knockdown of HDAC2 expression by valproic acid (VPA), BRD6688 or HDAC2 siRNA (small interfering RNA) not only attenuated paclitaxel-induced mechanical allodynia but also suppressed HDAC2 upregulation, glutamate accumulation and the corresponding changes in EAAT2/VGLUT/synaptophysin expression and HDAC2/YY1 interaction. These findings indicate that loss of the balance between glutamate release and reuptake due to dysregulation EAAT2/VGLUT2/synaptophysin cascade in the spinal dorsal horn plays an important role in the development of paclitaxel-induced neuropathic pain. HDAC2/YY1 interaction as a complex appears essential in regulating this pathway, which can potentially be a therapeutic target to relieve CIPN by reversing central sensitization of spinal nociceptive neurons.
... So we can conclude that the production of glutamate in the hippocampus depends on the duration of HU. VGLUTs control all steps of glutamatergic transmission, i.e., loading of glutamate into synaptic vesicular (SV), cargo SV to the presynaptic terminals, release of glutamate and recycling of SV (Santos et al. 2009;Hackett and Ueda 2015), and neurotransmitter synthesis directly correlates with vesicle loading Jin et al. 2003). Thus, our data revealed a depletion of glutamatergic system of the hippocampus caused by short-term HU. ...
Spaceflight and simulated microgravity both affect learning and memory, which are mostly controlled by the hippocampus. However, data about molecular alterations in the hippocampus in real or simulated microgravity conditions are limited. Adult Wistar rats were recruited in the experiments. Here we analyzed whether short-term simulated microgravity caused by 3-day hindlimb unloading (HU) will affect the glutamatergic and GABAergic systems of the hippocampus and how dynamic foot stimulation (DFS) to the plantar surface applied during HU can contribute in the regulation of hippocampus functioning. The results demonstrated a decreased expression of vesicular glutamate transporters 1 and 2 (VGLUT1/2) in the hippocampus after 3 days of HU, while glutamate decarboxylase 67 (GAD67) expression was not affected. HU also significantly induced Akt signaling and transcriptional factor CREB that are supposed to activate the neuroprotective mechanisms. On the other hand, DFS led to normalization of VGLUT1/2 expression and activity of Akt and CREB. Analysis of exocytosis proteins revealed the inhibition of SNAP-25, VAMP-2, and syntaxin 1 expression in DFS group proposing attenuation of excitatory neurotransmission. Thus, we revealed that short-term HU causes dysregulation of glutamatergic system of the hippocampus, but, at the same time, stimulates neuroprotective Akt-dependent mechanism. In addition, most importantly, we demonstrated positive effect of DFS on the hippocampus functioning that probably depends on the regulation of neurotransmitter exocytosis.
... The essential AZ scaffold-associated release factor (m)UNC13A (Bohme et al., 2016) also strongly accumulated upon loss of RAB2 and UNC13A aggregates again overlapped with the BRP accumulations ( Fig. S1G-I). SV proteins, here vesicular glutamate transporter protein (VGlut) (Santos et al., 2009), strongly accumulated in the rab2 -/motoneuron somata as well ( Fig. 1F, G, S1C). However, in contrast to RIM-BP and UNC13A, VGlut aggregates were 2-3fold larger than BRP aggregates (Fig. S1D) and, interestingly, localized rather adjacent than overlapping relative to the BRP accumulations. ...
Reliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals is prerequisite for faithful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. Here we show that in mutants of the small GTPase RAB2 active zone and synaptic vesicle proteins accumulated in the neuronal somata at the trans-Golgi network and were consequently depleted at synaptic terminals, provoking neurotransmission deficits. The ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40x60 nm and segregated in subfractions either positive for active zone proteins or co-positive for synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, rab2 behaved epistatic over arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we here identified a Golgi-associated assembly sequence in presynaptic precursor vesicle biogenesis controlled by RAB2 dependent membrane remodelling and protein sorting at the trans-Golgi.
... We also analyzed presynaptic glutamatergic protein, V-GLUT1, and found a similar increase after PhIP treatment. Glutamate is stored in the vesicles of the presynaptic terminals via the membrane-bound V-GLUT (Liguz-Lecznar and Skangiel-Kramska 2007;Santos et al. 2009;Wilson et al. 2005) and expression level of V-GLUTs reflects the amount of glutamate in vesicles (Herman et al. 2014;Ishikawa et al. 2002;Johnson et al. 2004;Wojcik et al. 2004). VGLUT1 expression is also reported to alter in AD and PD patients (Kashani et al. 2007;Poirel et al. 2018). ...
Alzheimer’s disease (AD) is a public health crisis due debilitating cognitive symptoms and lack of curative treatments, in the context of increasing prevalence. Thus, it is critical to identify modifiable risk factors. High levels of meat consumption may increase AD risk. Many toxins are formed during meat cooking such as heterocyclic aromatic amines (HAAs). Our prior studies have shown that HAAs produce dopaminergic neurotoxicity. Given the mechanistic and pathological overlap between AD and dopaminergic disorders we investigated whether exposure to 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), a prevalent dietary HAA formed during high-temperature meat cooking, may produce AD-relevant neurotoxicity. Here, C57BL/6 mice were treated with 100 or 200 mg/kg PhIP for 8 h or 75 mg/kg for 4 weeks and 16 weeks. PhIP exposure for 8 h produced oxidative damage, and AD-relevant alterations in hippocampal synaptic proteins, Amyloid-beta precursor protein (APP), and β-Site amyloid precursor protein cleaving enzyme 1 (BACE1). PhIP exposure for 4 weeks resulted in an increase in BACE1. PhIP exposure for 16 weeks resulted in increased hippocampal oxidative damage, APP, BACE1, Aβ aggregation, and tau phosphorylation. Quantification of intracellular nitrotyrosine revealed oxidative damage in cholinergic neurons after 8 h, 4 weeks and 16 weeks of PhIP exposure. Our study demonstrates that increase in oxidative damage, APP and BACE1 might be a possible mechanism by which PhIP promotes Aβ aggregation. Given many patients with AD or PD exhibit neuropathological overlap, our study suggests that HAA exposure should be further studied for roles in mediating pathogenic overlap.
... An in vitro study screened a series of SH3 domain--containing proteins, including multiple Src family tyrosine kinases and scaffolding/adaptor proteins, and showed several potential binding partners (e.g. Nck1, Nck2, ArgBP2, sorting nexin 9) (Santos et al., 2009). interacts endophilin A1 at PP2 domain. ...
Synaptic vesicles (SVs) are essential for neurotransmission, and more efforts are needed for better understanding their neurotransmitter content, release kinetics, distribution and mobility. SVs are not only clustered in presynaptic boutons, but also dynamically shared among multiple en passant presynaptic boutons, a phenomenon named SV super‐pool. Previous work from our laboratory suggested that the Vesicular GLUtamate Transporter 1 (VGLUT1) may play a role in regulating SV super-pool size beyond loading glutamate into SV. My Ph.D project is focused on SVs mobility in axons. Firstly, I generated a VGLUT1mEos2 knock-in (KI) mouse line, which provides extended possibilities to study the SV trafficking and characterize SV super‐pool. Secondly, I engaged in a thorough VGLUT1 structure‐function analysis. I identified that VGLUT1 tends to cluster SVs in the presynaptic boutons and reduce SVs exchange with the super‐pool via the second poly‐proline motif of its C‐terminus. Overall, my Ph.D work contributes to the knowledge of the role of VGLUT1 in regulating SVs mobility and provides new tools for the further investigations on SV super-pool physiology.
... Over decades, significant progress has been made to discover synaptic molecules that regulate neurotransmitter metabolism, synaptic vesicle formation, axonal trafficking, and neurotransmitter recycling 24 . On the one hand, researchers have sought molecules expressed in specific subtypes of DRG neurons and have developed their www.nature.com/scientificreports ...
Central sprouting of nociceptive afferents in response to neural injury enhances excitability of nociceptive pathways in the central nervous system, often causing pain. A reliable quantification of central projections of afferent subtypes and their synaptic terminations is essential for understanding neural plasticity in any pathological condition. We previously characterized central projections of cutaneous nociceptive A and C fibers, selectively labeled with cholera toxin subunit B (CTB) and Isolectin B4 (IB4) respectively, and found that they expressed a general synaptic molecule, synaptophysin, largely depending on afferent subtypes (A vs. C fibers) across thoracic dorsal horns. The current studies extended the central termination profiles of nociceptive afferents with synaptoporin, an isoform of synaptophysin, known to be preferentially expressed in C fibers in lumbar dorsal root ganglions. Our findings demonstrated that synaptophysin was predominantly expressed in both peptidergic and IB4-binding C fiber populations in superficial laminae of the thoracic dorsal horn. Cutaneous IB4-labeled C fibers showed comparable expression levels of both isoforms, while cutaneous CTB-labeled A fibers exclusively expressed synaptophysin. These data suggest that central expression of synaptophysin consistently represents synaptic terminations of projecting afferents, at least in part, including nociceptive A-delta and C fibers in the dorsal horn.
... Morphologically, interneurons can be divided into islet, central, radial and vertical (43), and according to their neurochemistry, in excitatory and inhibitory. Excitatory interneurons, expressing the vesicular glutamate transporter type 2 (VGLUT2) (44)(45), are a heterogeneous group of neurons under the control of different transcription factors (46), determining different subpopulations based on the expression of, for instance, protein kinase C gamma (PKCγ) (47), µ-opioid receptor (MOR) (48)(49)(50), neurotensin(51), somatostatine (52), and neuroquinin B (53).In contrast, inhibitory interneurons utilize GABA and/or glycine as principal neurotransmitters (54). Inhibitory interneurons can also be divided into subpopulations expressing the neuropeptide tyrosine (NPY), galanin, parvalbumin or neuronal nitric oxide synthase (55). ...
The most up-to-date description defines pain as an “unpleasant and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” (International Association for the Study of Pain (IASP)). Two words stand out: “experience” and “emotional.” In fact, the cognitive processing of painful sensations is strongly dependent on the remembrance of previous painful experiences (both own and as seen on others) and is influenced by, or can influence, emotional states. The IASP description also accounts for a massive neurobiology behind the processing of painful sensations and its impact on emotions. In fact, thousands of neurons in the peripheral and central nervous system are related to each other through very specific connections, and they participate in the conscious evoking of pain, through yet not totally understood mechanisms. Ultimately, the general role of pain is to assist us, in a sensorial fashion, in our interaction with the surrounding world and to avoid potential tissue damage. We could say that this is good pain, a pain we need. However, when pain becomes chronic, even in the absence of tissue damage, it triggers an experience that we certainly do not need, namely, suffering. Suffering involves the human person as a whole, urging us to find ways to face it appropriately, in a manner that may be accepted in terms of meaning. The present chapter aims to present pain encompassing both the neurobiological and experiential/emotional aspects that make it both a necessity and a challenge in human life.
... We also found that approximately half of all synaptophysin-ir terminals in the PVHmpd of both species contained VGluT2 (Fig. 4j). Only about one third of the VGluT3-ir structures in the rat PVHmpd co-occurred with synaptophysin, and about 10% of the synaptophysin co-occurred with VGluT3 (Fig. 4j), indicating that most VGluT3 is not situated in pre-synaptic terminals (Fig. 4j), which is consistent with previous findings [Seal and Edwards, 2006;Santos, 2009]. We also observed apparent VGlut3 in some axons and in the soma of some PVHpm magnocellular neurons (Fig. 3b), again consistent with the non-terminal expression of this transporter. ...
Virtually all rodent neuroendocrine corticotropin-releasing-hormone (CRH) neurons are in the dorsal medial parvicellular (mpd) part of the paraventricular nucleus of the hypothalamus (PVH). They form the final common pathway for adrenocortical stress responses. Their activity is controlled by sets of GABA-, glutamate-, and catecholamine-containing inputs arranged in an interactive pre-motor network. Defining the nature and arrangement of these inputs can help clarify how stressor type and intensity information is conveyed to neuroendocrine neurons. Here we use immunohistochemistry with high-resolution 3-dimensional image analyses to examine the arrangement of single- and co-occurring GABA, glutamate, and catecholamine markers in synaptophysin-defined pre-synaptic terminals in the PVHmpd of unstressed rats and Crh-IRES-Cre;Ai14 transgenic mice: respectively, vesicular glutamate transporter 2 (VGluT2), vesicular GABA transporter (VGAT), dopamine β-hydroxylase (DBH), and phenylethanolamine n-methyltransferase (PNMT). Just over half of all PVHmpd pre-synaptic terminals contain VGAT, with slightly less containing VGluT2. The vast majority of terminal appositions with mouse CRH neurons occur non-somatically. However, there are significantly more somatic VGAT than VGluT2 appositions. In the rat PVHmpd, about five times as many pre-synaptic terminals contain PNMT than DBH only. However, because epinephrine release has never been detected in the PVH, PNMT terminals may functionally be noradrenergic not adrenergic. PNMT and VGluT2 co-occur in some pre-synaptic terminals indicating the potential for co-transmission of glutamate and norepinephrine. Collectively, these results provide a structural basis for how GABA/glutamate/catecholamine interactions enable adrenocortical responses to fast-onset interosensory stimuli, and more broadly, how combinations of PVH neurotransmitters and neuromodulators interact dynamically to control adrenocortical activity.
... Usually two to four boutons, sometimes one bouton, attach to a dendritic shaft by multiple puncta adherentia junctions (PAJs) and wrap around a highly branched dendritic spine, known as thorny excrescences, where multiple synaptic junctions (SJs) are formed (Amaral & Dent 1981). Electron microscopically, the pools of synaptic vesicles (SVs) in the mossy fiber bouton can be classified into three types: the readily releasable pool in which SVs are docked to the active zones (AZs) or clustered around the AZs, the recycling pool in which SVs are relocated to the AZs by intense stimulation, and the reserve pool in which SVs are at a distance from the AZs and tethered by cytoskeletal elements (Santos et al. 2009;Chamberland & T oth 2016). Postsynaptic densities (PSDs) are located at the heads of the spine branches and face toward the AZs and a single mossy fiber bouton has approximately 20 AZs in mice. ...
A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure. We have previously shown using afadin-deficient mice that afadin plays multiple roles in the structural and functional differentiations of this synapse. We investigated here using a co-culture system with cultured hippocampal neurons and non-neuronal COS-7 cells expressing synaptogenic cell adhesion molecules (CAMs) whether afadin is involved in the presynaptic differentiation of hippocampal synapses. Postsynaptic CAMs NGL-3 (alias, a Lrrc4b gene product) and neuroligin induced presynaptic differentiation by trans-interacting with their respective presynaptic binding CAMs LAR (alias, a Ptprf gene product) and neurexin. This activity of NGL-3, but not neuroligin, was dependent on afadin, but not the afadin-binding presynaptic CAM nectin-1. The afadin-binding postsynaptic CAM nectin-3 did not induce presynaptic differentiation. Immunofluorescence and immunoelectron microscopy analyses showed that afadin was localized mainly at puncta adherentia junctions, but partly at synaptic junctions, of the mossy fiber synapse. β-Catenin and γ-catenin known to bind to LAR were co-immunoprecipitated with afadin from the lysate of mouse brain. These results suggest that afadin is involved in the NGL-3-LAR system-induced presynaptic differentiation of hippocampal neurons cooperatively with β-catenin and γ-catenin in a nectin-1-independent manner.
... The mossy fiber synapse has a large and complex structure in which two to four boutons, sometimes one bouton, attach to a dendritic shaft by multiple puncta adherentia junctions (PAJs) and wrap around a highly branched dendritic spine, known as thorny excrescences, where multiple neurotransmitter release sites and multiple synaptic junctions are formed (Amaral & Dent 1981). Electron microscopic analysis has shown three types of pools of the synaptic vesicles (SVs) in the mossy fiber bouton: the readily releasable pool in which SVs are docked to the active zones (AZs) or clustered around the AZs, the recycling pool in which SVs are recruited to the AZs by intense stimulation, and the reserve pool in which SVs are far from the AZs and tethered by cytoskeletal elements (Santos et al. 2009;Chamberland & T oth 2016). Postsynaptic densities (PSDs) are located at the heads of the spine branches and face toward the AZs. ...
A hippocampal mossy fiber synapse has a complex structure and is implicated in learning and memory. In this synapse, the mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions and wrap around a multiply-branched spine, forming synaptic junctions. We have recently shown using transmission electron microscopy, immunoelectron microscopy and serial block face-scanning electron microscopy that atypical puncta adherentia junctions are formed in the afadin-deficient mossy fiber synapse and that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities and the density of synaptic vesicles docked to active zones are decreased in the afadin-deficient synapse. We investigated here the roles of afadin in the functional differentiations of the mossy fiber synapse using the afadin-deficient mice. The electrophysiological studies showed that both the release probability of glutamate and the postsynaptic responsiveness to glutamate were markedly reduced, but not completely lost, in the afadin-deficient mossy fiber synapse, whereas neither long-term potentiation nor long-term depression was affected. These results indicate that afadin plays roles in the functional differentiations of the presynapse and the postsynapse of the hippocampal mossy fiber synapse.
... However, this TNF-glutaminase link, despite first being made a decade ago (above), does not yet appear to be common currency in neurodegenerative disease circles (e.g., [108]). Readers interested in the complexities of glutamate release, including its physiological control, are directed to the examples provided by references [109][110][111]. ...
The basic mechanism of the major neurodegenerative diseases, including neurogenic pain, needs to be agreed upon before rational treatments can be determined, but this knowledge is still in a state of flux. Most have agreed for decades that these disease states, both infectious and non-infectious, share arguments incriminating excitotoxicity induced by excessive extracellular cerebral glutamate. Excess cerebral levels of tumor necrosis factor (TNF) are also documented in the same group of disease states. However, no agreement exists on overarching mechanism for the harmful effects of excess TNF, nor, indeed how extracellular cerebral glutamate reaches toxic levels in these conditions. Here, we link the two, collecting and arguing the evidence that, across the range of neurodegenerative diseases, excessive TNF harms the central nervous system largely through causing extracellular glutamate to accumulate to levels high enough to inhibit synaptic activity or kill neurons and therefore their associated synapses as well. TNF can be predicted from the broader literature to cause this glutamate accumulation not only by increasing glutamate production by enhancing glutaminase, but in addition simultaneously reducing glutamate clearance by inhibiting re-uptake proteins. We also discuss the effects of a TNF receptor biological fusion protein (etanercept) and the indirect anti-TNF agents dithio-thalidomides, nilotinab, and cannabinoids on these neurological conditions. The therapeutic effects of 6-diazo-5-oxo-norleucine, ceptriaxone, and riluzole, agents unrelated to TNF but which either inhibit glutaminase or enhance re-uptake proteins, but do not do both, as would anti-TNF agents, are also discussed in this context. By pointing to excess extracellular glutamate as the target, these arguments greatly strengthen the case, put now for many years, to test appropriately delivered ant-TNF agents to treat neurodegenerative diseases in randomly controlled trials.
... However, we only detected a trend towards an increase in the number of NPY+ cells in CA1 ( Figure 4I). We also did not observe differences between control and EE rats upon immunostaining for a dendritic marker, Map2 ( Figure 4J) (Matesic and Lin, 1994, Folkerts et al., 1998, Hoskison et al., 2007, or a marker for glutamatergic synaptic transmission, Vglut1 ( Figure 4J) (Santos et al., 2009). Immunoblotting for Map2 in cortical extracts confirmed the lack of differences between control and EE rats ( Figure 4K). ...
Age-associated changes in cognition are mirrored by impairments in cellular models of memory and learning, such as long-term potentiation (LTP) and long-term depression (LTD). In young rodents, environmental enrichment (EE) can enhance memory, alter LTP and LTD, as well as reverse cognitive deficits induced by aging. Whether short-term EE can benefit cognition and synaptic plasticity in aged rodents is unclear. Here, we tested if short-term EE could overcome age-associated impairments in induction of LTP and LTD. LTP and LTD could not be induced in the CA1 region of hippocampal slices in control, aged rats using standard stimuli that are highly effective in young rats. However, exposure of aged littermates to EE for three weeks enabled successful induction of LTP and LTD. EE-facilitated LTP was dependent upon N-methyl-D-aspartate receptors (NMDARs). These alterations in synaptic plasticity occurred with elevated levels of phosphorylated cAMP response element-binding protein and vascular endothelial growth factor, but in the absence of changes in several other synaptic and cellular markers. Importantly, our study suggests that even a relatively short period of EE is sufficient to alter synaptic plasticity and molecular markers linked to cognitive function in aged animals.
... The transporter properties of the three VGLUT isoforms, when measured in recombinant systems, are largely similar, including a low (mM) affinity for glutamate, lack of aspartate uptake, dependence on vesicular membrane potential rather than pH gradient, biphasic dependence on extra-vesicular chloride levels and inhibition by dyes such as Evans blue (El Mestikawy et al., 2011;Fremeau et al., 2004a,b;Takamori, 2006). On the other hand, additional properties of the transporters, such as the unique regulatory mechanisms that control expression level or trafficking at the nerve terminal, may confer distinct functional roles (Santos et al., 2009;Voglmaier and Edwards, 2007;Edwards, 2007). VGLUT expression level and trafficking have been shown to influence the rate and extent of synaptic vesicle filling as well as the probability of synaptic vesicle release (Herman et al., 2014;Moechars et al., 2006;Voglmaier et al., 2006;Weston et al., 2011;Wojcik et al., 2004). ...
The somatosensory system transmits touch, temperature, itch and pain. Three vesicular glutamate transporter isoforms mediate the release of glutamate throughout the mammalian nervous system with largely non-overlapping distributions and unique roles at the synapse. This review discusses the contribution of each of these essential transporters to circuits underlying pain and other somatosensory behaviors throughout postnatal development and in the adult. A better understanding of the individual contributions of the VGLUT isoforms could provide new avenues for therapeutic intervention.
... Such synaptic contacts, which represent the minimal storage unit of information in the nervous system, are maintained through structural and functional coupling of a repertoire of the same and different proteins in these distinct compartments [94] . Many of these proteins are transported to terminals on kinesin motors, particularly during the initiation phase of synapse formation, while a great number of other proteins are locally translated during differentiation and maturation [95, 96] . In the latter case, the asymmetric localization of mRNAs helps to limit protein expression to these compartments. ...
Enriched KEGG pathways of mir-154 family targets. DIANA-miRPath v2.0 was used to predict all enriched KEGG pathways of mir-154 family targets. The target prediction threshold was set at 0.85. Benjamini-Hochberg [38] correction for multiple testing controlled the P-values.
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... The storage and release of glutamate in excitatory circuits in the mammalian brain is regulated by the vesicular glutamate transporters (VGLUTs) and glutamate receptors (Collingidge et al., 1989;Fremeau et al., 2004aFremeau et al., , 2004bFremeau et al., , 2001Gras et al., 2002;Herzog et al., 2001;Kaneko and Fujiyama, 2002;Kaneko et al., 2002;Takamori, 2006;Takamori et al., 2000Takamori et al., , 2001. VGLUTs accumulate glutamate into synaptic vesicles of glutamatergic neurons at the presynaptic terminals, and glutamate released from the vesicles binds to glutamate receptors on postsynaptic membranes (Newpher and Ehlers, 2008;Santos et al., 2009). Three types of VGLUTs have been identified in mammals: VGLUT1, VGLUT2, and VGLUT3. ...
... The receptors, or their subunits, recycle between cytoplasmic and membrane pools [6]. This cycling may allow fast, regulated changes in synaptic AMPA receptor concentration, thus enabling changes in synaptic strength [7]. Indirect evidence indicates a vesicular mechanism for this recycling [8]. ...
Vertebrate organisms adapt to a continuously changing environment by regulating the strength of synaptic connections between brain cells. Excitatory synapses are believed to increase their strength by vesicular insertion of transmitter glutamate receptors into the postsynaptic plasma membrane. These vesicles, however, have never been demonstrated or characterized. For the first time, we show the presence of small vesicles in postsynaptic spines, often closely adjacent to the plasma membrane and PSD (postsynaptic density). We demonstrate that they harbor vesicle-associated membrane protein 2 (VAMP2/synaptobrevin-2) and glutamate receptor subunit 1 (GluA1). Disrupting VAMP2 by tetanus toxin treatment reduces the concentration of GluA1 in the postsynaptic plasma membrane. GluA1/VAMP2-containing vesicles, but not GluA2/VAMP2-vesicles, are concentrated in postsynaptic spines relative to dendrites. Our results indicate that small postsynaptic vesicles containing GluA1 are inserted directly into the spine plasma membrane through a VAMP2-dependent mechanism.
... (2) Synapsins may be required for the efficient reuptake and processing of precursor vesicles exocytosed into the presynaptic plasma membrane (Bradke and Dotti, 2000;Santos et al., 2009). In this model, absence of synapsins would cause an accumulation of precursor vesicles in the membrane of the terminal, which we did not observe. ...
At chemical synapses synaptic vesicles are functionally segregated into distinct pools depending on their mobility and release probability in response to action potentials. At most synapses synapsin proteins cluster and immobilize reserve pool vesicles within the cytoskeletal network, distally from the active zones. Activity-dependent phosphorylation of synapsins releases synaptic vesicles from the complex meshwork of the cytoskeletal components and renders them freely mobile and able to undergo vesicle cycling. There are three synapsin genes in mammals and their alternative splicing results in more than ten isoforms whose functional differences in maintaining synaptic transmission are not fully elucidated. In this study we examined the involvement of synapsins in synaptic transmission using two independent approaches. First, we established the structure-function relationship at synapses, overexpressing synapsin I isoforms (synapsin Ia or synapsin Ib). Second, synaptic transmission and structural integrity of synapses in mice, lacking all three synapsin genes (triple knock-out (TKO)) were examined. The calyx of Held, a giant glutamatergic synapse located in the auditory brain stem, was utilized as a model system. Synapsin I isoforms overexpression was accomplished through transduction of globular bushy cells (GBCs), located in the ventral cochlear nucleus, with recombinant adeno- associated viral particles. The GBCs are projection neurons, which give rise to the calyx of Held in the medial nucleus of the trapezoid body. 10 days after the transduction, overexpression of synapsin I isoforms resulted in a redistribution of SV within the calyx of Held, without changing the size and the overall structure of the perturbed synapses. Ultrastructural analysis, using serial sectioning scanning electron microscopy (S3EM) revealed that synapsin Ia overexpression resulted in decreased numbers of SVs at the active zone without altering the total vesicle number. Therefore, we could conclude that synapsin Ia overexpression was followed by vesicle redistribution within the presynaptic terminal. On the functional level, the overexpression of both synapsin I isoforms had no effect on the properties of spontaneous and evoked EPSCs. However, repeated stimulation at frequencies exceeding 10 Hz led to accelerated short-term depression (STD). Overexpression of either isofroms led also to accelerated recovery from depression after strong stimulation. Brain lysates from synapsin TKO mice revealed a strong reduction in the level of several synaptic vesicle proteins, while proteins of the active zone cytomatrix or of the postsynaptic density remained unaffected. Accordingly, TKO calyces had lower amounts of vGluT1 immunoreactivity while the level of the active zone marker bassoon was unchanged Summary i as shown via 3D reconstructions of TKO calyces. The S3EM analysis confirmed these results revealing a 50 % reduction in the number of synaptic vesicles in TKO calyces. The structural alterations resulting in the absence of synapsins led to accelerated and more pronounced STD at stimulation at frequencies above 100 Hz. Synapsin deletion, contrary to synapsin overexpression, slowed down the recovery of depression. This might prove that synapsin- dependent SVs contribute to the faster replenishment of the readily releasable pool, which maintains synaptic transmission under basic conditions. Despite the structural defects and the alterations in the short-term depression, transmission failures were not observed during the high-frequency trains. These results reveal that in wild-type synapses the synapsin-dependent vesicles account only for a small fraction of the SVs that enter the RRP. In conclusion, synapsins maintain of a specific vesicle population at CNS synapses. However, these vesicles are dispensable from normal basal synaptic transmission and are recruited only when the synapse is exposed to long-lasting high-frequency activity. The synapsin-dependent vesicles are fed into the readily releasable pool to counteract the effects of presynaptic depression and aid the faster recovery of the synapse. Although synapsins may be required for normal synaptic vesicle biogenesis, trafficking and immobilization, they are not essential for sustained synaptic transmission at the calyx of Held.
... Sinapsinės pūslelės su glutamatu iš plaukuotųjų ląstelių išeina į sinapsinį plyšį ir jų turinys išsilieja ant nervo galūnėlės. VGLUT3 baltymo stoka sutrikdo glutamatų reabsorbciją iš sinapsinio tarpo bei jų atpalaidavimą iš sinapsinės pūslelės [47]. Otoferlinas, koduojamas OTOF geno, dalyvauja vėlyvame teorija ir praktika 2015 -T. ...
Congenital hearing loss is one of the most common defects diagnosed 1 in 1000 newborns. Prelingual hearing loss disturbs development of the child and it is one of disabling conditions in present day environment. It is a highly heterogeneous disorder, with the majority of cases having genetic etiology. Ear is a very complex organ, proper development of the tissues in macroscopic, microscopic and molecular levels and function are essencial for the perception of sound. These processes are influenced mostly by genetic factors. Recent advances in the gene identification techniques have revolutionized the clinical approach to congenital hearing loss. More than 400 and 100 genetic loci are associated with syndromic and nonsyndromic hearing loss respectively. High heterogeneity of hearing loss is important in genetic counselling – the disorder may be inherited in autosomal dominant, autosomal recessive, X recessive and mitochondrial manner. In this review we discuss the structure and the function of auditory organ, the pathogenic mechanisms of hearing loss and provide the clinical approach. The identification of genes implicated in pathogenesis of hearing loss is essential in establishing of genetic diagnosis and rehabilitation, opens new perspectives in treatment, and allows predicting the severity of the disorder, progression and effectiveness of rehabilitating measures.
... Diese Motive werden unter anderem als potentielle Bindestellen für Regulationsproteine der Endozytose (z.B. AP2) vermutet (Bonifacino & Traub, 2003;Jung & Haucke, 2007;Kim & Ryan, 2009;Santos et al., 2009;, was eine GST-Bindungsstudie zur Identifizierung von Interaktionspartnern interessant machte. ...
... Such synaptic contacts, which represent the minimal storage unit of information in the nervous system, are maintained through structural and functional coupling of a repertoire of the same and different proteins in these distinct compartments [94] . Many of these proteins are transported to terminals on kinesin motors, particularly during the initiation phase of synapse formation, while a great number of other proteins are locally translated during differentiation and maturation [95, 96] . In the latter case, the asymmetric localization of mRNAs helps to limit protein expression to these compartments. ...
In eukaryotic cells, gene activity is not directly reflected by protein levels because mRNA processing, transport, stability, and translation are co- and post-transcriptionally regulated. These processes, collectively known as the ribonome, are tightly controlled and carried out by a plethora of trans-acting RNA-binding proteins (RBPs) that bind to specific cis elements throughout the RNA sequence. Within the nervous system, the role of RBPs in brain function turns out to be essential due to the architectural complexity of neurons exemplified by a relatively small somal size and an extensive network of projections and connections. Thus far, RBPs have been shown to be indispensable for several aspects of neurogenesis, neurite outgrowth, synapse formation, and plasticity. Consequently, perturbation of their function is central in the etiology of an ever-growing spectrum of neurological diseases, including fragile X syndrome and the neurodegenerative disorders frontotemporal lobar degeneration and amyotrophic lateral sclerosis.
... It is unclear whether signals for the removal of proteins from nascent vesicles are necessary (Hannah et al, 1999;Prado & Prado, 2002). The interactions of the SV proteins with endocytosis or endosomal proteins in the Golgi apparatus are based on various sorting signals (Grote et al, 1995;West A et al, 1997), including both classical dileucine-based signals (Santos et al, 2009) or more specialized sorting motifs (Koo et al, 2011), whose discussion, however, is beyond the purpose of this review. ...
Synaptic vesicle recycling is one of the best-studied cellular pathways. Many of the proteins involved are known, and their interactions are becoming increasingly clear. However, as for many other pathways, it is still difficult to understand synaptic vesicle recycling as a whole. While it is generally possible to point out how synaptic reactions take place, it is not always easy to understand what triggers or controls them. Also, it is often difficult to understand how the availability of the reaction partners is controlled: how the reaction partners manage to find each other in the right place, at the right time. I present here an overview of synaptic vesicle recycling, discussing the mechanisms that trigger different reactions, and those that ensure the availability of reaction partners. A central argument is that synaptic vesicles bind soluble cofactor proteins, with low affinity, and thus control their availability in the synapse, forming a buffer for cofactor proteins. The availability of cofactor proteins, in turn, regulates the different synaptic reactions. Similar mechanisms, in which one of the reaction partners buffers another, may apply to many other processes, from the biogenesis to the degradation of the synaptic vesicle.
... Increased expression of CAMK2a in the cerebral cortex of humans compared with chimpanzees and rhesus monkeys was described in a previous study (Cáceres et al. 2003). Finally, SLC17A7 is a vesicular glutamate transporter bounded to membranes of synaptic vesicles and it is involved in the storage of glutamate as well in its biogenesis and recycling (Santos et al. 2009). One major feature of LTP is the requirement of activation of NMDA receptors, which has been hypothesized to be the molecular pathway underlying cognitive learning (Morris et al. 1986). ...
Increased relative brain size characterizes the evolution of primates, suggesting that enhanced cognition plays an important
part in the behavioral adaptations of this mammalian order. In addition to changes in brain anatomy, cognition can also be
regulated by molecular changes that alter synaptic function, but little is known about modifications of synapses in primate
brain evolution. The aim of the current study was to investigate the expression patterns and evolution of 20 synaptic genes
from the prefrontal cortex of 12 primate species. The genes investigated included glutamate receptors, scaffolding proteins,
synaptic vesicle components, as well as factors involved in synaptic vesicle release and structural components of the nervous
system. Our analyses revealed that there have been significant changes during primate brain evolution in the components of
the glutamatergic signaling pathway in terms of gene expression, protein expression, and promoter sequence changes. These
results could entail functional modifications in the regulation of specific genes related to processes underlying learning
and memory.
... However, it should be noted that glutamatergic neurotransmission, from a synaptic vesicle point of view, is influenced by a variety of additional factors, comprising: (1) several steps to produce mature synaptic vesicles, mostly at the axons, although some may already happen in the soma [235]; (2) an active and tight regulation of presynaptic vesicle and transmitter recycling at the level of the synaptic cleft, to counteract depletion in situations of high activity [19,216,236]; (3) the extravesicular/cytoplasmic glutamate concentration (regulated by the enzyme glutaminase) 2 to 3 times higher in the terminals than in the cell body [217] and crucial in defining intravesicular glutamate content [19,216]; (4) chloride conductance, along with the synaptic membrane potential, as also determining the glutamatergic content of synaptic vesicles and involving the participation of VGLUTs [237]; and (5) vesicular size, as it has been shown that there are different naturally occurring sizes that influence the quanta for different neurotransmitters, including glutamate [238]. How all these factors are challenged by peripheral nerve or tissue injury is not yet known. ...
Vesicular glutamate transporters (VGLUTs) are key molecules for the incorporation of glutamate in synaptic vesicles across the nervous system, and since their discovery in the early 1990s, research on these transporters has been intense and productive. This review will focus on several aspects of VGLUTs research on neurons in the periphery and the spinal cord. Firstly, it will begin with a historical account on the evolution of the morphological analysis of glutamatergic systems and the pivotal role played by the discovery of VGLUTs. Secondly, and in order to provide an appropriate framework, there will be a synthetic description of the neuroanatomy and neurochemistry of peripheral neurons and the spinal cord. This will be followed by a succinct description of the current knowledge on the expression of VGLUTs in peripheral sensory and autonomic neurons and neurons in the spinal cord. Finally, this review will address the modulation of VGLUTs expression after nerve and tissue insult, their physiological relevance in relation to sensation, pain, and neuroprotection, and their potential pharmacological usefulness.
Chemical synapses are commonly known as a structurally and functionally highly diverse class of cell-cell contacts specialized to mediate communication between neurons. They represent the smallest "computational" unit of the brain and are typically divided into excitatory and inhibitory as well as modulatory categories. These categories are subdivided into diverse types, each representing a different structure-function repertoire that in turn are thought to endow neuronal networks with distinct computational properties. The diversity of structure and function found among a given category of synapses is referred to as heterogeneity. The main building blocks for this heterogeneity are synaptic vesicles, the active zone, the synaptic cleft, the postsynaptic density, and glial processes associated with the synapse. Each of these five structural modules entails a distinct repertoire of functions, and their combination specifies the range of functional heterogeneity at mammalian excitatory synapses, which are the focus of this review. We describe synapse heterogeneity that is manifested on different levels of complexity ranging from the cellular morphology of the pre- and postsynaptic cells toward the expression of different protein isoforms at individual release sites. We attempt to define the range of structural building blocks that are used to vary the basic functional repertoire of excitatory synaptic contacts and discuss sources and general mechanisms of synapse heterogeneity. Finally, we explore the possible impact of synapse heterogeneity on neuronal network function.
Itch is a topic to which everyone can relate. The physiologic roles of itch are increasingly understood and appreciated. The pathophysiologic consequences of itch impact quality of life as much as pain. These dynamics have led to increasingly deep dives into the mechanisms that underlie and contribute to the sensation of itch. When the prior review on the Physiology of Itching was published in this journal, in 1941, itch was a black box of interest to a small number of neuroscientists and dermatologists. Itch is now appreciated as a complex and colorful Rubik's cube. Acute and chronic itch are being carefully scratched apart and reassembled by puzzle solvers across the biomedical spectrum. Mediators are being identified. Mechanisms blur boundaries of the circuitry that blend neuroscience and immunology. Measures involve psychophysics and behavioral psychology. The efforts associated with these approaches is positively impacting the care of itchy patients. There is now the potential to markedly alleviate itch, a condition that does not end life, but often ruins it. We review the itch field and provide a current understanding of the pathophysiology of itch. Itch is a disease, not only a symptom of disease.
System xc- exchanges extracellular cystine for intracellular glutamate across the plasma membrane of many cell types. One of the physiological roles of System xc- is to provide cystine for synthesis of the antioxidant glutathione. Here we report that hydrogen peroxide (H2O2) triggers the translocation of System xc- to the plasma membrane within 10 min of the initial exposure. Specifically, we observed a three-fold increase in 35S-l-cystine uptake following a 10 min exposure to 0.3 mM H2O2. This effect was dose-dependent with an EC50 for H2O2 of 65 μM. We then used cell surface biotinylation analysis to test the hypothesis that the increase in activity is due to an increased number of transporters on the plasma membrane. We demonstrated that the amount of transporter protein, xCT, localized to the plasma membrane doubles within 10 min of H2O2 exposure as a result of an increase in its delivery rate and a reduction in its internalization rate. In addition, we demonstrated that H2O2 triggered a rapid decrease in total cellular glutathione which recovered within 2 h of the oxidative insult. The kinetics of glutathione recovery matched the time course for the recovery of xCT cell surface expression and System xc- activity following removal of the oxidative insult. Collectively, these results suggest that oxidants acutely modulate the activity of System xc- by increasing its cell surface expression, and that this process may serve as an important mechanism to increase de novo glutathione synthesis during periods of oxidative stress.
This study investigated the effects of resveratrol feeding and exercise training on the skeletal muscle function and transcriptome of aged rats. Male SD rats (25 months old) were divided into the control group (Old), the daily exercise training group (Trained), and the resveratrol feeding group (Resveratrol). After 6 weeks of intervention, the body mass, grip strength, and gastrocnemius muscle mass were determined, and the muscle samples were analyzed by transcriptome sequencing. The differentially expressed genes were analyzed followed by GO enrichment analysis and KEGG analysis. The Old group showed positive increases in body mass, while both the Trained and Resveratrol groups showed negative growth. No significant differences in the gastrocnemius muscle index and absolute grip strength were found among the three groups. However, the relative grip strength was higher in the Trained group than in the Old group. Only 21 differentially expressed genes were identified in the Trained group vs. the Old group, and 12 differentially expressed genes were identified in the Resveratrol group vs. the Old group. The most enriched GO terms in the Trained group vs. the Old group were mainly associated with RNA metabolic processes and transmembrane transporters, and the significantly upregulated KEGG pathways included mucin-type O-glycan biosynthesis, drug metabolism, and pyrimidine metabolism. The most enriched GO terms in the Resveratrol group vs. the Old group were primarily associated with neurotransmitter transport and synaptic vesicle, and the upregulated KEGG pathways included synaptic vesicle cycle, nicotine addiction, retinol metabolism, insulin secretion, retrograde endocannabinoid signaling, and glutamatergic synapse. Neither exercise training nor resveratrol feeding has a notable effect on skeletal muscle function and related gene expression in aged rats. However, both exercise training and resveratrol feeding have strong effects on weight loss, which is beneficial for reducing the exercise loads of the elderly.
Although the use of nanoparticles for neuro-diagnostic and neurotherapeutic purposes provides superior benefits than the conventional approaches, it may be potentially toxic in central nervous system. In this respect, nanotechnological research focuses on nanoneurotoxicity-nanoneurosafety concepts. Despite these efforts, nanoparticles (NPs) may cause neurotoxicity, neuroinflammation, and neurodegeneration by penetrating the brain-olfactory route and blood-brain barrier (BBB). Indeed, due to their unique structures nanomaterials can easily cross biological barriers, thus avoid drug delivery problems. Despite the advancement of nanotechnology for designing therapeutic agents, toxicity of these nanomaterials is still a concern. Activation of neurons by astrocytic glutamate is a result of NPs-mediated astrocyte-neuron crosstalk. Increased extracellular glutamate levels due to enhanced synthesis and reduced reuptake may induce neuronal damage by abnormal activation of extrasynaptic N-methyl D-aspartate receptor (NMDAR) subunits. NMDAR is the key factor that mediates the disturbances in intracellular calcium homeostasis, mitochondrial dysfunction and generation of reactive oxygen species in NPs exposed neurons. While some NPs cause neuronal death by inducing NMDARs, others may be neurotoxic through the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors or protect the neurons via blocking NMDARs. However, mechanisms of dual effects of NPs, neurotoxicity or neuroprotection are not precisely known. Some NPs present neuroprotective effect either by selectively inhibiting extrasynaptic subunit of NMDARs or by attenuating oxidative stress. NPs-related proinflammatory activation of microglia contributes to the dysfunction and cytotoxicity in neurons. Therefore, investigation of the interaction of NPs with the neuronal signaling molecules and neuronal receptors is necessary for the better understanding of the neurotoxicity or neurosafety of nanomaterials.
The area of Computer Networks requires an educational model based on real market practices that can provide students with consistent technological training. However, it should be noted that a Network Project is subject to a wide range of restrictions (such as time constraints, budgeting, and the use of other necessary resources) which means there is a need to work with real cases and in the presence of a client. The purpose of this paper is to describe the effects of the application of PBL‐Maestro in the area of Network Design. PBL‐Maestro consists of a LMS (Learning Management System) that has been designed to underpin a methodological workflow for implementing PBL (Problem‐Based Learning) in the teaching of Computing, called xPBL, which provides support for Authentic Assessment. By taking xPBL as a benchmark, the course management can be carried out by following the dynamics of the cycle and stages. The topics was taught in an entirely ̈hands‐on̈ format with the use of real equipment installed inside the classroom, and it included situations and problems arising from real circumstances. Clients took part in the whole process and took on the responsibility of making requests, monitoring, guiding, and evaluating the development of the problem solution, together with the teacher. In addition, the fact that they were immersed in a practical environment and could make use of the devices employed in companies enabled them to solve problems when requested, with more facility and assertiveness. Semi‐structured interviews were conducted with the users who used the LMS.
Microtubule motor proteins – kinesins and dyneins – play an important role in intracellular transport. Impairments to axon transport can influence neurotransmitter release and short-term presynaptic plasticity. Impairments to dendritic transport, particularly recycling of synaptic receptors, affect postsynaptic plasticity. This review seeks to follow the link between microtubule motor proteins and the mechanisms of synaptic plasticity from the point of view of their involvement in transporting proteins and organelles, where their role in the mechanisms of synaptic plasticity has been demonstrated.
Synaptic efficacy is determined by various factors, including the quantal size, which is dependent on the amount of neurotransmitters in synaptic vesicles at the presynaptic terminal. It is essential for stable synaptic transmission that the quantal size is kept within a constant range and that synaptic efficacy during and after repetitive synaptic activation is maintained by replenishing release sites with synaptic vesicles. However, the mechanisms for these fundamental properties have still been undetermined. We found that the active zone protein CAST (cytomatrix at the active zone structural protein) played pivotal roles in both presynaptic regulation of quantal size and recycling of endocytosed synaptic vesicles. In the CA1 region of hippocampal slices of the CAST knockout mice, miniature excitatory synaptic responses were increased in size and synaptic depression after prolonged synaptic activation was larger, which was attributable to selective impairment of synaptic vesicle trafficking via the endosome in the presynaptic terminal likely mediated by Rab6. Therefore, CAST serves as a key molecule that regulates dynamics and neurotransmitter contents of synaptic vesicles in the excitatory presynaptic terminal in the central nervous system. This article is protected by copyright. All rights reserved.
Significance statement:
The amygdala-the brain's center for emotions-is strongly linked with the orbital cortex, a region associated with social interactions. This study provides evidence that a robust pathway from the amygdala reaches neurons in the thalamus that link directly with the orbital cortex, forming a tight tripartite network. The dual pathways from the amygdala to the orbital cortex and to the thalamus are distinct by morphology, neurochemistry, and function. This tightly linked network suggests the presence of fool-proof avenues for emotions to influence high-order cortical areas associated with affective reasoning. Specific nodes of this tripartite network are disrupted in psychiatric diseases, divorcing areas that integrate emotions and thoughts for decisions and flexible behavior.
Nicotinamide adenine dinucleotide (NAD(+)) is an enzyme cofactor or cosubstrate in many essential biological pathways. To date, the primary source of neuronal NAD(+) has been unclear. NAD(+) can be synthesized from several different precursors, among which nicotinamide is the substrate predominantly used in mammals. The rate-limiting step in the NAD(+) biosynthetic pathway from nicotinamide is performed by nicotinamide phosphoribosyltransferase (Nampt). Here, we tested the hypothesis that neurons use intracellular Nampt-mediated NAD(+) biosynthesis by generating and evaluating mice lacking Nampt in forebrain excitatory neurons (CaMKIIαNampt(-/-) mice). CaMKIIαNampt(-/-) mice showed hippocampal and cortical atrophy, astrogliosis, microgliosis, and abnormal CA1 dendritic morphology by 2-3 months of age. Importantly, these histological changes occurred with altered intrahippocampal connectivity and abnormal behavior; including hyperactivity, some defects in motor skills, memory impairment, and reduced anxiety, but in the absence of impaired sensory processes or long-term potentiation of the Schaffer collateral pathway. These results clearly demonstrate that forebrain excitatory neurons mainly use intracellular Nampt-mediated NAD(+) biosynthesis to mediate their survival and function. Studying this particular NAD(+) biosynthetic pathway in these neurons provides critical insight into their vulnerability to pathophysiological stimuli and the development of therapeutic and preventive interventions for their preservation.
Accumulating evidence suggests that glutamatergic system plays a crucial role in methamphetamine (METH) addiction. In the glutamatergic transmission, vesicular glutamate transporters (VGLUTs) are responsible for transporting glutamate into synaptic vesicles and affect the glutamate concentrations in the synaptic cleft. It is well documented that VGLUTs play an essential role in pathophysiology of several psychiatric and neurological diseases, however, whether VGLUTs also have a role in addiction caused by psychostimulant drugs is still unknown. The present study was underwent to investigate the effect of inhibition of VGLUTs on METH -induced induce conditioned place preference in rats. Rats were induced to conditioned place preference with METH (0.5, 1.0 and 2.0mg/kg) by intraperitoneal injection. Intracerebroventricular administration of 1.0 or 5.0μg Chicago sky blue 6B (CSB6B), a VGLUTs inhibitor, and 2.5h prior to METH was to observe its effect on METH-induced conditioned place preference in rats. The rats receiving METH showed stronger place preference at the dose of 1.0mg/kg than that of other doses. The intracerebroventricular administration of CSB6B (1.0, 5.0μg) 2.5h prior to the exposure to METH attenuated the acquisition of METH -induced conditioned place preference, while CSB6B itself had no effect on place preference. These results indicate that VGLUTs are involved in the effect of METH-induced conditioned place preference and may be a new target against METH addiction.
Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is
maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclindependent
kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitro, which are identical to its endogenous
phosphorylation sites in vivo. Cdk5 antagonists and expression of dominant-negative Cdk5 block phosphorylation of dynamin I,
but not of amphiphysin or AP180, in nerve terminals and inhibit SVE. Thus Cdk5 has an essential role in SVE and is the first
dephosphin kinase identified in nerve terminals.
Heterotetrameric adaptor complexes vesiculate donor mem- branes. One of the adaptor protein complexes, AP-3, is present in two forms; one form is expressed in all tissues of the body, whereas the other is restricted to brain. Mice lacking both the ubiquitous and neuronal forms of AP-3 exhibit neurological disorders that are not observed in mice that are mutant only in the ubiquitous form. To begin to understand the role of neuronal AP-3 in neurological disease, we investigated its function in in vitro assays as well as its localization in neural tissue. In the presence of GTPS both ubiquitous and neuronal forms of AP-3 can bind to purified synaptic vesicles. However, only the neuronal form of AP-3 can produce synaptic vesicles from endosomes in vitro. We also identified that the expression of neuronal AP-3 is limited to varicosities of neuronal-like pro- cesses and is expressed in most axons of the brain. Although the AP-2/clathrin pathway is the major route of vesicle produc- tion and the relatively minor neuronal AP-3 pathway is not necessary for viability, the absence of the latter could lead to the neurological abnormalities seen in mice lacking the expres- sion of AP-3 in brain. In this study we have identified the first brain-specific function for a neuronal adaptor complex.
The nerve axon is a good model system for studying the molecular mechanism of organelle transport in cells. Recently, the
new kinesin superfamily proteins (KIFs) have been identified as candidate motor proteins involved in organelle transport.
Among them KIF1A, a murine homologue of unc-104 gene of Caenorhabditis elegans, is a unique monomeric neuron– specific microtubule plus end–directed motor and has been proposed as a transporter of synaptic
vesicle precursors (Okada, Y., H. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell. 81:769–780). To elucidate the function of KIF1A in vivo, we disrupted the KIF1A gene in mice. KIF1A mutants died mostly
within a day after birth showing motor and sensory disturbances. In the nervous systems of these mutants, the transport
of synaptic vesicle precursors showed a specific and significant decrease. Consequently, synaptic vesicle density decreased
dramatically, and clusters of clear small vesicles accumulated in the cell bodies. Furthermore, marked neuronal degeneration
and death occurred both in KIF1A mutant mice and in cultures of mutant neurons. The neuronal death in cultures was blocked
by coculture with wild-type neurons or exposure to a low concentration of glutamate. These results in cultures suggested
that the mutant neurons might not sufficiently receive afferent stimulation, such as neuronal contacts or neurotransmission,
resulting in cell death. Thus, our results demonstrate that KIF1A transports a synaptic vesicle precursor and that KIF1A-mediated
axonal transport plays a critical role in viability, maintenance, and function of neurons, particularly mature neurons.
Synaptic vesicles, which have been a paradigm for the fusion of a vesicle with its target membrane, also serve as a model for understanding the formation of a vesicle from its donor membrane. Synaptic vesicles, which are formed and recycled at the periphery of the neuron, contain a highly restricted set of neuronal proteins. Insight into the trafficking of synaptic vesicle proteins has come from studying not only neurons but also neuroendocrine cells, which form synaptic-like microvesicles (SLMVs). Formation and recycling of synaptic vesicles/SLMVs takes place from the early endosome and the plasma membrane. The cytoplasmic machinery of synaptic vesicle/SLMV formation and recycling has been studied by a variety of experimental approaches, in particular using cell-free systems. This has revealed distinct machineries for membrane budding and fission. Budding is mediated by clathrin and clathrin adaptors, whereas fission is mediated by dynamin and its interacting protein SH3p4, a lysophosphatidic acid acyl transferase.
The nerve axon is a good model system for studying the molecular mechanism of organelle transport in cells. Recently, the new kinesin superfamily proteins (KIFs) have been identified as candidate motor proteins involved in organelle transport. Among them KIF1A, a murine homologue of unc-104 gene of Caenorhabditis elegans, is a unique monomeric neuron- specific microtubule plus end-directed motor and has been proposed as a transporter of synaptic vesicle precursors (Okada, Y., H. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell. 81:769-780). To elucidate the function of KIF1A in vivo, we disrupted the KIF1A gene in mice. KIF1A mutants died mostly within a day after birth showing motor and sensory disturbances. In the nervous systems of these mutants, the transport of synaptic vesicle precursors showed a specific and significant decrease. Consequently, synaptic vesicle density decreased dramatically, and clusters of clear small vesicles accumulated in the cell bodies. Furthermore, marked neuronal degeneration and death occurred both in KIF1A mutant mice and in cultures of mutant neurons. The neuronal death in cultures was blocked by coculture with wild-type neurons or exposure to a low concentration of glutamate. These results in cultures suggested that the mutant neurons might not sufficiently receive afferent stimulation, such as neuronal contacts or neurotransmission, resulting in cell death. Thus, our results demonstrate that KIF1A transports a synaptic vesicle precursor and that KIF1A-mediated axonal transport plays a critical role in viability, maintenance, and function of neurons, particularly mature neurons.
In mature neurons synaptic vesicles (SVs) undergo cycles of exo-endocytosis at synapses. It is currently unknown whether SV exocytosis and recycling occurs also in developing axons prior to synapse formation. To address this question, we have developed an immunocytochemical assay to reveal SV exo-endocytosis in hippocampal neurons developing in culture. In this assay antibodies directed against the lumenal domain of synaptotagmin I (Syt I), an intrinsic membrane protein of SVs, are used to reveal exposure of SV membranes at the cell surface. Addition of antibodies to the culture medium of living neurons for 1 hr at 37 degrees C resulted in their rapid and specific internalization by all neuronal processes and, particularly, by axons. Double immunofluorescence and electron microscopy immunocytochemistry indicated that the antibodies were retained within SVs in cell processes and underwent cycles of exo-endocytosis in parallel with SV membranes. In contrast, another endocytotic marker, wheat germ agglutinin, was rapidly cleared from the processes and transported to the cell body. Antibody-labeled SVs were still present in axons several days after antibody loading and became clustered at presynaptic sites in parallel with synaptogenesis. These results demonstrate that SVs undergo multiple cycles of exo-endocytosis in developing neuronal processes irrespective of the presence of synaptic contacts.
We have reported previously that the synaptic vesicle (SV) protein synaptophysin, when expressed in fibroblastic CHO cells, accumulates in a population of recycling microvesicles. Based on preliminary immunofluorescence observations, we had suggested that synaptophysin is targeted to the preexisting population of microvesicles that recycle transferrin (Johnston, P. A., P. L. Cameron, H. Stukenbrok, R. Jahn, P. De Camilli, and T. C. Südhof. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:2863-2872). In contrast to our results, another group reported that expression of synaptophysin in cells which normally do not express SV proteins results in the generation of a novel population of microvesicles (Leube, R. E., B. Wiedenmann, and W. W. Franke. 1989. Cell. 59:433-446). We report here a series of morphological and biochemical studies conclusively demonstrating that synaptophysin and transferrin receptors are indeed colocalized on the same vesicles in transfected CHO cells. These observations prompted us to investigate whether an overlap between the distribution of the two proteins also occurs in endocrine cell lines that endogenously express synaptophysin and other SV proteins. We have found that endocrine cell lines contain two pools of membranes positive for synaptophysin and other SV proteins. One of the two pools also contains transferrin receptors and migrates faster during velocity centrifugation. The other pool is devoid of transferrin receptors and corresponds to vesicles with the same sedimentation characteristics as SVs. These findings suggest that in transfected CHO cells and in endocrine cell lines, synaptophysin follows the same endocytic pathway as transferrin receptors but that in endocrine cells, at some point along this pathway, synaptophysin is sorted away from the recycling receptors into a specialized vesicle population. Finally, using immunofluorescent analyses, we found an overlap between the distribution of synaptophysin and transferrin receptors in the dendrites of hippocampal neurons in primary cultures before synapse formation. Axons were enriched in synaptophysin immunoreactivity but did not contain detectable levels of transferrin receptor immunoreactivity. These results suggest that SVs may have evolved from, as well as coexist with, a constitutively recycling vesicular organelle found in all cells.
The dependence of glutamate uptake on ATP-generated proton electrochemical potential was studied in a highly purified preparation of synaptic vesicles from rat brain. At low chloride concentration (4 mM), the proton pump present in synaptic vesicles generated a large membrane potential (inside-positive), associated with only minor acidification. Under these conditions, the rate of L-[3H]glutamate uptake was maximal. In addition, L-glutamate induced acidification of the vesicle interior. D-Glutamate produced only 40% of the effect, and L-aspartate or gamma-aminobutyric acid produced less than 5%. The initial rate of glutamate-induced acidification increased with increasing glutamate concentration. It was saturable and showed first-order kinetics (KM = 0.32 mM). Correspondingly, L-glutamate induced a small reduction in the membrane potential. The rate of ATP hydrolysis was unaffected. In comparison, glutamate had no effect on acidification or membrane potential in resealed membranes of chromaffin granules. At high chloride concentration (150 mM), the vesicular proton pump generated a large pH difference, associated with a small change in membrane potential. Under these conditions, uptake of L-[3H]glutamate by synaptic vesicles was low. For reconstitution, vesicle proteins were solubilized with the detergent sodium cholate, supplemented with brain phospholipids, and incorporated into liposomes. Proton pump and glutamate uptake activities of the proteoliposomes showed properties similar to those of intact vesicles indicating that the carrier was reconstituted in a functionally active form. It is concluded that glutamate uptake by synaptic vesicles is dependent on the membrane potential and that all components required for uptake are integral parts of the vesicle membrane.
Curarized cutaneous pectoris nerve-muscle preparations from frogs were stimulated at 10/s or at 2/s for periods ranging from 20 min to 4 h. End plate potential were recorded intracellularly and used to estimate the quantity of transmitter secreted during the period of stimulation. At the ends of the periods of stimulation the preparations were either fixed for electron microscopy or treated with black widow spider venom to determine the quantities of transmitter remainind in the terminal. Horseradish peroxidase or dextran was added to the bathing solution and used as a tracer to detect the formation of vesicles from the axolemma. During 4 h of stimulation at 2/s many new vesicles were formed from the axolemma and the quantity of transmitter secreted was several times greater than the quantity in the initial store. After this period of stimulation, the terminals were severely depleted of transmitter, but not of vesicles, and their general morphological organization was normal. During 20 min of stimulation at 10/s the nerve terminals swelled and were severely depleted both of vesicles and of transmitter. During a subsequent hour of rest the changes in morphology were largely reversed, many new vesicles were formed from the axolemma and the stores of transmitter were partially replenished. These results suggest (a) that synaptic vesicles fuse with, and re-form from, the membrane of the nerve terminal during and after stimulation and (b), that the re-formed vesicles can store and release transmitter.
When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae.
To identify the structures to be rapidly transported through the axons, we developed a new method to permit local cooling of mouse saphenous nerves in situ without exposing them. By this method, both anterograde and retrograde transport were successfully interrupted, while the structural integrity of the nerves was well preserved. Using radioactive tracers, anterogradely transported proteins were shown to accumulate just proximal to the cooled site, and retrogradely transported proteins just distal to the cooled site. Where the anterogradely transported proteins accumulated, the vesiculotubular membranous structures increased in amount inside both myelinated and unmyelinated axons. Such accumulated membranous structures showed a relatively uniform diameter of 50--80 nm, and some of them seemed to be continuous with the axonal smooth endoplasmic reticulum (SER). Thick sections of nerves selectively stained for the axonal membranous structures revealed that the network of the axonal SER was also packed inside axons proximal to the cooled site. In contrast, large membranous bodies of varying sizes accumulated inside axons just distal to the cooled site, where the retrogradely transported proteins accumulated. These bodies were composed mainly of multivesicular bodies and lamellated membranous structures. When horseradish peroxidase was administered in the distal end of the nerve, membranous bodies showing this activity accumulated, together with unstained membranous bodies. Hence, we are led to propose that, besides mitochondria, the membranous components in the axon can be classified into two systems from the viewpoint of axonal transport: "axonal SER and vesiculotubular structures" in the anterograde direction and "large membranous bodies" in the retrograde direction.
Frog nerve-muscle preparations were quick-frozen at various times after a single electrical stimulus in the presence of 4-aminopyridine (4-AP), after which motor nerve terminals were visualized by freeze-fracture. Previous studies have shown that such stimulation causes prompt discharge of 3,000-6,000 synaptic vesicles from each nerve terminal and, as a result, adds a large amount of synaptic vesicle membrane to its plasmalemma. In the current experiments, we sought to visualize the endocytic retrieval of this vesicle membrane back into the terminal, during the interval between 1 s and 2 min after stimulation. Two distinct types of endocytosis were observed. The first appeared to be rapid and nonselective. Within the first few seconds after stimulation, relatively large vacuoles (approximately 0.1 micron) pinched off from the plasma membrane, both near to and far away from the active zones. Previous thin-section studies have shown that such vacuoles are not coated with clathrin at any stage during their formation. The second endocytic process was slower and appeared to be selective, because it internalized large intramembrane particles. This process was manifest first by the formation of relatively small (approximately 0.05 micron) indentations in the plasma membrane, which occurred everywhere except at the active zones. These indentations first appeared at 1 s, reached a peak abundance of 5.5/micron2 by 30 s after the stimulus, and disappeared almost completely by 90 s. Previous thin-section studies indicate that these indentations correspond to clathrin-coated pits. Their total abundance is comparable with the number of vesicles that were discharged initially. These endocytic structures could be classified into four intermediate forms, whose relative abundance over time suggests that, at this type of nerve terminal, endocytosis of coated vesicles has the following characteristics: (a) the single endocytotic event is short lived relative to the time scale of two minutes; (b) earlier forms last longer than later forms; and (c) a single event spends a smaller portion of its lifetime in the flat configuration soon after the stimulus than it does later on.
Synaptic vesicles are synthesized at a rapid rate in nerve terminals to compensate for their rapid loss during neurotransmitter release. Their biogenesis involves endocytosis of synaptic vesicle membrane proteins from the plasma membrane and requires two steps, the segregation of synaptic vesicle membrane proteins from other cellular proteins, and the packaging of those unique proteins into vesicles of the correct size. By labeling an epitope-tagged variant of a synaptic vesicle protein, VAMP (synaptobrevin), at the cell surface of the neuroendocrine cell line PC12, synaptic vesicle biogenesis could be followed with considerable precision, quantitatively and kinetically. Epitope-tagged VAMP was recovered in synaptic vesicles within a few minutes of leaving the cell surface. More efficient targeting was obtained by using the VAMP mutant, del 61-70. Synaptic vesicles did not form at 15 degrees C although endocytosis still occurred. Synaptic vesicles could be generated in vitro from a homogenate of cells labeled at 15 degrees C. The newly formed vesicles are identical to those formed in vivo in their sedimentation characteristics, the presence of the synaptic vesicle protein synaptophysin, and the absence of detectable transferrin receptor. Brain, but not fibroblast cytosol, allows vesicles of the correct size to form. Vesicle formation is time and temperature-dependent, requires ATP, is calcium independent, and is inhibited by GTP-gamma S. Thus, two key steps in synaptic vesicle biogenesis have been reconstituted in vitro, allowing direct analysis of the proteins involved.
Synaptobrevin, a membrane protein of synaptic vesicles that plays a key role in exocytosis, occurs in two closely related isoforms, synaptobrevin I and II. We have analyzed the axonal transport of both isoforms in sciatic nerve and spinal roots. When fast axonal transport was interrupted by crushing, the proteins accumulated continuously proximal to the crush. Accumulation also was observed distal to the crush, but to a lesser extent (47 and 63% of the proximal accumulation for synaptobrevin I and II, respectively). Immunoelectron microscopy revealed that, proximal to the crush, synaptobrevin I and II were associated with small clear vesicles reminiscent of typical synaptic vesicles. Distal to the crush, membranes positive for synaptobrevin I or II were more heterogeneous, including larger membrane profiles that may represent endosomes. In spinal cord, synaptobrevin I and II were colocalized in many terminals. However, labeling for synaptobrevin I was more intense whereas labeling for synaptobrevin II was stronger in dorsal than in ventral horn terminals. Motor endplates contained only synaptobrevin I. In the sciatic nerve, synaptobrevin I was present predominantly in large, myelinated axons, whereas synoptobrevin II was virtually absent but abundant in small- and medium-sized axons. Lumbar sympathectomy, ventral rhizotomy, and double-labeling studies confirmed that synaptobrevin I is present predominantly in motor neurons whereas synaptobrevin II is present in adrenergic and sensory neurons. We conclude that synaptobrevin I and II are transported bidirectionally by fast axonal transport and are expressed heterogeneously in different neurons in the peripheral nervous system of the adult rat, suggesting that these isoforms have special functional roles in different sets of neurons.
After synaptic vesicles fuse with the plasma membrane and release their contents, vesicle membrane proteins recycle by endocytosis and are targeted to newly formed synaptic vesicles. The membrane traffic of an epitope-tagged form of VAMP-2 (VAMP-TAg) was observed in transfected cells to identify sequence requirements for recycling of a synaptic vesicle membrane protein. In the neuroendocrine PC12 cell line VAMP-TAg is found not only in synaptic vesicles, but also in endosomes and on the plasma membrane. Endocytosis of VAMP-TAg is a rapid and saturable process. At high expression levels VAMP-TAg accumulates at the cell surface. Rapid endocytosis of VAMP-TAg also occurs in transfected CHO cells and is therefore independent of other synaptic proteins. The majority of the measured endocytosis is not directly into synaptic vesicles since mutations in VAMP-TAg that enhance synaptic vesicle targeting did not affect endocytosis. Nonetheless, mutations that inhibited synaptic vesicle targeting, in particular replacement of methionine-46 by alanine, inhibited endocytosis by 85% in PC12 cells and by 35% in CHO cells. These results demonstrate that the synaptic vesicle targeting signal is also used for endocytosis and can be recognized in cells lacking synaptic vesicles.
Glutamate uptake into synaptic vesicles is driven by an electrochemical proton gradient formed across the membrane by a vacuolar H+-ATPase. Chloride has a biphasic effect on glutamate transport, which it activates at low concentrations (2-8 mM) and inhibits at high concentrations (>20 mM). Stimulation with 4 mM chloride was due to an increase in the Vmax of transport, whereas inhibition by high chloride concentrations was related to an increase in Km to glutamate. Both stimulation and inhibition by Cl- were observed in the presence of A23187 or (NH4)2SO4, two substances that dissipate the proton gradient (deltapH). With the use of these agents, we show that the transmembrane potential regulates the apparent affinity for glutamate, whereas the deltapH antagonizes the effect of high chloride concentrations and is important for retaining glutamate inside the vesicles. Selective dissipation of deltapH in the presence of chloride led to a significant glutamate efflux from the vesicles and promoted a decrease in the velocity of glutamate uptake. The H+-ATPase activity was stimulated when the deltapH component was dissipated. Glutamate efflux induced by chloride was saturable, and half-maximal effect was attained in the presence of 30 mM Cl-. The results indicate that: (i) both transmembrane potential and deltapH modulate the glutamate uptake at different levels and (ii) chloride affects glutamate transport by two different mechanisms. One is related to a change of the proportions between the transmembrane potential and the deltapH components of the electrochemical proton gradient, and the other involves a direct interaction of the anion with the glutamate transporter.
Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.
In this paper, evidence is presented that two distinct synaptic vesicle recycling pathways exist within a single terminal. One pathway emanates from the active zone, has a fast time course, involves no intermediate structures, and is blocked by exposure to high Mg2+/low Ca2+ saline, while the second pathway emanates at sites away from the active zone, has a slower time course, involves an endosomal intermediate, and is not sensitive to high Mg2+/low Ca2+. To visualize these two recycling pathways, the temperature-sensitive Drosophila mutant, shibire, in which vesicle recycling is normal at 19 degrees C but is blocked at 29 degrees C, was used. With exposure to 29 degrees C, complete vesicle depletion occurs as exocytosis proceeds while endocytosis is blocked. When the temperature is lowered to 26 degrees C, vesicle recycling membrane begins to accumulate as invaginations of the plasmalemma, but pinch-off is blocked. Under these experimental conditions, it was possible to distinguish the two separate pathways by electron microscopic analysis. These two pathways were further characterized by observing the normal recycling process at the permissive temperature, 19 degrees C. It is suggested that the function of these two recycling pathways might be to produce two distinct vesicle populations: the active zone and nonactive zone populations. The possibility that these two populations have different release characteristics and functions is discussed.
rsec6 and rsec8 are two components of a 17S complex in mammalian brain that is homologous to the yeast 834 kDa Sec6/8/15 complex which is essential for exocytosis. Purification and partial amino acid sequencing of the mammalian rsec6/8 complex reveals that it is composed of eight novel proteins with a combined molecular weight of 743 kDa. The complex is broadly expressed in brain and displays a plasma membrane localization in nerve terminals. Membrane associated rsec6/8 complex coimmunoprecipitates with syntaxin, a plasma membrane protein critical for neurotransmission. These data suggest a role for the mammalian rsec6/8 complex in neurotransmitter release via interactions with the core vesicle docking and fusion apparatus.
The biogenesis of synaptic‐like microvesicles (SLMVs) in neuroendocrine cells was investigated by studying the traffic of newly synthesized synaptophysin to SLMVs in PC12 cells. Synaptophysin was found to be sulfated, which facilitated the determination of its exit route from the trans‐Golgi network (TGN). Virtually all [35S]sulfate‐labeled synaptophysin was found to leave the TGN in vesicles which were indistinguishable from constitutive secretory vesicles but distinct from immature secretory granules and SLMVs. [35S]sulfate‐labeled synaptophysin was rapidly transported from the TGN to the cell surface, with a t1/2 of approximately 10 min in resting cells. After arrival at the cell surface, [35S]sulfate‐labeled synaptophysin cycled for at least 1 h between the plasma membrane and an intracellular compartment likely to be the early endosome. Up to approximately 40% of the [35S]sulfate‐labeled synaptophysin eventually (after 3 h and later) reached SLMVs, which could be distinguished from the other post‐TGN compartments by their lower buoyant density in a sucrose gradient and their selective inclusion upon permeation chromatography using a controlled‐pore glass column. Our results suggest that newly synthesized membrane proteins of SLMVs in neuroendocrine cells, and possibly of small synaptic vesicles in neurons, reach these organelles via the TGN‐‐‐‐plasma membrane‐‐‐‐early endosome.
Views about neurotransmission are strongly conditioned by information about secretory processes in gland cells, whose geometry and technical accessibility have permitted more thorough studies of several facets of the storage and release of secretions than have yet proved feasible with nervous tissue. This chapter discusses the features of gland cells directly pertinent to neurons. Although there are still a few dissenters, most cell biologists agree that the secretions of typical gland cells follow more or less the same general path through the cell. Protein components are thought to be synthesized on the ribosomes of the rough or granular endoplasmic reticulum (ER) and to enter the membrane delimited cavities of the cisternae of the ER. In cells secreting lipoproteins, the lipids are synthesized by the endoplasmic reticulum. It is often assumed that the agranular or smooth (ribosome-free) ER is especially important for this as in cells such as hepatocytes, the agranular reticulum is seen to accumulate the small globules believed to represent liproproteins.
The therapeutic effect of a course of antidepressant treatment is believed to involve a cascade of neuroadaptive changes in gene expression leading to increased neural plasticity. Because glutamate is linked to mechanisms of neural plasticity, this transmitter may play a role in these changes. This study investigated the effect of antidepressant treatment on expression of the vesicular glutamate transporters, VGLUT1-3 in brain regions of the rat. Repeated treatment with fluoxetine, paroxetine or desipramine increased VGLUT1 mRNA abundance in frontal, orbital, cingulate and parietal cortices, and regions of the hippocampus. Immunoautoradiography analysis showed that repeated antidepressant drug treatment increased VGLUT1 protein expression. Repeated electroconvulsive shock (ECS) also increased VGLUT1 mRNA abundance in regions of the cortex and hippocampus compared to sham controls. The antidepressant drugs and ECS did not alter VGLUT1 mRNA abundance after acute administration, and no change was detected after repeated treatment with the antipsychotic agents, haloperidol and chlorpromazine. In contrast to VGLUT1, the different antidepressant treatments did not commonly increase the expression of VGLUT2 or VGLUT3 mRNA. These data suggest that a course of antidepressant drug or ECS treatment increases expression of VGLUT1, a key gene involved in the regulation of glutamate secretion.
We have reported previously that the synaptic vesicle (SV) protein synaptophysin, when expressed in fibroblastic CHO cells, accumulates in a population of recycling microvesicles. Based on preliminary immunofluorescence observations, we had suggested that synaptophysin is targeted to the preexisting population of microvesicles that recycle transferrin (Johnston, P. A., P. L. Cameron, H. Stukenbrok, R. Jahn, P. De Camilli, and T. C. Sudhof. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:2863-2872). In contrast to our results, another group reported that expression of synaptophysin in cells which normally do not express SV proteins results in the generation of a novel population of microvesicles (Leube, R. E., B. Wiedenmann, and W. W. Franke. 1989. Cell. 59:433-446). We report here a series of morphological and biochemical studies conclusively demonstrating that synaptophysin and transferrin receptors are indeed colocalized on the same vesicles in transfected CHO cells. These observations prompted us to investigate whether an overlap between the distribution of the two proteins also occurs in endocrine cell lines that endogenously express synaptophysin and other SV proteins. We have found that endocrine cell lines contain two pools of membranes positive for synaptophysin and other SV proteins. One of the two pools also contains transferrin receptors and migrates faster during velocity centrifugation. The other pool is devoid of transferrin receptors and corresponds to vesicles with the same sedimentation characteristics as SVs. These findings suggest that in transfected CHO cells and in endocrine cell lines, synaptophysin follows the same endocytic pathway as transferrin receptors but that in endocrine cells, at some point along this pathway, synaptophysin is sorted away from the recycling receptors into a specialized vesicle population. Finally, using immunofluorescent analyses, we found an overlap between the distribution of synaptophysin and transferrin receptors in the dendrites of hippocampal neurons in primary cultures before synapse formation. Axons were enriched in synaptophysin immunoreactivity but did not contain detectable levels of transferrin receptor immunoreactivity. These results suggest that SVs may have evolved from, as well as coexist with, a constitutively recycling vesicular organelle found in all cells.
The optic nerve, as a part of the central nervous system (CNS), has been used to study axonal transport for decades. The present study has concentrated on the axonal transport of synaptic vesicle proteins in the optic nerve, using the “stop-flow/nerve crush” method. After blocking fast axonal transport, distinct accumulations of synaptic vesicle proteins developed during the first hour after crush-operation and marked increases were observed up to 8 h postoperative. Semiquantitative analysis, using cytofluorimetric scanning (CFS) of immunoincubated sections, revealed that the ratio between distal accumulations (organelles in retrograde transport) and proximal accumulations (organelles in anterograde transport) was much higher (up to 80–90%) for the transmembrane proteins than that for surface adsorbed proteins (only 10–20%). The pattern of axonal transport in the optic nerve was comparable to that in the sciatic nerve. However, clathrin and Rab3a immunoreactivities were accumulated in much lower amounts than that in the sciatic nerve. Most synaptic vesicle proteins were colocalized in the axons proximal to the crush. A differential distribution of synaptobrevin I and II, however, was observed in the optic nerve axons; synaptobrevin I was present in large-sized axons, while synaptobrevin II immunoreactivity was present in most axons, including the large ones. The two isoforms were, thus, partially colocalized. The results demonstrate that (1) cytofluorimetric scanning techniques could be successfully used to study axonal transport not only in peripheral nerves, but also in the CNS; (2) synaptic vesicles are transported with fast axonal transport in this nerve; and (3) some differences were noted compared with the sciatic nerve, especially for Rab3a and clathrin. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 237–250, 1997.
Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.
In mature neurons synaptic vesicles (SVs) undergo cycles of exo-endocytosis at synapses. It is currently unknown whether SV exocytosis and recycling occurs also in developing axons prior to synapse formation. To address this question, we have developed an immunocytochemical assay to reveal SV exo-endocytosis in hippocampal neurons developing in culture. In this assay antibodies directed against the lumenal domain of synaptotagmin I (Syt I), an intrinsic membrane protein of SVs, are used to reveal exposure of SV membranes at the cell surface. Addition of antibodies to the culture medium of living neurons for 1 hr at 37 degrees C resulted in their rapid and specific internalization by all neuronal processes and, particularly, by axons. Double immunofluorescence and electron microscopy immunocytochemistry indicated that the antibodies were retained within SVs in cell processes and underwent cycles of exo-endocytosis in parallel with SV membranes. In contrast, another endocytotic marker, wheat germ agglutinin, was rapidly cleared from the processes and transported to the cell body. Antibody-labeled SVs were still present in axons several days after antibody loading and became clustered at presynaptic sites in parallel with synaptogenesis. These results demonstrate that SVs undergo multiple cycles of exo-endocytosis in developing neuronal processes irrespective of the presence of synaptic contacts.
Abstract Amphiphysin and synaptojanin are two nerve terminal proteins with a putative role in synaptic vesicle endocytosis and recycling. We have investigated the intraneuronal dynamics and distribution of these two proteins, using nerve crush techniques in combination with immunofluorescence, cytofluorimetric scanning (CFS), confocal laser scanning microscopy and immuno-electron microscopy (EM). Accumulations of amphiphysin and synaptojanin immunoreactivities at the crush site were detected as short as 1 h after the lesion, indicating that a pool of these two partially cytosolic proteins moves along the axon by fast axoplasmic transport. The amount of proximal accumulation increased linearly between 1 and 8 h. CFS analysis demonstrated that only 30% of fast anterogradely transported amphiphysin and synaptojanin was returned by fast retrograde transport, in contrast to the 70% value observed for synaptophysin, a transmembrane protein. This indicates that the majority of amphiphysin and synaptojanin is degraded/metabolized in the nerve terminals. Immuno-EM showed that both amphiphysin and synaptojanin are primarily associated with heterogeneous membrane profiles in the crushed sciatic nerve and the immunoperoxidase reaction product is concentrated in the nerve terminal cytomatrix of the spinal cord. Both proteins were differentially distributed in subsets of nerve terminals, indicating heterogeneous expression in neurons.
In most models of endocytosis, the endocytic machinery is recruited from the cytoplasm by cytoplasmic tails of the plasma membrane proteins that are to be internalized. This does not appear to be true at synapses where the endocytic machinery required for synaptic vesicle recycling is localized to membrane-associated 'hot spots' [1,2]. In Drosophila neuromuscular junctions, the multi-domain protein Dap160 is also localized to hot spots [3] and has some characteristics expected of an anchoring protein. Anchoring the endocytic machinery to the plasma membrane might help contribute to the remarkable speed of synaptic vesicle recycling [4]. Here, we report that the endocytic machinery surrounds sites that are believed to be sites of exocytosis. We propose that the radial distribution of the synaptic vesicle recycling machinery already present on the plasma membrane in unstimulated nerve terminals is a fundamental unit of pre-synaptic organization and allows the nerve terminal to extract maximum recycling efficiency out of conventional endocytic machinery. Concentration of the endocytic machinery into a honeycomb matrix Dynamin [5,6] and AP-2 [2] are required for synaptic vesicle recycling in Drosophila. Both proteins were shown by confocal immunofluorescence to co-localize with Dap160 in third instar larval neuromuscular junctions (J.R. and R.B.K., unpublished observations). Dap160 was the first member of a family of proteins now called the inter-sectins [7–10] that have two Eps15 homology domains, multiple Src-homology 3 (SH3) groups and are involved in the formation of endocytic clathrin-coated vesicles [11]. We examined the localization of proteins involved in endocytosis in unstimulated nerve terminals of Drosophila neuromuscular junctions using a deconvolution micro-scope. The images acquired were refined by applying deconvolution algorithms to reduce out-of-focus haze that is generally present in confocal sections. The images of single sections of a synaptic bouton revealed that the endo-cytic machinery had holes of almost constant diameter that give the terminal a 'honeycomb' appearance (Figure 1).
Synaptic vesicles are recycled with remarkable speed and precision in nerve terminals. A major recycling pathway involves clathrin-mediated endocytosis at endocytic zones located around sites of release. Different ‘accessory’ proteins linked to this pathway have been shown to alter the shape and composition of lipid membranes, to modify membrane–coat protein interactions, and to influence actin polymerization. These include the GTPase dynamin, the lysophosphatidic acid acyl transferase endophilin, and the phosphoinositide phosphatase synaptojanin. Protein perturbation studies in living nerve terminals are now beginning to link the actions of these proteins with morphologically defined steps of endocytosis.
In most models of endocytosis, the endocytic machinery is recruited from the cytoplasm by cytoplasmic tails of the plasma membrane proteins that are to be internalized. This does not appear to be true at synapses where the endocytic machinery required for synaptic vesicle recycling is localized to membrane-associated 'hot spots' [1] [2]. In Drosophila neuromuscular junctions, the multi-domain protein Dap160 is also localized to hot spots [3] and has some characteristics expected of an anchoring protein. Anchoring the endocytic machinery to the plasma membrane might help contribute to the remarkable speed of synaptic vesicle recycling [4]. Here, we report that the endocytic machinery surrounds sites that are believed to be sites of exocytosis. We propose that the radial distribution of the synaptic vesicle recycling machinery already present on the plasma membrane in unstimulated nerve terminals is a fundamental unit of pre-synaptic organization and allows the nerve terminal to extract maximum recycling efficiency out of conventional endocytic machinery.
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Views about neurotransmission are strongly conditioned by information about secretory processes in gland cells, whose geometry and technical accessibility have permitted more thorough studies of several facets of the storage and release of secretions than have yet proved feasible with nervous tissue. This chapter discusses the features of gland cells directly pertinent to neurons. Although there are still a few dissenters, most cell biologists agree that the secretions of typical gland cells follow more or less the same general path through the cell. Protein components are thought to be synthesized on the ribosomes of the rough or granular endoplasmic reticulum (ER) and to enter the membrane delimited cavities of the cisternae of the ER. In cells secreting lipoproteins, the lipids are synthesized by the endoplasmic reticulum. It is often assumed that the agranular or smooth (ribosome-free) ER is especially important for this as in cells such as hepatocytes, the agranular reticulum is seen to accumulate the small globules believed to represent liproproteins.
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The biogenesis of synaptic-like microvesicles (SLMVs) in neuroendocrine cells was investigated by studying the traffic of newly synthesized synaptophysin to SLMVs in PC12 cells. Synaptophysin was found to be sulfated, which facilitated the determination of its exit route from the trans-Golgi network (TGN). Virtually all [35S]sulfate-labeled synaptophysin was found to leave the TGN in vesicles which were indistinguishable from constitutive secretory vesicles but distinct from immature secretory granules and SLMVs. [35S]sulfate-labeled synaptophysin was rapidly transported from the TGN to the cell surface, with a t1/2 of approximately 10 min in resting cells. After arrival at the cell surface, [35S]sulfate-labeled synaptophysin cycled for at least 1 h between the plasma membrane and an intracellular compartment likely to be the early endosome. Up to approximately 40% of the [35S]sulfate-labeled synaptophysin eventually (after 3 h and later) reached SLMVs, which could be distinguished from the other post-TGN compartments by their lower buoyant density in a sucrose gradient and their selective inclusion upon permeation chromatography using a controlled-pore glass column. Our results suggest that newly synthesized membrane proteins of SLMVs in neuroendocrine cells, and possibly of small synaptic vesicles in neurons, reach these organelles via the TGN----plasma membrane----early endosome.
The distribution of a cholinergic synaptic vesicle-specific transmembrane glycoprotein (Buckley and Kelly, 1985, J. Cell Biol. 100, 1284-1294) was investigated in the entire electromotor neuron of Torpedo marmorata using a monoclonal antibody and immunocytochemistry at the light- and electron-microscopical level (immunoperoxidase, colloidal gold). In the nerve, terminal binding of immunogold particles is restricted to synaptic vesicles. In the axon a number of additional membrane compartments like multivesicular bodies, vesiculotubular structures, lamellar bodies and electron-dense granules share the surface located synaptic vesicle-specific transmembrane glycoprotein-epitope. Membranous structures likely to represent the axoplasmic reticulum inside axons and nerve terminals are not labelled. Antibody-binding membrane compartments are accumulated at nodes of Ranvier. In the perikaryon the tubules of the trans-Golgi network as well as multivesicular bodies, lamellar bodies, electron-lucent vesicles, granules with electron-dense core and peroxisomes are labelled. Immunotransfer blots of isolated synaptic vesicles and tissue extracts of electric organ display a 100,000 mol. wt band of broad electrophoretic mobility typical of the synaptic vesicle-specific transmembrane glycoprotein. Extracts of electromotor nerve and electric lobe contain in addition a strong band at 85,000 mol. wt and a few lower molecular weight bands. We suggest that the synaptic vesicle originates directly from the trans-Golgi network. The endoplasmic reticulum is not involved in vesicle formation or retrieval. On retrograde transport the vesicle membrane compartment is likely to fuse with other intra-axonal (endosomal?) organelles.
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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.
1. Electrical activity of neuromuscular junctions of the frog was studied in a medium (Ca‐Ringer) whose Na ions had been entirely replaced by Ca.
2. Spontaneous miniature end‐plate potentials (m.e.p.p.s) of reduced amplitude are recorded in this abnormal ionic environment, and graded end‐plate potentials can be elicited by applying depolarizing current pulses to the pre‐junctional parts of the nerve.
3. Addition of 5 m M tetraethylammonium (TEA) to the Ca‐Ringer causes the appearance, in almost all‐or‐none fashion, of very large e.p.p.s (up to 45 mV in amplitude) in response to nerve stimulation.
4. These ‘giant’ e.p.p.s occur despite the curarizing action of TEA (and its depressing effect on the amplitude of m.e.p.p.s) and they persist after application of tetrodotoxin.
5. After several hours exposure to Ca‐Ringer, spontaneous end‐plate activity gradually declines, and eventually evoked e.p.p. responses fail. On return to normal Na‐Ringer, spontaneous end‐plate activity is quickly resumed, but the potentials have an abnormal, very wide, amplitude distribution.
6. The results are discussed, in conjunction with relevant work on the squid giant synapse, in terms of the ‘calcium hypothesis’ of transmitter release.
A nerve process grows by inserting new membrane material at its advancing tip, the growth cone. In embryonic cell culture and in embryos of Xenopus laevis, many growth cones establish functional synaptic transmission within minutes after contact with muscle cells. The rapidity of synapse formation suggests that the growth cone may have already acquired the appropriate neurotransmitter and the machinery for transmitter release before encountering the target cell. Here, we have used a patch of outside-out embryonic muscle membrane formed with gigaohm seal at the tip of a micropipette as an extracellular probe for the presence of channel-activating substances near the growth cones of the isolated Xenopus embryonic neurones in culture. We report that single-channel activity resembling that of muscle acetylcholine receptor channels was induced when the probe was positioned near the growth cones of 50% of the neurones, suggesting the spontaneous release of acetylcholine (ACh) from these growth cones. The release of material from growth cones may occur as a consequence of the incorporation of new membrane during neurite extension; it may also have a role in the interaction between the growth cone and its immediate environment.
Synaptic vesicles from the electric organ of the marine ray Narcine brasiliensis, purified to at least 90% homogeneity, were analyzed for the lipid and fatty acid content of their membranes. The major lipids (mol %) were phosphatidylcholine (32.3%), phosphatidylethanolamine (20.5%), phosphatidylserine (6.1%), sphingomyelin (3.0%), and cholesterol (33.3%), a composition which did not differ greatly from that of the parent electric organ. While the number of double bonds per fatty acid molecule was similar for both synaptic vesicle and whole electric organ phospholipids, the vesicles were highly enriched in docosahexenoic acid (22:6). Reaction with the amine labeling reagents isethionylacetimidate and trinitrobenzenesulfonic acid indicated that 40% of the phosphatidylserine and 60% of the phosphatidylethanolamine are present on the external (cytoplasmic) surface of the synaptic vesicle. These data on a natural fusing membrane have relevance to models of membrane fusion, which have been based largely on studies of in vitro fusion using synthetic membranes.
Axonal transport has been intensively examined as a good model for studying the mechanism of organelle transport in cells, but it is still unclear how different types of membrane organelles are transported through the nerve axon. To elucidate the function of this mechanism, we have cloned KIF1A, a novel neuron-specific kinesin superfamily motor that was discovered to be a monomeric, globular molecule and that had the fastest reported anterograde motor activity (1.2 microns/s). To identify its cargo, membranous organelles were isolated from the axon. KIF1A was associated with organelles that contained synaptic vesicle proteins such as synaptotagmin, synaptophysin, and Rab3A. However, this organelle did not contain SV2, another synaptic vesicle protein, nor did it contain presynaptic membrane proteins, such as syntaxin 1A or SNAP-25, or other known anterograde motor proteins, such as kinesin and KIF3. Thus, we suggest that the membrane proteins are sorted into different classes of transport organelles in the cell body and are transported by their specific motor proteins through the axon.
VAMP is a synaptic vesicle membrane protein required for fusion. Synaptic vesicle targeting was measured for mutants of an epitope-tagged form of VAMP in transfected PC12 cells. A signal within a predicted amphipathic alpha helix is essential for targeting to synaptic vesicles. Cellubrevin, a nonneural VAMP homolog, contains this signal and is also targeted to synaptic vesicles. Amino acid substitutions within the synaptic vesicle targeting signal either enhance or inhibit sorting of VAMP to synaptic vesicles, but do not affect the ability of VAMP to form complexes with syntaxin and SNAP-25.
The mechanisms through which synaptic vesicle membranes are reinternalized after exocytosis remain a matter of debate. Because several vesicular transport steps require GTP hydrolysis, GTP-gamma S may help identify intermediates in synaptic vesicle recycling. In GTP-gamma S-treated nerve terminals, we observed tubular invaginations of the plasmalemma that were often, but not always, capped by a clathrin-coated bud. Strikingly, the walls of these tubules were decorated by transverse electron-dense rings that were morphologically similar to structures formed by dynamin around tubular templates. Dynamin is a GTPase implicated in synaptic vesicle endocytosis and here we show that the walls of these membranous tubules, but not their distal ends, were positive for dynamin immunoreactivity. These findings demonstrate that dynamin and clathrin act at different sites in the formation of endocytic vesicles. They strongly support a role for dynamin in the fission reaction and suggest that stabilization of the GTP-bound conformation of dynamin leads to tubule formation by progressive elongation of the vesicle stalk.
Because synaptic vesicles and secretory granules are simple in composition and easy to purify, many of their protein components have been identified and often sequenced. Attempts are underway to link the small number of membrane proteins to the small number of functions the vesicles perform. The discovery of sequence homologies has helped greatly with this. In addition, techniques that have begun to prove successful involve microinjection, identification of proteins that bind synaptic vesicle proteins, DNA transfection into cells and oocytes, and more recently, in vitro reconstitution of exocytosis, endocytosis, and vesicle biogenesis. Advances in the latter areas have been strongly influenced by the breakthroughs in our knowledge of membrane traffic in nonneuronal cells. The budding reactions involved in making synaptic vesicles and secretory granules resemble in many ways the generation of carrier vesicles from the ER and the Golgi complex. Finally, exocytosis in neurons may closely resemble fusion of carrier vesicles with target organelles in nonneuronal cells, using complexes of peripheral membrane proteins, GTP hydrolysis, and integral membrane proteins with fusogenic domains. The usefulness of in vitro reconstitution, reverse genetics, and the parallels with better understood systems compensates in part for a major weakness in the field, namely the difficulty in obtaining viable mutants that are defective in the storage and release of secretory vesicle content.
We have isolated a brain-specific cDNA that encodes a Na(+)-dependent inorganic phosphate (Pi) cotransporter (BNPI). The nucleotide sequence of BNPI predicts a protein of 560 amino acids with 6-8 putative transmembrane-spanning segments that is approximately 32% identical to the rabbit kidney Na(+)-dependent Pi cotransporter. Expression of BNPI mRNA in Xenopus oocytes results in Na(+)-dependent Pi transport similar to that reported for the recombinantly expressed or native kidney Na(+)-dependent cotransporter. RNA blot analysis reveals that BNPI mRNA is expressed predominantly (if not exclusively) in brain, and in situ hybridization histochemistry reveals BNPI transcripts in neurons of the cerebral cortex, hippocampus, and cerebellum. Furthermore, we have confirmed the presence of saturable Na(+)-dependent Pi cotransport in cultured cerebellar granule cells. Together, these data demonstrate the presence of a specific neuronal Na(+)-dependent transport system for Pi in brain.
Axonal transport and targeting of the t-SNAREs SNAP-25 and syntaxin 1 were investigated in the rat peripheral nervous system using a stop-flow (crush) technique. In crush-operated sciatic nerves, accumulations of SNAP-25 and syntaxin 1 immunoreactivities were detected as early as 1 h after operation, indicating fast axonal transport. The amounts increased on the proximal side of the crush with time after crushing. Distal accumulations of SNAP-25, representing recycling to the cell body, were less than 10% of the proximal accumulations, but 40% for syntaxin 1, 50% for synaptobrevin II and 70% for synaptophysin. Immunoelectron microscopic studies demonstrated that SNAP-25 and syntaxin 1 are present on pleiotropic membranes within a diameter of 50 to 100 nm in axons proximal to a crush. Distal to the crush, labeling for syntaxin 1 and SNAP-25 were sparse and barely detectable, respectively. In addition, the two proteins were found in the axolemma. In nerve terminals of the spinal cord, both proteins were concentrated around small synaptic vesicles (about 50 nm in diameter), whereas only very few gold particles were observed near the presynaptic membrane or the active zones.
Rapid increases in Ca2+ concentration, produced by photolysis of caged Ca2+, triggered exocytosis in squid nerve terminals. This exocytosis was transient in nature, decaying with a time constant of approximately 30 ms. The decay could not be explained by a decline in presynaptic Ca2+ concentration, depletion of synaptic vesicles, or desensitization of postsynaptic receptors. Experiments in which Ca2+ was increased either in a series of steps or continuously at different rates suggested that the decay is caused by adaptation of the exocytotic Ca2+ receptor to higher levels of Ca2+. This adjustable sensitivity to Ca2+ represents a novel property of the triggering mechanism that can be used to evaluate molecular models of exocytosis. Adaptation can limit the amount of transmitter released by a nerve terminal and permit the speed of a presynaptic Ca2+ rise to serve as a critical determinant of synaptic efficacy.