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1.
Microsc Res Tech ; 87(7): 1647-1653, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38461470

RESUMEN

The synaptic basal lamina of the electrocytes was disclosed to be electron-translucent to some extent when viewed in an en-face direction in embedment-free section transmission electron microscopy (EFS-TEM), and synaptic vesicles located close to the presynaptic membrane were seen through the synaptic basal lamina together with the presynaptic and postsynaptic membranes. This feature of translucency has the potential to analyze possible spatial interrelations in situ between bioactive molecules in the synaptic basal lamina and the synaptic vesicles in further studies. The synaptic basal lamina, appearing as an electron-dense line sandwiched by two parallel lines representing the presynaptic and postsynaptic membranes in ultrathin sections cut right to the synaptic junctional plane in conventional TEM, was not fully continuous but randomly intermittent along its trajectory. Compatible with the intermittent line appearance, the en-face 3D view in embedment-free section TEM revealed for the first time partial irregular defects of the synaptic basal lamina. Considering the known functional significance of several molecules contained in the synaptic basal lamina in the maintenance and exertion of the synapse, its partial defects may not represent its rigid structural features, but its immature structure under remodeling or its dynamic changes in consistency such as the sol/gel transition, whose validity needs further examination. RESEARCH HIGHLIGHTS: In embedment-free section TEM, a 3D en-face view of synaptic basal lamina in situ is reliably possible. The basal lamina en-face is electron-translucent, which makes it possible to analyze spatial interrelation between pre- and post-synaptic components. Partial irregular defects in the basal lamina are revealed in Torpedo electrocytes, suggesting its remodeling or dynamic changes in consistency.


Asunto(s)
Microscopía Electrónica de Transmisión , Animales , Microscopía Electrónica de Transmisión/métodos , Vesículas Sinápticas/ultraestructura , Sinapsis Eléctricas/ultraestructura , Sinapsis Eléctricas/fisiología , Sinapsis/ultraestructura , Membranas Sinápticas/ultraestructura , Imagenología Tridimensional/métodos
2.
Neuropharmacology ; 196: 108711, 2021 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-34271021

RESUMEN

Glutamate is by far the most abundant neurotransmitter used by excitatory synapses in the vertebrate central nervous system. Once released into the synaptic cleft, it depolarises the postsynaptic membrane and activates downstream signalling pathways resulting in the propagation of the excitatory signal. Initial depolarisation is primarily mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. These ion channels are the first ones to be activated by released glutamate and their kinetics, dynamics and abundance on the postsynaptic membrane defines the strength of the postsynaptic response. This review focuses on native AMPA receptors and synaptic environment they inhabit and considers structural and functional properties of the receptors obtained in heterologous systems in the light of spatial and temporal constraints of the synapse. This article is part of the special Issue on 'Glutamate Receptors - AMPA receptors'.


Asunto(s)
Ácido Glutámico/metabolismo , Receptores AMPA/metabolismo , Sinapsis/metabolismo , Membranas Sinápticas/metabolismo , Animales , Humanos , Receptores AMPA/ultraestructura , Sinapsis/ultraestructura , Membranas Sinápticas/ultraestructura , Transmisión Sináptica , Factores de Tiempo
3.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-33875591

RESUMEN

Long-term potentiation (LTP) is a cellular mechanism of learning and memory that results in a sustained increase in the probability of vesicular release of neurotransmitter. However, previous work in hippocampal area CA1 of the adult rat revealed that the total number of vesicles per synapse decreases following LTP, seemingly inconsistent with the elevated release probability. Here, electron-microscopic tomography (EMT) was used to assess whether changes in vesicle density or structure of vesicle tethering filaments at the active zone might explain the enhanced release probability following LTP. The spatial relationship of vesicles to the active zone varies with functional status. Tightly docked vesicles contact the presynaptic membrane, have partially formed SNARE complexes, and are primed for release of neurotransmitter upon the next action potential. Loosely docked vesicles are located within 8 nm of the presynaptic membrane where SNARE complexes begin to form. Nondocked vesicles comprise recycling and reserve pools. Vesicles are tethered to the active zone via filaments composed of molecules engaged in docking and release processes. The density of tightly docked vesicles was increased 2 h following LTP compared to control stimulation, whereas the densities of loosely docked or nondocked vesicles congregating within 45 nm above the active zones were unchanged. The tethering filaments on all vesicles were shorter and their attachment sites shifted closer to the active zone. These findings suggest that tethering filaments stabilize more vesicles in the primed state. Such changes would facilitate the long-lasting increase in release probability following LTP.


Asunto(s)
Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Vesículas Sinápticas/ultraestructura , Animales , Encéfalo/metabolismo , Encéfalo/fisiología , Citoesqueleto , Tomografía con Microscopio Electrónico/métodos , Hipocampo/metabolismo , Potenciación a Largo Plazo/genética , Masculino , Neurotransmisores , Terminales Presinápticos/metabolismo , Terminales Presinápticos/fisiología , Ratas , Ratas Long-Evans , Sinapsis/fisiología , Membranas Sinápticas/fisiología , Membranas Sinápticas/ultraestructura , Vesículas Sinápticas/fisiología
4.
Nat Commun ; 12(1): 858, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558502

RESUMEN

Synaptic vesicles are storage organelles for neurotransmitters. They pass through a trafficking cycle and fuse with the pre-synaptic membrane when an action potential arrives at the nerve terminal. While molecular components and biophysical parameters of synaptic vesicles have been determined, our knowledge on the protein interactions in their membranes is limited. Here, we apply cross-linking mass spectrometry to study interactions of synaptic vesicle proteins in an unbiased approach without the need for specific antibodies or detergent-solubilisation. Our large-scale analysis delivers a protein network of vesicle sub-populations and functional assemblies including an active and an inactive conformation of the vesicular ATPase complex as well as non-conventional arrangements of the luminal loops of SV2A, Synaptophysin and structurally related proteins. Based on this network, we specifically target Synaptobrevin-2, which connects with many proteins, in different approaches. Our results allow distinction of interactions caused by 'crowding' in the vesicle membrane from stable interaction modules.


Asunto(s)
Reactivos de Enlaces Cruzados/química , Espectrometría de Masas , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Encéfalo/metabolismo , Fusión de Membrana , Unión Proteica , Mapas de Interacción de Proteínas , Proteolípidos , Proteoma/metabolismo , Ratas , Membranas Sinápticas/ultraestructura , Vesículas Sinápticas/ultraestructura , Sinaptofisina/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismo
5.
Neuron ; 108(5): 843-860.e8, 2020 12 09.
Artículo en Inglés | MEDLINE | ID: mdl-32991831

RESUMEN

Electron microscopy can resolve synapse ultrastructure with nanometer precision, but the capture of time-resolved, activity-dependent synaptic membrane-trafficking events has remained challenging, particularly in functionally distinct synapses in a tissue context. We present a method that combines optogenetic stimulation-coupled cryofixation ("flash-and-freeze") and electron microscopy to visualize membrane trafficking events and synapse-state-specific changes in presynaptic vesicle organization with high spatiotemporal resolution in synapses of cultured mouse brain tissue. With our experimental workflow, electrophysiological and "flash-and-freeze" electron microscopy experiments can be performed under identical conditions in artificial cerebrospinal fluid alone, without the addition of external cryoprotectants, which are otherwise needed to allow adequate tissue preservation upon freezing. Using this approach, we reveal depletion of docked vesicles and resolve compensatory membrane recycling events at individual presynaptic active zones at hippocampal mossy fiber synapses upon sustained stimulation.


Asunto(s)
Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/fisiología , Hipocampo/ultraestructura , Membranas Sinápticas/fisiología , Membranas Sinápticas/ultraestructura , Animales , Técnicas de Sustitución del Gen/métodos , Ratones , Ratones Transgénicos , Microscopía Electrónica/métodos , Microtomía/métodos , Técnicas de Cultivo de Órganos , Transporte de Proteínas/fisiología
6.
Brain Struct Funct ; 225(1): 403-425, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31875262

RESUMEN

The motor outflow for the pupillary light reflex originates in the preganglionic motoneuron subdivision of the Edinger-Westphal nucleus (EWpg), which also mediates lens accommodation. Despite their importance for vision, the morphology, ultrastructure and luminance-related inputs of these motoneurons have not been fully described in primates. In macaque monkeys, we labeled EWpg motoneurons from ciliary ganglion and orbital injections. Both approaches indicated preganglionic motoneurons occupy an EWpg organized as a unitary, ipsilateral cell column. When tracers were placed in the pretectal complex, labeled terminals targeted the ipsilateral EWpg and reached contralateral EWpg by crossing both above and below the cerebral aqueduct. They also terminated in the lateral visceral column, a ventrolateral periaqueductal gray region containing neurons projecting to the contralateral pretectum. Combining olivary pretectal and ciliary ganglion injections to determine whether a direct pupillary light reflex projection is present revealed a labeled motoneuron subpopulation that displayed close associations with labeled pretectal terminal boutons. Ultrastructurally, this subpopulation received synaptic contacts from labeled pretectal terminals that contained numerous clear spherical vesicles, suggesting excitation, and scattered dense-core vesicles, suggesting peptidergic co-transmitters. A variety of axon terminal classes, some of which may serve the near response, synapsed on preganglionic motoneurons. Quantitative analysis indicated that pupillary motoneurons receive more inhibitory inputs than lens motoneurons. To summarize, the pupillary light reflex circuit utilizes a monosynaptic, excitatory, bilateral pretectal projection to a distinct subpopulation of EWpg motoneurons. Furthermore, the interconnections between the lateral visceral column and olivary pretectal nucleus may provide pretectal cells with bilateral retinal fields.


Asunto(s)
Núcleo de Edinger-Westphal/ultraestructura , Neuronas Motoras/ultraestructura , Reflejo Pupilar/fisiología , Sinapsis/ultraestructura , Animales , Femenino , Macaca fascicularis , Macaca mulatta , Masculino , Vías Nerviosas/ultraestructura , Técnicas de Trazados de Vías Neuroanatómicas , Sustancia Gris Periacueductal/ultraestructura , Terminales Presinápticos/ultraestructura , Área Pretectal/ultraestructura , Membranas Sinápticas/ultraestructura , Vesículas Sinápticas/ultraestructura
7.
PLoS Biol ; 17(10): e3000466, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31658245

RESUMEN

The pre- and postsynaptic membranes comprising the synaptic junction differ in protein composition. The membrane trafficking mechanisms by which neurons control surface polarization of synaptic receptors remain poorly understood. The sorting receptor Sortilin-related CNS expressed 1 (SorCS1) is a critical regulator of trafficking of neuronal receptors, including the presynaptic adhesion molecule neurexin (Nrxn), an essential synaptic organizer. Here, we show that SorCS1 maintains a balance between axonal and dendritic Nrxn surface levels in the same neuron. Newly synthesized Nrxn1α traffics to the dendritic surface, where it is endocytosed. Endosomal SorCS1 interacts with the Rab11 GTPase effector Rab11 family-interacting protein 5 (Rab11FIP5)/Rab11 interacting protein (Rip11) to facilitate the transition of internalized Nrxn1α from early to recycling endosomes and bias Nrxn1α surface polarization towards the axon. In the absence of SorCS1, Nrxn1α accumulates in early endosomes and mispolarizes to the dendritic surface, impairing presynaptic differentiation and function. Thus, SorCS1-mediated sorting in dendritic endosomes controls Nrxn axonal surface polarization required for proper synapse development and function.


Asunto(s)
Proteínas de Unión al Calcio/genética , Corteza Cerebral/metabolismo , Moléculas de Adhesión de Célula Nerviosa/genética , Neuronas/metabolismo , Receptores de Superficie Celular/genética , Membranas Sinápticas/metabolismo , Transmisión Sináptica/genética , Animales , Proteínas de Unión al Calcio/metabolismo , Polaridad Celular , Corteza Cerebral/citología , Embrión de Mamíferos , Endocitosis , Endosomas/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/ultraestructura , Cultivo Primario de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Ratas , Ratas Wistar , Receptores de Superficie Celular/metabolismo , Membranas Sinápticas/ultraestructura , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo
8.
Int J Mol Sci ; 20(11)2019 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-31159267

RESUMEN

Synaptic vesicles dock on the presynaptic plasma membrane of axon terminals and become ready to fuse with the presynaptic membrane or primed. Fusion of the vesicle membrane and presynaptic membrane results in the formation of a pore between the membranes, through which the vesicle's neurotransmitter is released into the synaptic cleft. A recent electron tomography study on frog neuromuscular junctions fixed at rest showed that there is no discernible gap between or merging of the membrane of docked synaptic vesicles with the presynaptic membrane, however, the extent of the contact area between the membrane of docked synaptic vesicles and the presynaptic membrane varies 10-fold with a normal distribution. The study also showed that when the neuromuscular junctions are fixed during repetitive electrical nerve stimulation, the portion of large contact areas in the distribution is reduced compared to the portion of small contact areas, suggesting that docked synaptic vesicles with the largest contact areas are greatly primed to fuse with the membrane. Furthermore, the finding of several hemifused synaptic vesicles among the docked vesicles was briefly reported. Here, the spatial relationship of 81 synaptic vesicles with the presynaptic membrane at active zones of the neuromuscular junctions fixed during stimulation is described in detail. For the most of the vesicles, the combined thickness of each of their contact sites was not different from the sum of the membrane thicknesses of the vesicle membrane and presynaptic membrane, similar to the docked vesicles at active zones of the resting neuromuscular junctions. However, the combined membrane thickness of a small portion of the vesicles was considerably less than the sum of the membrane thicknesses, indicating that the membranes at their contact sites were fixed in a state of hemifusion. Moreover, the hemifused vesicles were found to have large contact areas with the presynaptic membrane. These findings support the recently proposed hypothesis that, at frog neuromuscular junctions, docked synaptic vesicles with the largest contact areas are most primed for fusion with the presynaptic membrane, and that hemifusion is a fusion intermediate step of the vesicle membrane with the presynaptic membrane for synaptic transmission.


Asunto(s)
Unión Neuromuscular/metabolismo , Membranas Sinápticas/metabolismo , Transmisión Sináptica , Vesículas Sinápticas/metabolismo , Animales , Anuros , Modelos Biológicos , Unión Neuromuscular/ultraestructura , Membranas Sinápticas/ultraestructura , Vesículas Sinápticas/ultraestructura
9.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-31052288

RESUMEN

A critical aim in neuroscience is to obtain a comprehensive view of how regulated neurotransmission is achieved. Our current understanding of synapses relies mainly on data from electrophysiological recordings, imaging, and molecular biology. Based on these methodologies, proteins involved in a synaptic vesicle (SV) formation, mobility, and fusion at the active zone (AZ) membrane have been identified. In the last decade, electron tomography (ET) combined with a rapid freezing immobilization of neuronal samples opened a window for understanding the structural machinery with the highest spatial resolution in situ. ET provides significant insights into the molecular architecture of the AZ and the organelles within the presynaptic nerve terminal. The specialized sensory ribbon synapses exhibit a distinct architecture from neuronal synapses due to the presence of the electron-dense synaptic ribbon. However, both synapse types share the filamentous structures, also commonly termed as tethers that are proposed to contribute to different steps of SV recruitment and exocytosis. In this review, we discuss the emerging views on the role of filamentous structures in SV exocytosis gained from ultrastructural studies of excitatory, mainly central neuronal compared to ribbon-type synapses with a focus on inner hair cell (IHC) ribbon synapses. Moreover, we will speculate on the molecular entities that may be involved in filament formation and hence play a crucial role in the SV cycle.


Asunto(s)
Citoesqueleto/metabolismo , Exocitosis , Células Receptoras Sensoriales/metabolismo , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Citoesqueleto/ultraestructura , Humanos , Células Receptoras Sensoriales/ultraestructura , Membranas Sinápticas/ultraestructura , Transmisión Sináptica , Vesículas Sinápticas/ultraestructura
10.
Behav Brain Res ; 360: 209-215, 2019 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-30552946

RESUMEN

Glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) carry the bulk of excitatory synaptic transmission. Their modulation plays key roles in synaptic plasticity, which underlies hippocampal learning and memory. A dysfunctional glutamatergic system may negatively affect learning abilities and underlie symptoms of attention-deficit/hyperactivity disorder (ADHD). The aim of this study was to investigate whether the expression and function of AMPARs were altered in ADHD. We recorded AMPAR mediated synaptic transmission at hippocampal excitatory synapses and quantified immunogold labelling density of AMPAR subunits GluA1 and GluA2/3 in a rat model for ADHD; the spontaneously hypertensive rat (SHR). Electrophysiological recordings showed significantly reduced AMPAR mediated synaptic transmission at the CA3-to-CA1 pyramidal cell synapses in stratum radiatum and stratum oriens in SHRs compared to control rats. Electronmicroscopic immunogold quantifications did not show any statistically significant changes in labelling densities of the GluA1 subunit of the AMPAR on dendritic spines in stratum radiatum or in stratum oriens. However, there was a significant increase of the GluA2/3 subunit intracellularly in stratum oriens in SHR compared to control, interpreted as a compensatory effect. The proportion of synapses lacking AMPAR subunit labelling was the same in the two genotypes. In addition, electronmicroscopic examination of tissue morphology showed the density of this type of synapse (i.e., asymmetric synapses on spines), and the average size of the synaptic membranes, to be the same. AMPAR dysfunction, possibly involving molecular changes, in hippocampus may in part reflect altered learning in individuals with ADHD.


Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad/patología , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/metabolismo , Hipocampo/patología , Receptores AMPA/metabolismo , Animales , Animales Recién Nacidos , Trastorno por Déficit de Atención con Hiperactividad/genética , Espinas Dendríticas , Modelos Animales de Enfermedad , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/efectos de los fármacos , Inmunohistoquímica , Técnicas In Vitro , Masculino , Microscopía Electrónica , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Células Piramidales/ultraestructura , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores AMPA/ultraestructura , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestructura
11.
J Neurosci Res ; 96(3): 467-480, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29231975

RESUMEN

Discs-large (Dlg) plays important roles in nerve tissue and epithelial tissue in Drosophila. However, the precise positioning of Dlg in the neuromuscular junction remains to be confirmed using an optimized labeling method. In this study, we improved the method of pre-embedding immunogold electron microscopy without the osmic tetroxide procedure, and we found that Lowicryl K4 M resin and low temperature helped to preserve the authenticity of the labeling signal with relatively good contrast. Dlg was strongly expressed in the entire subsynaptic reticulum (SSR) membrane of type Ib boutons, expressed in parts of the SSR membrane of type Is boutons, weakly expressed in axon terminals and axons, and not expressed in pre- or postsynaptic membranes of type Is boutons. In muscle cells and stratum corneum cells, Dlg was expressed both in the cytoplasm and in organelles with biomembranes. The precise location of Dlg in SSR membranes, rather than in postsynaptic membranes, shows that Dlg, with its multiple domains, acts as a remote or indirect regulator in postsynaptic signal transduction.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/ultraestructura , Inmunohistoquímica/métodos , Larva/ultraestructura , Microscopía Inmunoelectrónica/métodos , Proteínas Supresoras de Tumor/metabolismo , Resinas Acrílicas , Animales , Drosophila/metabolismo , Larva/metabolismo , Células Musculares/metabolismo , Células Musculares/ultraestructura , Unión Neuromuscular/ultraestructura , Tetróxido de Osmio/toxicidad , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Reticulum/ultraestructura , Retículo Sarcoplasmático/ultraestructura , Sinapsis , Membranas Sinápticas/ultraestructura
12.
Int J Mol Sci ; 18(11)2017 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-29084181

RESUMEN

The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (cGK) signaling pathway regulates the clustering and the recruitment of proteins and vesicles to the synapse, thereby adjusting the exoendocytic cycle to the intensity of activity. Accordingly, this pathway can accelerate endocytosis following large-scale exocytosis, and pre-synaptic cGK type II (cGKII) plays a major role in this process, controlling the homeostatic balance of vesicle exocytosis and endocytosis. We have studied synaptic vesicle recycling in cerebellar granule cells from mice lacking cGKII under strong and sustained stimulation, combining imaging techniques and ultrastructural analyses. The ultrastructure of synapses in the adult mouse cerebellar cortex was also examined in these animals. The lack of cGKII provokes structural changes to synapses in cultured cells and in the cerebellar cortex. Moreover, endocytosis is slowed down in a subset of boutons in these cells when they are stimulated strongly. In addition, from the results obtained with the selective inhibitor of cGKs, KT5823, it can be concluded that cGKI also regulates some aspects of vesicle cycling. Overall, these results confirm the importance of the cGMP pathway in the regulation of vesicle cycling following strong stimulation of cerebellar granule cells.


Asunto(s)
Cerebelo/citología , Cerebelo/metabolismo , GMP Cíclico/metabolismo , Neuronas/metabolismo , Proteínas Quinasas/metabolismo , Membranas Sinápticas/metabolismo , Animales , Endocitosis , Exocitosis , Técnica del Anticuerpo Fluorescente , Potenciales de la Membrana , Ratones , Ratones Noqueados , Imagen Molecular , Neuronas/ultraestructura , Proteínas Quinasas/genética , Vesículas Secretoras/metabolismo , Membranas Sinápticas/ultraestructura
13.
Sci Rep ; 7(1): 13768, 2017 10 23.
Artículo en Inglés | MEDLINE | ID: mdl-29061992

RESUMEN

Both excitatory and inhibitory synaptic contacts display activity dependent dynamic changes in their efficacy that are globally termed synaptic plasticity. Although the molecular mechanisms underlying glutamatergic synaptic plasticity have been extensively investigated and described, those responsible for inhibitory synaptic plasticity are only beginning to be unveiled. In this framework, the ultrastructural changes of the inhibitory synapses during plasticity have been poorly investigated. Here we combined confocal fluorescence microscopy (CFM) with high resolution scanning electron microscopy (HRSEM) to characterize the fine structural rearrangements of post-synaptic GABAA Receptors (GABAARs) at the nanometric scale during the induction of inhibitory long-term potentiation (iLTP). Additional electron tomography (ET) experiments on immunolabelled hippocampal neurons allowed the visualization of synaptic contacts and confirmed the reorganization of post-synaptic GABAAR clusters in response to chemical iLTP inducing protocol. Altogether, these approaches revealed that, following the induction of inhibitory synaptic potentiation, GABAAR clusters increase in size and number at the post-synaptic membrane with no other major structural changes of the pre- and post-synaptic elements.


Asunto(s)
Microscopía Electrónica de Rastreo/métodos , Microscopía Fluorescente/métodos , Plasticidad Neuronal , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Sinapsis/fisiología , Membranas Sinápticas/metabolismo , Animales , Hipocampo/fisiología , Hipocampo/ultraestructura , Potenciación a Largo Plazo , Ratones , Ratones Endogámicos C57BL , Sinapsis/ultraestructura , Membranas Sinápticas/ultraestructura
14.
Proc Natl Acad Sci U S A ; 114(34): 9110-9115, 2017 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-28739947

RESUMEN

Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca2+-triggered release.


Asunto(s)
Proteínas de la Fusión de la Membrana/metabolismo , Fusión de Membrana , Neuronas/metabolismo , Membranas Sinápticas/metabolismo , Animales , Calcio/metabolismo , Microscopía por Crioelectrón/métodos , Proteínas de la Fusión de la Membrana/química , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Dominios Proteicos , Proteolípidos/metabolismo , Proteolípidos/ultraestructura , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Membranas Sinápticas/ultraestructura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura , Sinaptotagmina I/química , Sinaptotagmina I/metabolismo
15.
Methods Mol Biol ; 1609: 33-41, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28660571

RESUMEN

Sucrose gradient centrifugation is a very useful technique for isolating specific membrane types based on their size and density. This is especially useful for detecting fatty acids and lipid molecules that are targeted to specialized membranes. Without fractionation, these types of molecules could be below the levels of detection after being diluted out by the more abundant lipid molecules with a more ubiquitous distribution throughout the various cell membranes. Isolation of specific membrane types where these lipids are concentrated allows for their detection and analysis. We describe herein our synaptic membrane isolation protocol that produces excellent yield and clear resolution of five major membrane fractions from a starting neural tissue homogenate: P1 (Nuclear), P2 (Cytoskeletal), P3 (Neurosynaptosomal), PSD (Post-synaptic Densities), and SV (Synaptic Vesicle).


Asunto(s)
Centrifugación por Gradiente de Densidad , Neuronas/metabolismo , Sacarosa , Membranas Sinápticas/química , Membranas Sinápticas/metabolismo , Centrifugación por Gradiente de Densidad/métodos , Lípidos de la Membrana/química , Lípidos de la Membrana/aislamiento & purificación , Membranas Sinápticas/ultraestructura
16.
J Vis Exp ; (123)2017 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-28518090

RESUMEN

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, "flash-and-freeze," that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.


Asunto(s)
Neuronas/fisiología , Membranas Sinápticas/fisiología , Animales , Channelrhodopsins/fisiología , Congelación , Hipocampo/citología , Luz , Ratones , Microscopía Electrónica , Neuronas/ultraestructura , Optogenética/métodos , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Membranas Sinápticas/ultraestructura , Transmisión Sináptica/fisiología , Vesículas Sinápticas/fisiología , Vesículas Sinápticas/ultraestructura
17.
PLoS One ; 12(3): e0174895, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28362857

RESUMEN

Identification of synaptic cleft components has been hampered by the lack of a suitable preparation enriched in synaptic junctions devoid of adjoining peripheral membranes. Prior strategies for the isolation of synaptic junctions, relying on detergents for the removal of peripheral membranes, resulted in substantial loss of membranes lining the cleft. Here, a novel, detergent-free method is described for the preparation of a synaptic junction (SJ) fraction, using phospholipase A2. Limited digestion of synaptic plasma membrane (SPM) fraction with phospholipase A2 followed by centrifugation over a sucrose cushion results in selective removal of membranes peripheral to the cleft while junctional membranes remain relatively intact as observed by electron microscopy. Enrichment in synaptic junctional structures and loss of membranes peripheral to the junctional area are further verified by demonstrating enrichment in PSD-95 and loss in mGluR5, respectively. The SJ fraction is enriched in neuroligins and neurexins, in agreement with immuno-electron microscopy data showing their selective localization to the junctional area. Among additional cell adhesion molecules tested, N-cadherin and specific isoforms of the SynCAM and SALM families also show marked enrichment in the SJ fraction, suggesting preferential localization at the synaptic cleft while others show little enrichment or decrease, suggesting that they are not restricted to or concentrated at the synaptic cleft. Treatment of the SJ fraction with glycosidases results in electrophoretic mobility shifts of all cell adhesion molecules tested, indicating glycosylation at the synaptic cleft. Biochemical and ultrastructural data presented indicate that the novel synaptic junction preparation can be used as a predictive tool for the identification and characterization of the components of the synaptic cleft.


Asunto(s)
Sinapsis/metabolismo , Animales , Western Blotting , Adhesión Celular/fisiología , Homólogo 4 de la Proteína Discs Large , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Inmunoelectrónica , Fosfolipasas A2/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor del Glutamato Metabotropico 5/metabolismo , Sinapsis/ultraestructura , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestructura
18.
Proc Natl Acad Sci U S A ; 113(8): E1098-107, 2016 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-26858418

RESUMEN

The priming of a docked synaptic vesicle determines the probability of its membrane (VM) fusing with the presynaptic membrane (PM) when a nerve impulse arrives. To gain insight into the nature of priming, we searched by electron tomography for structural relationships correlated with fusion probability at active zones of axon terminals at frog neuromuscular junctions. For terminals fixed at rest, the contact area between the VM of docked vesicles and PM varied >10-fold with a normal distribution. There was no merging of the membranes. For terminals fixed during repetitive evoked synaptic transmission, the normal distribution of contact areas was shifted to the left, due in part to a decreased number of large contact areas, and there was a subpopulation of large contact areas where the membranes were hemifused, an intermediate preceding complete fusion. Thus, fusion probability of a docked vesicle is related to the extent of its VM-PM contact area. For terminals fixed 1 h after activity, the distribution of contact areas recovered to that at rest, indicating the extent of a VM-PM contact area is dynamic and in equilibrium. The extent of VM-PM contact areas in resting terminals correlated with eccentricity in vesicle shape caused by force toward the PM and with shortness of active zone material macromolecules linking vesicles to PM components, some thought to include Ca(2+) channels. We propose that priming is a variable continuum of events imposing variable fusion probability on each vesicle and is regulated by force-generating shortening of active zone material macromolecules in dynamic equilibrium.


Asunto(s)
Canales de Calcio/metabolismo , Tomografía con Microscopio Electrónico , Membranas Sinápticas , Vesículas Sinápticas , Animales , Rana pipiens , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestructura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura
19.
Mol Neurobiol ; 53(10): 7137-7157, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-26680419

RESUMEN

The nanoscale three-dimensional structures of neurosynapses are unknown, and the neuroanatomical basis of epilepsy remains to be elucidated. Here, we studied the nanoscale three-dimensional synapses between hippocampal neurons, and membranous conjunctions between neurons were found with atomic force microscopy (AFM) and confirmed by transmission electron microscope (TEM), and their pathophysiological significance was primarily investigated. The neurons and dendrites were marked by MAP-2, axons by neurofilament 200, and synapses by synapsin I immunological staining. In the synapsin I-positive neurite ends of the neurons positively stained with MAP-2 and neurofilament 200, neurosynapses with various nanoscale morphology and structure could be found by AFM. The neurosynapses had typical three-dimensional structures of synaptic triplet including the presynaptic neurite end, synaptic cleft of 30 ∼ 40 in chemical synapses and 2 ∼ 6 nm in electrical ones, the postsynaptic neurite or dendrite spine, the typical neurite end button, the distinct pre- and postsynaptic membranes, and the obvious thickening of the postsynaptic membranes or neurites. Some membranous connections including membrane-like junctions (MLJ) and fiber-tube links (FTL) without triplet structures and cleft were found between neurons. The development frequencies of the two membranous conjunctions increased while those of the synaptic conjunctions decreased between the neurons from Otx1 knock-out mice in comparison with those between the neurons from normal mice. These results suggested that the neuroanatomical basis of Otx1 knock-out epilepsy is the combination of the decreased synaptic conjunctions and the increased membranous conjunctions.


Asunto(s)
Epilepsia/patología , Hipocampo/patología , Nanopartículas/ultraestructura , Neuronas/ultraestructura , Membranas Sinápticas/ultraestructura , Animales , Células Cultivadas , Filamentos Intermedios/metabolismo , Ratones Noqueados , Microscopía de Fuerza Atómica , Neuroglía/metabolismo , Ratas Wistar , Sinapsinas/metabolismo
20.
Neuron ; 88(4): 735-48, 2015 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-26590345

RESUMEN

Synapses are often far from their cell bodies and must largely independently cope with dysfunctional proteins resulting from synaptic activity and stress. To identify membrane-associated machines that can engulf synaptic targets destined for degradation, we performed a large-scale in vitro liposome-based screen followed by functional studies. We identified a presynaptically enriched chaperone Hsc70-4 that bends membranes based on its ability to oligomerize. This activity promotes endosomal microautophagy and the turnover of specific synaptic proteins. Loss of microautophagy slows down neurotransmission while gain of microautophagy increases neurotransmission. Interestingly, Sgt, a cochaperone of Hsc70-4, is able to switch the activity of Hsc70-4 from synaptic endosomal microautophagy toward chaperone activity. Hence, Hsc70-4 controls rejuvenation of the synaptic protein pool in a dual way: either by refolding proteins together with Sgt, or by targeting them for degradation by facilitating endosomal microautophagy based on its membrane deforming activity.


Asunto(s)
Autofagia/genética , Proteínas del Choque Térmico HSC70/genética , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Drosophila , Proteínas de Drosophila/genética , Tomografía con Microscopio Electrónico , Endosomas/metabolismo , Endosomas/ultraestructura , Escherichia coli , Proteínas de Escherichia coli , Microscopía Fluorescente , Chaperonas Moleculares , Polimerizacion , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Sinapsis/metabolismo , Sinapsis/ultraestructura , Membranas Sinápticas/ultraestructura , Transmisión Sináptica , Vesículas Sinápticas/ultraestructura
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