RESUMEN
Synaptic proteins need to be replaced regularly, to maintain function and to prevent damage. It is unclear whether this process, known as protein turnover, relates to synaptic morphology. To test this, we relied on nanoscale secondary ion mass spectrometry, to detect newly synthesized synaptic components in the brains of young adult (6 mo old) and aged mice (24 mo old), and on transmission electron microscopy, to reveal synapse morphology. Several parameters correlated to turnover, including pre- and postsynaptic size, the number of synaptic vesicles and the presence of a postsynaptic nascent zone. In aged mice, the turnover of all brain compartments was reduced by â¼20%. The turnover rates of the pre- and postsynapses correlated well in aged mice, suggesting that they are subject to common regulatory mechanisms. This correlation was poorer in young adult mice, in line with their higher synaptic dynamics. We conclude that synapse turnover is reflected by synaptic morphology.
Asunto(s)
Encéfalo , Sinapsis , Vesículas Sinápticas , Animales , Ratones , Sinapsis/metabolismo , Encéfalo/metabolismo , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura , Masculino , Envejecimiento/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Microscopía Electrónica de Transmisión , Ratones Endogámicos C57BLRESUMEN
A Disintegrin And Metalloproteinase 10 (ADAM10) plays a pivotal role in shaping neuronal networks by orchestrating the activity of numerous membrane proteins through the shedding of their extracellular domains. Despite its significance in the brain, the specific cellular localization of ADAM10 remains not well understood due to a lack of appropriate tools. Here, using a specific ADAM10 antibody suitable for immunostainings, we observed that ADAM10 is localized to presynapses and especially enriched at presynaptic vesicles of mossy fiber (MF)-CA3 synapses in the hippocampus. These synapses undergo pronounced frequency facilitation of neurotransmitter release, a process that play critical roles in information transfer and neural computation. We demonstrate, that in conditional ADAM10 knockout mice the ability of MF synapses to undergo this type of synaptic plasticity is greatly reduced. The loss of facilitation depends on the cytosolic domain of ADAM10 and association with the calcium sensor synaptotagmin 7 rather than ADAM10's proteolytic activity. Our findings unveil a new role of ADAM10 in the regulation of synaptic vesicle exocytosis.
Asunto(s)
Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide , Proteínas de la Membrana , Ratones Noqueados , Plasticidad Neuronal , Vesículas Sinápticas , Animales , Proteína ADAM10/metabolismo , Proteína ADAM10/genética , Plasticidad Neuronal/fisiología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones , Vesículas Sinápticas/metabolismo , Ratones Endogámicos C57BL , Sinapsis/metabolismo , Fibras Musgosas del Hipocampo/metabolismo , Hipocampo/metabolismo , Exocitosis/fisiología , Terminales Presinápticos/metabolismo , Transmisión Sináptica , Sinaptotagminas/metabolismo , Sinaptotagminas/genéticaRESUMEN
Synaptotagmin-1 (Syt1) is a calcium sensor that regulates synaptic vesicle fusion in synchronous neurotransmitter release. Syt1 interacts with negatively charged lipids and the SNARE complex to control the fusion event. However, it remains incompletely understood how Syt1 mediates Ca2+-trigged synaptic vesicle fusion. Here, we discovered that Syt1 undergoes liquid-liquid phase separation (LLPS) to form condensates both in vitro and in living cells. Syt1 condensates play a role in vesicle attachment to the PM and efficiently recruit SNAREs and complexin, which may facilitate the downstream synaptic vesicle fusion. We observed that Syt1 condensates undergo a liquid-to-gel-like phase transition, reflecting the formation of Syt1 oligomers. The phase transition can be blocked or reversed by Ca2+, confirming the essential role of Ca2+ in Syt1 oligomer disassembly. Finally, we showed that the Syt1 mutations causing Syt1-associated neurodevelopmental disorder impair the Ca2+-driven phase transition. These findings reveal that Syt1 undergoes LLPS and a Ca2+-sensitive phase transition, providing new insights into Syt1-mediated vesicle fusion.
Asunto(s)
Calcio , Vesículas Sinápticas , Sinaptotagmina I , Sinaptotagmina I/metabolismo , Sinaptotagmina I/genética , Calcio/metabolismo , Humanos , Animales , Vesículas Sinápticas/metabolismo , Multimerización de Proteína , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Transición de Fase , Mutación/genética , Células HEK293 , Fusión de Membrana , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Separación de FasesRESUMEN
The quantal content of an evoked postsynaptic response is typically determined by dividing it by the average spontaneous miniature response. However, this approach is challenged by the notion that different synaptic vesicle pools might drive spontaneous and evoked release. Here, we "silence" synaptic vesicles through pharmacological alkalinization and subsequently rescue them by optogenetic acidification. We find that such silenced synaptic vesicles, retrieved during evoked or spontaneous activity, cross-deplete the complementary release mode in a fully reversible manner. A fluorescently tagged version of the endosomal SNARE protein Vti1a, which has been suggested to identify a separate pool of spontaneously recycling synaptic vesicles, is trafficked to synaptic vesicles significantly only upon overexpression but not when endogenously tagged by CRISPR-Cas9. Thus, both release modes draw synaptic vesicles from the same readily releasable pool.
Asunto(s)
Vesículas Sinápticas , Vesículas Sinápticas/metabolismo , Animales , Transmisión Sináptica , Ratas , OptogenéticaRESUMEN
To achieve the functional polarization that underlies brain computation, neurons sort protein material into distinct compartments. Ion channel composition, for example, differs between axons and dendrites, but the molecular determinants for their polarized trafficking remain obscure. Here, we identify mechanisms that target voltage-gated Ca2+ channels (CaVs) to distinct subcellular compartments. In hippocampal neurons, CaV2s trigger neurotransmitter release at the presynaptic active zone, and CaV1s localize somatodendritically. After knockout of all three CaV2s, expression of CaV2.1, but not CaV1.3, restores neurotransmitter release. We find that chimeric CaV1.3s with CaV2.1 intracellular C-termini localize to the active zone, mediate synaptic vesicle exocytosis, and render release sensitive to CaV1 blockers. This dominant targeting function of the CaV2.1 C-terminus requires the first EF hand in its proximal segment, and replacement of the CaV2.1 C-terminus with that of CaV1.3 abolishes CaV2.1 active zone localization and function. We conclude that CaV intracellular C-termini mediate compartment-specific targeting.
Asunto(s)
Hipocampo , Animales , Hipocampo/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio/metabolismo , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo N/genética , Neuronas/metabolismo , Humanos , Ratones , Ratas , Vesículas Sinápticas/metabolismo , Exocitosis , Células HEK293RESUMEN
α-Synuclein (α-Syn) aggregation is closely associated with Parkinson's disease neuropathology. Physiologically, α-Syn promotes synaptic vesicle (SV) clustering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly. However, the underlying structural and molecular mechanisms are uncertain and it is not known whether this function affects the pathological aggregation of α-Syn. Here we show that the juxtamembrane region of vesicle-associated membrane protein 2 (VAMP2)-a component of the SNARE complex that resides on SVs-directly interacts with the carboxy-terminal region of α-Syn through charged residues to regulate α-Syn's function in clustering SVs and promoting SNARE complex assembly by inducing a multi-component condensed phase of SVs, α-Syn and other components. Moreover, VAMP2 binding protects α-Syn against forming aggregation-prone oligomers and fibrils in these condensates. Our results suggest a molecular mechanism that maintains α-Syn's function and prevents its pathological amyloid aggregation, the failure of which may lead to Parkinson's disease.
Asunto(s)
Vesículas Sinápticas , Proteína 2 de Membrana Asociada a Vesículas , alfa-Sinucleína , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/genética , Vesículas Sinápticas/metabolismo , Animales , Humanos , Unión Proteica , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Ratones , Ratas , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Neuronas/metabolismo , Neuronas/patología , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genéticaRESUMEN
Copper homeostasis is critical to the functioning of the brain, and its breakdown is linked with many brain diseases. Copper is also known to interact with the negatively charged lipid, phosphatidylserine (PS), as well as α-synuclein, an aggregation-prone protein enriched in the synapse, which plays a role in synaptic vesicle docking and fusion. However, the interplay between copper, PS lipid, and α-synuclein is not known. Herein, we report a detailed and predominantly kinetic study of the interactions among these three components pertinent to copper homeostasis and neurotransmission. We found that synaptic vesicle-mimicking small unilamellar vesicles (SUVs) can sequester any excess free Cu2+ within milliseconds, and bound Cu2+ on SUVs can be reduced to Cu+ by GSH at a nearly constant rate under physiological conditions. Moreover, we revealed that SUV-bound Cu2+ does not affect the binding between wild-type α-synuclein and SUVs but affect that between N-terminal acetylated α-synuclein and SUVs. In contrast, Cu2+ can effectively displace both types of α-synuclein from the vesicles. Our results suggest that synaptic vesicles may mediate copper transfer in the brain, while copper could participate in synaptic vesicle docking to the plasma membrane via its regulation of the interaction between α-synuclein and synaptic vesicle.
Asunto(s)
Cobre , Homeostasis , Fosfatidilserinas , Vesículas Sinápticas , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Fosfatidilserinas/metabolismo , Vesículas Sinápticas/metabolismo , Cobre/metabolismo , Homeostasis/fisiología , Humanos , Transmisión Sináptica/fisiología , AnimalesRESUMEN
Members of the synaptophysin and synaptogyrin family are vesicle proteins with four transmembrane domains. In spite of their abundance in synaptic vesicle (SV) membranes, their role remains elusive and only mild defects at the cellular and organismal level are observed in mice lacking one or more family members. Here, we show that coexpression with synapsin in fibroblasts of each of the four brain-enriched members of this family-synaptophysin, synaptoporin, synaptogyrin 1, and synaptogyrin 3-is sufficient to generate clusters of small vesicles in the same size range of SVs. Moreover, mice lacking all these four proteins have larger SVs. We conclude that synaptophysin and synaptogyrin family proteins play an overlapping function in the biogenesis of SVs and in determining their small size.
Asunto(s)
Vesículas Sinápticas , Sinaptogirinas , Sinaptofisina , Animales , Sinaptofisina/metabolismo , Sinaptofisina/genética , Vesículas Sinápticas/metabolismo , Ratones , Sinaptogirinas/metabolismo , Sinaptogirinas/genética , Sinapsinas/metabolismo , Sinapsinas/genética , Ratones Noqueados , Fibroblastos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratas , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genéticaRESUMEN
Major burdens for patients suffering from stroke are cognitive co-morbidities and epileptogenesis. Neural network disinhibition and deficient inhibitive pulses for fast network activities may result from impaired presynaptic release of the inhibitory neurotransmitter GABA. To test this hypothesis, a cortical photothrombotic stroke was induced in Sprague Dawley rats, and inhibitory currents were recorded seven days later in the peri-infarct blood-brain barrier disrupted (BBBd) hippocampus via patch-clamp electrophysiology in CA1 pyramidal cells (PC). Miniature inhibitory postsynaptic current (mIPSC) frequency was reduced to about half, and mIPSCs decayed faster in the BBBd hippocampus. Furthermore, the paired-pulse ratio of evoked GABA release was increased at 100 Hz, and train stimulations with 100 Hz revealed that the readily releasable pool (RRP), usually assumed to correspond to the number of tightly docked presynaptic vesicles, is reduced by about half in the BBBd hippocampus. These pathophysiologic changes are likely to contribute significantly to disturbed fast oscillatory activity, like cognition-associated gamma oscillations or sharp wave ripples and epileptogenesis in the BBBd hippocampus.
Asunto(s)
Barrera Hematoencefálica , Hipocampo , Potenciales Postsinápticos Inhibidores , Ratas Sprague-Dawley , Ácido gamma-Aminobutírico , Animales , Barrera Hematoencefálica/metabolismo , Ratas , Ácido gamma-Aminobutírico/metabolismo , Hipocampo/metabolismo , Masculino , Células Piramidales/metabolismo , Vesículas Sinápticas/metabolismo , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/fisiopatología , Transmisión SinápticaRESUMEN
At chemical synapses, voltage-gated Ca2+ channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca2+ microdomains they elicit must be located precisely to primed SVs to evoke rapid transmitter release. Localization is mediated by Rab3-interacting molecule (RIM) and RIM-binding proteins, which interact and bind to the C terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction of Caenorhabditis elegans hermaphrodites. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Although postsynaptic depolarization was reduced, rimb-1 mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition (E-I) balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABAA or GABAB receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus, RIMB-1 function differentially affects excitation-inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission within circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the rimb-1 defects, and similar phenotypes resulted from acute degradation of the CaV2 ß-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet it does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerge from VGCC regulation.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Unión Neuromuscular , Transmisión Sináptica , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Transmisión Sináptica/fisiología , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiología , Vesículas Sinápticas/metabolismo , Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Sinapsis/metabolismo , Sinapsis/fisiología , Red Nerviosa/fisiología , Red Nerviosa/metabolismo , Mutación , Proteínas Portadoras , Proteínas de la MembranaRESUMEN
The release of neurotransmitters (NTs) at central synapses is dependent on a cascade of protein interactions, specific to the presynaptic compartment. Among those dedicated molecules, the cytosolic complexins play an incompletely defined role as synaptic transmission regulators. Complexins are multidomain proteins that bind soluble N-ethylmaleimide sensitive factor attachment protein receptor complexes, conferring both inhibitory and stimulatory functions. Using systematic mutagenesis and comparing reconstituted in vitro membrane fusion assays with electrophysiology in cultured neurons from mice of either sex, we deciphered the function of the N-terminus of complexin (Cpx) II. The N-terminus (amino acid 1-27) starts with a region enriched in hydrophobic amino acids (1-12), which binds lipids. Mutants maintaining this hydrophobic character retained the stimulatory function of Cpx, whereas exchanges introducing charged residues perturbed both spontaneous and evoked exocytosis. Mutants in the more distal region of the N-terminal domain (amino acid 11-18) showed a spectrum of effects. On the one hand, mutation of residue A12 increased spontaneous release without affecting evoked release. On the other hand, replacing D15 with amino acids of different shapes or hydrophobic properties (but not charge) not only increased spontaneous release but also impaired evoked release. Most surprising, this substitution reduced the size of the readily releasable pool, a novel function for Cpx at mammalian synapses. Thus, the exact amino acid composition of the Cpx N-terminus fine-tunes the degree of spontaneous and evoked NT release.
Asunto(s)
Proteínas del Tejido Nervioso , Vesículas Sinápticas , Animales , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/genética , Ratones , Masculino , Femenino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/química , Mutación , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Fusión de Membrana/fisiología , Fusión de Membrana/genética , Células Cultivadas , Fenotipo , Neuronas/metabolismo , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , Ratones Endogámicos C57BL , Exocitosis/fisiología , Exocitosis/genéticaRESUMEN
Neurons are highly polarized cells that are composed of a single axon and multiple dendrites. Axon-dendrite polarity is essential for proper tissue formation and brain functions. Intracellular protein transport plays an important role in the establishment of neuronal polarity. However, the regulatory mechanism of polarized transport remains unclear. Here, we show that Rab6, a small GTPase that acts on the regulation of intracellular vesicular trafficking, plays key roles in neuronal polarization and brain development. Central nervous system-specific Rab6a/b double knock-out (Rab6 DKO) mice of both sexes exhibit severe dysplasia of the neocortex and the cerebellum. In the Rab6 DKO neocortex, impaired axonal extension of neurons results in hypoplasia of the intermediate zone. In vitro, deletion of Rab6a and Rab6b in cultured neurons from both sexes causes the abnormal accumulation of synaptic vesicle precursors (SVPs) adjacent to the Golgi apparatus, which leads to defects in axonal extension and the loss of axon-dendrite polarity. Moreover, Rab6 DKO causes significant expansion of lysosomes in the soma in neurons. Overall, our results reveal that Rab6-mediated polarized transport of SVPs is crucial for neuronal polarization and subsequent brain formation.
Asunto(s)
Encéfalo , Polaridad Celular , Ratones Noqueados , Neuronas , Vesículas Sinápticas , Proteínas de Unión al GTP rab , Animales , Polaridad Celular/fisiología , Ratones , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Neuronas/metabolismo , Femenino , Masculino , Vesículas Sinápticas/metabolismo , Encéfalo/metabolismo , Encéfalo/embriología , Encéfalo/citología , Células CultivadasRESUMEN
Phospholipids (PLs) are asymmetrically distributed at the plasma membrane. This asymmetric lipid distribution is transiently altered during calcium-regulated exocytosis, but the impact of this transient remodeling on presynaptic function is currently unknown. As phospholipid scramblase 1 (PLSCR1) randomizes PL distribution between the two leaflets of the plasma membrane in response to calcium activation, we set out to determine its role in neurotransmission. We report here that PLSCR1 is expressed in cerebellar granule cells (GrCs) and that PLSCR1-dependent phosphatidylserine egress occurred at synapses in response to neuron stimulation. Synaptic transmission is impaired at GrC Plscr1 -/- synapses, and both PS egress and synaptic vesicle (SV) endocytosis are inhibited in Plscr1 -/- cultured neurons from male and female mice, demonstrating that PLSCR1 controls PL asymmetry remodeling and SV retrieval following neurotransmitter release. Altogether, our data reveal a novel key role for PLSCR1 in SV recycling and provide the first evidence that PL scrambling at the plasma membrane is a prerequisite for optimal presynaptic performance.
Asunto(s)
Cerebelo , Proteínas de Transferencia de Fosfolípidos , Sinapsis , Transmisión Sináptica , Vesículas Sinápticas , Animales , Vesículas Sinápticas/metabolismo , Transmisión Sináptica/fisiología , Ratones , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Femenino , Masculino , Cerebelo/citología , Sinapsis/metabolismo , Sinapsis/fisiología , Células Cultivadas , Ratones Noqueados , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/fisiología , Endocitosis/fisiologíaRESUMEN
After exocytosis, release sites are cleared of vesicular residues to replenish with transmitter-filled vesicles. Endocytic and scaffold proteins are thought to underlie this site-clearance mechanism. However, the physiological significance of this mechanism at diverse mammalian central synapses remains unknown. Here, we tested this in a physiologically optimized condition using action potential evoked EPSCs at fast calyx synapse and relatively slow hippocampal CA1 synapse, in post-hearing mice brain slices at 37°C and in 1.3 mM [Ca2+]. Pharmacological block of endocytosis enhanced synaptic depression at the calyx synapse, whereas it attenuated synaptic facilitation at the hippocampal synapse. Block of scaffold protein activity likewise enhanced synaptic depression at the calyx but had no effect at the hippocampal synapse. At the fast calyx synapse, block of endocytosis or scaffold protein activity significantly enhanced synaptic depression as early as 10 ms after the stimulation onset. Unlike previous reports, neither endocytic blockers nor scaffold protein inhibitors prolonged the recovery from short-term depression. We conclude that the release-site clearance by endocytosis can be a universal phenomenon supporting vesicle replenishment at both fast and slow synapses, whereas the presynaptic scaffold mechanism likely plays a specialized role in vesicle replenishment predominantly at fast synapses.
Asunto(s)
Endocitosis , Vesículas Sinápticas , Endocitosis/fisiología , Animales , Ratones , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/fisiología , Sinapsis/fisiología , Hipocampo/fisiología , Exocitosis , Región CA1 Hipocampal/fisiologíaRESUMEN
The release of neurotransmitter from a single synaptic vesicle generates a quantal response, which at excitatory synapses in voltage-clamped neurons is referred to as a miniature excitatory postsynaptic current (mEPSC). We analyzed mEPSCs in cultured mouse hippocampal neurons and in HEK cells expressing postsynaptic proteins enabling them to receive synaptic inputs from cocultured neurons. mEPSC amplitudes and rise-times varied widely within and between cells. In neurons, mEPSCs with larger amplitudes had longer rise-times, and this correlation was stronger in neurons with longer mean rise-times. In HEK cells, this correlation was weak and unclear. Standard mechanisms thought to govern mEPSCs cannot account for these results. We therefore developed models to simulate mEPSCs and assess their dependence on different factors. Modeling indicated that longer diffusion times for transmitters released by larger vesicles to reach more distal receptors cannot account for the correlation between rise-time and amplitude. By contrast, incorporating the vesicle size dependence of fusion pore expulsion time recapitulated experimental results well. Larger vesicles produce mEPSCs with larger amplitudes and also take more time to lose their content. Thus, fusion pore flux directly contributes to mEPSC rise-time. Variations in fusion pores account for differences among neurons, between neurons and HEK cells, and the correlation between rise-time and the slope of rise-time versus amplitude plots. Plots of mEPSC amplitude versus rise-time are sensitive to otherwise inaccessible properties of a synapse and offer investigators a means of assessing the role of fusion pores in synaptic release.
Asunto(s)
Hipocampo , Neuronas , Vesículas Sinápticas , Animales , Ratones , Humanos , Neuronas/fisiología , Neuronas/metabolismo , Células HEK293 , Vesículas Sinápticas/metabolismo , Hipocampo/fisiología , Hipocampo/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Sinapsis/fisiología , Sinapsis/metabolismo , Células Cultivadas , Fusión de Membrana/fisiología , Potenciales Postsinápticos Miniatura/fisiologíaRESUMEN
During electrochemical signal transmission through synapses, triggered by an action potential (AP), a stochastic number of synaptic vesicles (SVs), called the "quantal content," release neurotransmitters in the synaptic cleft. It is widely accepted that the quantal content probability distribution is a binomial based on the number of ready-release SVs in the presynaptic terminal. But the latter number itself fluctuates due to its stochastic replenishment, hence the actual distribution of quantal content is unknown. We show that exact distribution of quantal content can be derived for general stochastic AP inputs in the steady state. For fixed interval AP train, we prove that the distribution is a binomial, and corroborate our predictions by comparison with electrophysiological recordings from MNTB-LSO synapses of juvenile mice. For a Poisson train, we show that the distribution is nonbinomial. Moreover, we find exact moments of the quantal content in the Poisson and other general cases, which may be used to obtain the model parameters from experiments.
Asunto(s)
Modelos Neurológicos , Transmisión Sináptica , Vesículas Sinápticas , Transmisión Sináptica/fisiología , Animales , Ratones , Vesículas Sinápticas/fisiología , Vesículas Sinápticas/metabolismo , Potenciales de Acción/fisiología , Procesos Estocásticos , Distribución de PoissonRESUMEN
The intricate molecular and structural sequences guiding the formation and consolidation of memories within neuronal circuits remain largely elusive. In this study, we investigate the roles of two pivotal presynaptic regulators, the small GTPase Rab3, enriched at synaptic vesicles, and the cell adhesion protein Neurexin-1, in the formation of distinct memory phases within the Drosophila mushroom body Kenyon cells. Our findings suggest that both proteins play crucial roles in memory-supporting processes within the presynaptic terminal, operating within distinct plasticity modules. These modules likely encompass remodeling and maturation of existing active zones (AZs), as well as the formation of new AZs.
Asunto(s)
Proteínas de Drosophila , Memoria , Cuerpos Pedunculados , Terminales Presinápticos , Proteínas de Unión al GTP rab3 , Animales , Cuerpos Pedunculados/fisiología , Cuerpos Pedunculados/metabolismo , Terminales Presinápticos/fisiología , Terminales Presinápticos/metabolismo , Proteínas de Drosophila/metabolismo , Memoria/fisiología , Proteínas de Unión al GTP rab3/metabolismo , Proteínas de Unión al GTP rab3/genética , Proteínas del Tejido Nervioso/metabolismo , Drosophila , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/fisiologíaRESUMEN
The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to glutamate release at the synapse. Fluorescence microscopy imaging revealed that the postsynaptic density (PSD) and scaffolding proteins in the presynaptic active zone (AZ) align across the synapse to form a trans-synaptic "nanocolumn," but the relation to synaptic vesicle release sites is uncertain. Here, we employ focused-ion beam (FIB) milling and cryoelectron tomography to image synapses under near-native conditions. Improved image contrast, enabled by FIB milling, allows simultaneous visualization of supramolecular nanoclusters within the AZ and PSD and synaptic vesicles. Surprisingly, membrane-proximal synaptic vesicles, which fuse to release glutamate, are not preferentially aligned with AZ or PSD nanoclusters. These synaptic vesicles are linked to the membrane by peripheral protein densities, often consistent in size and shape with Munc13, as well as globular densities bridging the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by our tomograms predict that clustering AMPARs within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. Together, our data support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites.
Asunto(s)
Tomografía con Microscopio Electrónico , Vesículas Sinápticas , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura , Tomografía con Microscopio Electrónico/métodos , Animales , Ratas , Densidad Postsináptica/metabolismo , Densidad Postsináptica/ultraestructura , Microscopía por Crioelectrón/métodos , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
Synapsins are highly abundant presynaptic proteins that play a crucial role in neurotransmission and plasticity via the clustering of synaptic vesicles. The synapsin III isoform is usually downregulated after development, but in hippocampal mossy fiber boutons, it persists in adulthood. Mossy fiber boutons express presynaptic forms of short- and long-term plasticity, which are thought to underlie different forms of learning. Previous research on synapsins at this synapse focused on synapsin isoforms I and II. Thus, a complete picture regarding the role of synapsins in mossy fiber plasticity is still missing. Here, we investigated presynaptic plasticity at hippocampal mossy fiber boutons by combining electrophysiological field recordings and transmission electron microscopy in a mouse model lacking all synapsin isoforms. We found decreased short-term plasticity, i.e., decreased facilitation and post-tetanic potentiation, but increased long-term potentiation in male synapsin triple knock-out (KO) mice. At the ultrastructural level, we observed more dispersed vesicles and a higher density of active zones in mossy fiber boutons from KO animals. Our results indicate that all synapsin isoforms are required for fine regulation of short- and long-term presynaptic plasticity at the mossy fiber synapse.
Asunto(s)
Ratones Noqueados , Fibras Musgosas del Hipocampo , Plasticidad Neuronal , Terminales Presinápticos , Sinapsinas , Animales , Sinapsinas/metabolismo , Sinapsinas/genética , Fibras Musgosas del Hipocampo/fisiología , Masculino , Plasticidad Neuronal/fisiología , Terminales Presinápticos/fisiología , Terminales Presinápticos/metabolismo , Ratones Endogámicos C57BL , Ratones , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura , Potenciales Postsinápticos Excitadores/fisiologíaRESUMEN
Synaptic vesicles are organelles with a precisely defined protein and lipid composition1,2, yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single-particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains3. The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the proton gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters4,5. Synaptophysin6 and its paralogues synaptoporin7 and synaptogyrin8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin-knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin-knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin.