RESUMEN
With the increasing popularity of cryo-electron tomography (cryo-ET) in recent years, the quest to establish a method for growing primary neurons directly on electron microscopy grids (EM grids) has been ongoing. Here we describe a straightforward way to establish a mature neuronal network on EM grids, which includes formation of synaptic contacts. These synapses were thin enough to allow for direct visualization of small filaments such as SNARE proteins tethering the synaptic vesicle (SV) to the active zone plasma membrane on a Titan Krios without prior focused ion-beam milling.
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Astrocitos , Sinapsis , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , NeuronasRESUMEN
Sub-diffraction imaging of plasma membrane localized proteins, such as the SNARE (Soluble NSF Attachment Protein Receptor) proteins involved in exocytosis, in fixed cells have resulted in images with high spatial resolution, at the expense of dynamical information. Here, we have imaged localized fluorescence bursts of DRONPA-fused SNAP-25 molecules in live chromaffin cells by Total Internal Reflection Fluorescence (TIRF) imaging. We find that this method allows tracking protein cluster dynamics over relatively long times (â¼20 min.), partly due to the diffusion into the TIRF field of fresh molecules, making possible the simultaneous identification of cluster size, location and temporal evolution. The results indicate that the DRONPA-fused SNAP-25 clusters display rich dynamics, going from staying constant to disappearing and reappearing in specific cluster domains within minutes.
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Glándulas Suprarrenales/metabolismo , Células Cromafines/metabolismo , Proteínas Luminiscentes/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Animales , Difusión , Proteínas Luminiscentes/genética , Ratones Noqueados , Microscopía Fluorescente , Proteína 25 Asociada a Sinaptosomas/genética , Factores de TiempoRESUMEN
Here, we present a protocol for quantifying pyramidal neuron hyperexcitability in a mouse model of STXBP1 neurodevelopmental encephalopathy (Stxbp1hap). We describe steps for preparing brain slices, positioning electrodes, and performing an excitability test to investigate microcircuit failures. This protocol is based on recording layer 2/3 cortical pyramidal neurons in response to stimulation of two independent sets of excitatory axons that recruit feedforward inhibition microcircuits. For complete details on the use and execution of this protocol, please refer to Dos Santos et al.1.
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Modelos Animales de Enfermedad , Células Piramidales , Animales , Ratones , Trastornos del Neurodesarrollo/fisiopatologíaRESUMEN
SNAP25 is one of three neuronal SNAREs driving synaptic vesicle exocytosis. We studied three mutations in SNAP25 that cause epileptic encephalopathy: V48F, and D166Y in the synaptotagmin-1 (Syt1)-binding interface, and I67N, which destabilizes the SNARE complex. All three mutations reduced Syt1-dependent vesicle docking to SNARE-carrying liposomes and Ca2+-stimulated membrane fusion in vitro and when expressed in mouse hippocampal neurons. The V48F and D166Y mutants (with potency D166Y > V48F) led to reduced readily releasable pool (RRP) size, due to increased spontaneous (miniature Excitatory Postsynaptic Current, mEPSC) release and decreased priming rates. These mutations lowered the energy barrier for fusion and increased the release probability, which are gain-of-function features not found in Syt1 knockout (KO) neurons; normalized mEPSC release rates were higher (potency D166Y > V48F) than in the Syt1 KO. These mutations (potency D166Y > V48F) increased spontaneous association to partner SNAREs, resulting in unregulated membrane fusion. In contrast, the I67N mutant decreased mEPSC frequency and evoked EPSC amplitudes due to an increase in the height of the energy barrier for fusion, whereas the RRP size was unaffected. This could be partly compensated by positive charges lowering the energy barrier. Overall, pathogenic mutations in SNAP25 cause complex changes in the energy landscape for priming and fusion.
Neurons in the brain communicate with one another by passing molecules called neurotransmitters across the synapse connecting them together. Mutations in the machinery that controls neurotransmitter release can lead to epilepsy or developmental delays in early childhood, but how exactly is poorly understood. Neurotransmitter release is primarily controlled by three proteins that join together to form the SNARE complex, and another protein called synaptotagmin-1. This assembly of proteins primes vesicles containing neurotransmitter molecules to be released from the neuron. When calcium ions bind to synaptotagmin-1, this triggers vesicles in this readily releasable pool to then fuse with the cell membrane and secrete their contents into the small gap between the communicating neurons. Mutations associated with epilepsy and developmental delays have been found in all components of this release machinery. Here, Kádková, Murach, Østergaard et al. set out to find how three of these mutations, which are found in a protein in the SNARE complex called SNAP25, lead to aberrant neurotransmitter release. Two of these mutations are located in the interface between the SNARE complex and synaptotagmin-1, while the other is found within the bundle of proteins that make up the SNARE complex. In vitro and ex vivo experiments in mice revealed that the two interface mutations led to defects in vesicle priming, while at the same time bypassing the control by synaptotagmin-1, resulting in vesicles spontaneously fusing with the cell membrane in an unregulated manner. These mutations therefore combine loss-of-function and gain-of-function features. In contrast, the bundle mutation did not impact the number of vesicles in the releasable pool but reduced spontaneous and calcium ion evoked vesicle fusion. This was due to the mutation destabilizing the SNARE complex, which reduced the amount of energy available for merging vesicles to the membrane. These findings reveal how SNAP25 mutations can have different effects on synapse activity, and how these defects disrupt the release of neurotransmitters. This experimental framework could be used to study how other synaptic mutations lead to diseases such as epilepsy. Applying this approach to human neurons and live model organisms may lead to the discovery of new therapeutic targets for epilepsy and delayed development.
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Fusión de Membrana , Transmisión Sináptica , Animales , Ratones , Exocitosis , Mutación , Proteínas SNARE/genéticaRESUMEN
De novo mutations in STXBP1 are among the most prevalent causes of neurodevelopmental disorders and lead to haploinsufficiency, cortical hyperexcitability, epilepsy, and other symptoms in people with mutations. Given that Munc18-1, the protein encoded by STXBP1, is essential for excitatory and inhibitory synaptic transmission, it is currently not understood why mutations cause hyperexcitability. We find that overall inhibition in canonical feedforward microcircuits is defective in a P15-22 mouse model for Stxbp1 haploinsufficiency. Unexpectedly, we find that inhibitory synapses formed by parvalbumin-positive interneurons were largely unaffected. Instead, excitatory synapses fail to recruit inhibitory interneurons. Modeling confirms that defects in the recruitment of inhibitory neurons cause hyperexcitation. CX516, an ampakine that enhances excitatory synapses, restores interneuron recruitment and prevents hyperexcitability. These findings establish deficits in excitatory synapses in microcircuits as a key underlying mechanism for cortical hyperexcitability in a mouse model of Stxbp1 disorder and identify compounds enhancing excitation as a direction for therapy.
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Encefalopatías , Animales , Humanos , Ratones , Encefalopatías/genética , Encefalopatías/metabolismo , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Mutación , Neuronas/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/genéticaRESUMEN
Munc13 proteins are priming factors for SNARE-dependent exocytosis, which are activated by diacylglycerol (DAG)-binding to their C1-domain. Several Munc13 paralogs exist, but their differential roles are not well understood. We studied the interdependence of phorbolesters (DAG mimics) with Munc13-1 and ubMunc13-2 in mouse adrenal chromaffin cells. Although expression of either Munc13-1 or ubMunc13-2 stimulated secretion, phorbolester was only stimulatory for secretion when ubMunc13-2 expression dominated, but inhibitory when Munc13-1 dominated. Accordingly, phorbolester stimulated secretion in wildtype cells, or cells overexpressing ubMunc13-2, but inhibited secretion in Munc13-2/Unc13b knockout (KO) cells or in cells overexpressing Munc13-1. Phorbolester was more stimulatory in the Munc13-1/Unc13a KO than in WT littermates, showing that endogenous Munc13-1 limits the effects of phorbolester. Imaging showed that ubMunc13-2 traffics to the plasma membrane with a time-course matching Ca2+-dependent secretion, and trafficking is independent of Synaptotagmin-7 (Syt7). However, in the absence of Syt7, phorbolester became inhibitory for both Munc13-1 and ubMunc13-2-driven secretion, indicating that stimulatory phorbolester x Munc13-2 interaction depends on functional pairing with Syt7. Overall, DAG/phorbolester, ubMunc13-2 and Syt7 form a stimulatory triad for dense-core vesicle priming.
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Diglicéridos , Ésteres del Forbol , Animales , Ratones , Vesículas de Núcleo Denso , Exocitosis , Proteínas SNARE/metabolismo , SinaptotagminasRESUMEN
Fusion of vesicles with the plasma membrane and liberation of their contents is a multistep process involving several proteins. Correctly assigning the role of specific proteins and reactions in this cascade requires a measurement method with high temporal resolution. Patch-clamp recordings of cell membrane capacitance in combination with calcium measurements, calcium uncaging, and carbon-fiber amperometry allow for the accurate determination of vesicle pool sizes, their fusion kinetics, and their secreted oxidizable content. Here, we will describe this method in a model system for neurosecretion, the adrenal chromaffin cells, which secrete adrenaline.
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Calcio/metabolismo , Células Cromafines/metabolismo , Exocitosis/genética , Técnicas de Placa-Clamp/métodos , Glándulas Suprarrenales/metabolismo , Animales , Señalización del Calcio/genética , Capacidad Eléctrica , Cinética , Potenciales de la Membrana/genética , RatonesRESUMEN
Synaptotagmins confer calcium-dependence to the exocytosis of secretory vesicles, but how coexpressed synaptotagmins interact remains unclear. We find that synaptotagmin-1 and synaptotagmin-7 when present alone act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-1 and synaptotagmin-7 are found in largely non-overlapping clusters on dense-core vesicles. Synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming, and it promotes ubMunc13-2- and phorbolester-dependent priming, especially at low resting calcium concentrations. The priming effect of synaptotagmin-7 increases the number of vesicles fusing via synaptotagmin-1, while negatively affecting their fusion speed, indicating both synergistic and competitive interactions between synaptotagmins. Synaptotagmin-7 places vesicles in close membrane apposition (<6 nm); without it, vesicles accumulate out of reach of the fusion complex (20-40 nm). We suggest that a synaptotagmin-7-dependent movement toward the membrane is involved in Munc13-2/phorbolester/Ca2+-dependent priming as a prelude to fast and slow exocytosis triggering.
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Calcio/metabolismo , Membrana Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vesículas Secretoras/metabolismo , Sinaptotagminas/metabolismo , Animales , Células Cromafines/metabolismo , Tomografía con Microscopio Electrónico/métodos , Exocitosis , Fusión de Membrana , Ratones , Ratones Endogámicos C57BLRESUMEN
Chemical synaptic transmission relies on the Ca2+-induced fusion of transmitter-laden vesicles whose coupling distance to Ca2+ channels determines synaptic release probability and short-term plasticity, the facilitation or depression of repetitive responses. Here, using electron- and super-resolution microscopy at the Drosophila neuromuscular junction we quantitatively map vesicle:Ca2+ channel coupling distances. These are very heterogeneous, resulting in a broad spectrum of vesicular release probabilities within synapses. Stochastic simulations of transmitter release from vesicles placed according to this distribution revealed strong constraints on short-term plasticity; particularly facilitation was difficult to achieve. We show that postulated facilitation mechanisms operating via activity-dependent changes of vesicular release probability (e.g. by a facilitation fusion sensor) generate too little facilitation and too much variance. In contrast, Ca2+-dependent mechanisms rapidly increasing the number of releasable vesicles reliably reproduce short-term plasticity and variance of synaptic responses. We propose activity-dependent inhibition of vesicle un-priming or release site activation as novel facilitation mechanisms.
Cells in the nervous system of all animals communicate by releasing and sensing chemicals at contact points named synapses. The 'talking' (or pre-synaptic) cell stores the chemicals close to the synapse, in small spheres called vesicles. When the cell is activated, calcium ions flow in and interact with the release-ready vesicles, which then spill the chemicals into the synapse. In turn, the 'listening' (or post-synaptic) cell can detect the chemicals and react accordingly. When the pre-synaptic cell is activated many times in a short period, it can release a greater quantity of chemicals, allowing a bigger reaction in the post-synaptic cell. This phenomenon is known as facilitation, but it is still unclear how exactly it can take place. This is especially the case when many of the vesicles are not ready to respond, for example when they are too far from where calcium flows into the cell. Computer simulations have been created to model facilitation but they have assumed that all vesicles are placed at the same distance to the calcium entry point: Kobbersmed et al. now provide evidence that this assumption is incorrect. Two high-resolution imaging techniques were used to measure the actual distances between the vesicles and the calcium source in the pre-synaptic cells of fruit flies: this showed that these distances are quite variable some vesicles sit much closer to the source than others. This information was then used to create a new computer model to simulate facilitation. The results from this computing work led Kobbersmed et al. to suggest that facilitation may take place because a calcium-based mechanism in the cell increases the number of vesicles ready to release their chemicals. This new model may help researchers to better understand how the cells in the nervous system work. Ultimately, this can guide experiments to investigate what happens when information processing at synapses breaks down, for example in diseases such as epilepsy.
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Canales de Calcio/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Drosophila/metabolismoRESUMEN
Doc2B is a cytosolic protein with binding sites for Munc13 and Tctex-1 (dynein light chain), and two C2-domains that bind to phospholipids, Ca2+ and SNAREs. Whether Doc2B functions as a calcium sensor akin to synaptotagmins, or in other calcium-independent or calcium-dependent capacities is debated. We here show by mutation and overexpression that Doc2B plays distinct roles in two sequential priming steps in mouse adrenal chromaffin cells. Mutating Ca2+-coordinating aspartates in the C2A-domain localizes Doc2B permanently at the plasma membrane, and renders an upstream priming step Ca2+-independent, whereas a separate function in downstream priming depends on SNARE-binding, Ca2+-binding to the C2B-domain of Doc2B, interaction with ubMunc13-2 and the presence of synaptotagmin-1. Another function of Doc2B - inhibition of release during sustained calcium elevations - depends on an overlapping protein domain (the MID-domain), but is separate from its Ca2+-dependent priming function. We conclude that Doc2B acts as a vesicle priming protein.
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Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Células Cromafines/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas SNARE/metabolismo , Sinaptotagmina I/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Células Cultivadas , Expresión Génica , Técnicas de Inactivación de Genes , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Vesículas Secretoras/metabolismoRESUMEN
Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.
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Exocitosis , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Células Cromafines/metabolismo , Técnicas Citológicas/métodos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Sinaptotagmina I/metabolismoRESUMEN
Optogenetics is an emergent technology that combines light-sensitive proteins derived from algae, so-called opsins, with genetics. Viral vectors encoding opsins are injected into selective brain regions whereby specific cell populations can be controlled with high precision light pulses delivered via implanted optical fibres. This review focuses on explaining basic principles of optogenetics and describes important insights into neuropsychiatric mechanisms provided by the technology.
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Optogenética , Humanos , Trastornos Mentales/terapia , Enfermedades del Sistema Nervioso/terapia , Neuronas/metabolismo , Opsinas/metabolismo , Canales de Sodio/metabolismoRESUMEN
The energy required to fuse synaptic vesicles with the plasma membrane ('activation energy') is considered a major determinant in synaptic efficacy. From reaction rate theory, we predict that a class of modulations exists, which utilize linear modulation of the energy barrier for fusion to achieve supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced by hypertonic solutions. We show that complexinI/II deficiency or phorbol ester stimulation indeed affects responses to hypertonic solution in a supralinear manner. An additive vs multiplicative relationship between activation energy and fusion rate provides a novel explanation for previously observed non-linear effects of genetic/pharmacological perturbations on synaptic transmission and a novel interpretation of the cooperative nature of Ca(2+)-dependent release.
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Calcio/metabolismo , Fusión de Membrana/efectos de los fármacos , Neuronas/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Transporte Biológico , Expresión Génica , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Cinética , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Concentración Osmolar , Técnicas de Placa-Clamp , Ésteres del Forbol/farmacología , Cultivo Primario de Células , Sacarosa/farmacología , Sinapsis/efectos de los fármacos , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/metabolismo , TermodinámicaRESUMEN
Calcium-triggered exocytosis of neurotransmitter or hormone-filled vesicles has developed as the main mechanism for cell-to-cell communication in animals. Consequently, in the course of evolution this form of exocytosis has been optimized for speed. Since many of the maturation processes of vesicles are intrinsically slow, the solution has been to develop a pool of vesicles that are fully matured and can be fused very rapidly upon stimulation. Vesicles in this readily releasable pool are characterized by very low release rate constants at the resting cytosolic [Ca2+] ([Ca2+]i) and very high release rate constants at stimulated [Ca2+]i. Here I review the kinetic and molecular requirements for the existence of such a pool of vesicles, focusing on chromaffin cells of the adrenal medulla. I discuss how the use of assay methods with different time resolution may lead to fundamentally different conclusions about the role of proteins in exocytosis. Finally, I review recent evidence that the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, formed between proteins residing in the vesicle and the plasma membrane, is involved in formation and stabilization of the readily releasable vesicle pool, whereas synaptotagmin, a Ca2+- and phospholipid-binding vesicular protein, is involved in setting the Ca2+ dependence of the fusion process itself. Future studies are likely to focus on the interaction between these two classes of proteins.
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Exocitosis/fisiología , Vesículas Secretoras/fisiología , Proteínas de Transporte Vesicular/fisiología , Animales , Calcio/fisiología , Membrana Celular/fisiología , HumanosRESUMEN
Our previous mathematical model of solute-coupled water transport through the intestinal epithelium is extended for dealing with electrolytes rather than electroneutral solutes. A 3Na+-2K+ pump in the lateral membranes provides the energy-requiring step for driving transjunctional and translateral flows of water across the epithelium with recirculation of the diffusible ions maintained by a 1Na+-1K+-2Cl- cotransporter in the plasma membrane facing the serosal compartment. With intracellular non-diffusible anions and compliant plasma membranes, the model describes the dependence on membrane permeabilities and pump constants of fluxes of water and electrolytes, volumes and ion concentrations of cell and lateral intercellular space (lis), and membrane potentials and conductances. Simulating physiological bioelectrical features together with cellular and paracellular fluxes of the sodium ion, computations predict that the concentration differences between lis and bathing solutions are small for all three ions. Nevertheless, the diffusion fluxes of the ions out of lis significantly exceed their mass transports. It is concluded that isotonic transport requires recirculation of all three ions. The computed sodium recirculation flux that is required for isotonic transport corresponds to that estimated in experiments on toad small intestine. This result is shown to be robust and independent of whether the apical entrance mechanism for the sodium ion is a channel, a SGLT1 transporter driving inward uphill water flux, or an electroneutral Na+-K+-2Cl- cotransporter.