Gli autori ringraziano i membri del laboratorio Harata per utili discussioni durante l&#39;esecuzione di questo lavoro. Questo lavoro è stato finanziato da sovvenzioni dal American Heart Association, la distonia Fondazione di ricerca medica, l&#39;Edward Mallinckrodt, Jr. Foundation, la National Science Foundation e la Fondazione Whitehall a NCH

Gli autori dichiarano interessi finanziari concorrenti.

Abbiamo descritto protocolli per la colorazione e decolorazione vescicole sinaptiche in risposta evocata, attività sinaptica spontanea e miniatura, e per l&#39;imaging durante la fase di decolorazione. Oltre ai protocolli esistenti, abbiamo incluso un nuovo protocollo di osservare il decolorante FM basato su attività sinaptica miniatura. L&#39;utilizzo di questi protocolli, abbiamo precedentemente identificato anomalie nei neuroni in coltura da un modello murino della malattia movimento distonia. In confronto alle lor...

La funzionalità di vescicole sinaptiche è un importante determinante della trasmissione sinaptica. Tali vescicole rilasciano neurotrasmettitori quando si fondono con la membrana plasmatica presinaptica (esocitosi), e sono pronte per un altro ciclo di stampa dopo essere rigenerata dalla membrana plasmatica (endocitosi) e ricaricato con neurotrasmettitore. La ricerca sulla dinamica dei meccanismi sottostanti e riciclo delle vescicole sinaptiche è stata notevolmente accelerato dall&#39;introduzione di coloranti styryl FM 1. Queste molecole anfipatiche, che hanno carica positiva gruppi di testa idrofile e code idrofobiche (più coloranti in figura 1A, stereoview di FM1-43 in Figura 1B), possono reversibilmente entrare e uscire membrane lipidiche senza permea. Gruppi di coloranti FM condividono caratteristiche simili che influenzano la gamma di luce che essi emettono. Ad esempio, FM2-10, FM1-43, e FM1-84 hanno un doppio legame tra due composti ciclici e show emissione verde. La differenza tra loro è la lunghezza della coda idrofoba, che determina la sua idrofobicità e quindi il tasso di uscita dalla membrana (departitioning). Nei casi di FM5-95 e FM4-64, tre doppi legami collegano i composti ciclici, e mostrano emissione rossa. Questi coloranti differiscono rispetto alle loro parti idrofile. In tutti i coloranti FM, l&#39;intensità di fluorescenza aumenta quando inseriti nelle membrane biologiche, a causa di un aumento della resa quantica nell&#39;ambiente idrofobo rispetto all&#39;ambiente idrofilo. Così i cambiamenti di intensità FM rappresentano le variazioni del fatturato membrana. I diversi colori (spettri di emissione) e idrofobicità rendono la FM tinge uno strumento di ricerca versatile nel riciclo delle vescicole sinaptiche. Sulla base di queste caratteristiche, i coloranti FM sono principalmente utilizzati secondo il seguente schema nell&#39;analisi riciclo delle vescicole sinaptiche (Figura 2). Neurons sono immersi in una soluzione extracellulare contenente il colorante FM, permettendogli di essere ripreso in vescicole sinaptiche (SVs), in quanto costituiscono via endocitosi (colorazione). Il colorante viene poi lavata applicando una soluzione extracellulare priva di coloranti; questo rivela terminazioni nervose funzionali, cioè solo quelli sottoposti attivamente endocitosi conterrà un cluster di vescicole sinaptiche che vengono caricati con il colorante (Figura 2 in basso). Esocitosi successiva porta alla perdita del colorante FM allo spazio extracellulare e una perdita concomitante di fluorescenza (decolorante, dovuto sia alla departitioning ad un ambiente idrofilo e diffusione lontano dal sito di esocitosi). Pertanto le variazioni di intensità di fluorescenza FM sono indicatori di vescicole sinaptiche eso-ed endocitosi. Coloranti FM sono stati utilizzati per macchiare e Destain le vescicole sinaptiche in vari organismi e preparati 2,3. Gli esempi includono neur mammifericulture onal 4-9, mammiferi fettine di cervello 10,11, giunzioni neuromuscolari 12,13, retina neuroni bipolari 14,15 e le cellule ciliate della coclea 16. Tipicamente in tali esperimenti, sia di colorazione e decolorazione sono innescati da ampiamente stimolando i neuroni (attività evocata). Recentemente, tuttavia, riciclo delle vescicole sinaptiche in risposta alla stimolazione debole è stato anche analizzato, come ha riciclo in assenza di uno stimolo esterno (attività sinaptica spontanea e miniatura) 9,17-19. Attività sinaptiche spontanee e miniaturizzati sono definiti come quelle che si verificano in assenza di stimoli esterni, le prime riguardano la scarica spontanea dei potenziali d&#39;azione (Figura 3). Queste attività sinaptiche deboli sono associati a piccoli cambiamenti nei segnali FM rispetto a quelli attivati ​​dalla stimolazione ampio. La misurazione richiede che le variazioni fl FMIntensità mento riflette accuratamente synaptic esocitosi delle vescicole o endocitosi ma le modifiche non artefatti di intensità. Una causa del manufatto è la presenza di marcatura non specifica della membrana plasmatica da coloranti FM. Washout graduale di questo componente comporti un cambiamento graduale nell&#39;intensità di fluorescenza misurata, che sarà erroneamente attribuita ad attività sinaptica. Questo fattore può essere ridotto con metodi appropriati (vedi Protocol). La causa più notevole del manufatto è il photobleaching di colorante FM trattenuto all&#39;interno vescicole sinaptiche. I cambiamenti Fotodecolorazione relativi a intensità FM devono essere piccoli in confronto ai biologiche (Synaptic) le modifiche che vengono misurati. Il recente sviluppo di telecamere sensibili, per esempio la fotocamera del dispositivo ad accoppiamento di carica-elettroni moltiplicando (EMCCD), permette di minimizzare photobleaching accorciando il tempo di esposizione e indebolendo l&#39;intensità della luce usata per eccitare il fluoroforo. Un&#39;altra causa del manufatto è a i driftn il livello di messa a fuoco del microscopio ottico. La deriva fuoco durante una sessione di imaging può essere causato da effetti meccanici o termici, e verrà erroneamente portare ad un cambiamento nell&#39;intensità di fluorescenza misurata. Qui si descrive protocolli e apparecchiature che consentono di utilizzare coloranti FM analizzare riciclo delle vescicole sinaptiche anche nell&#39;ambito di stimolazione debole o non, in particolare, l&#39;attività sinaptica miniatura. Mostriamo esempi di colorazione e decolorazione delle vescicole sinaptiche durante gli eventi evocati e spontanei, utilizzando colture di neuroni dell&#39;ippocampo roditori, e di imaging la fase di decolorazione. Dimostriamo anche il modo di valutare il grado di FM colorante photobleaching, in assenza di eventuali perdite colorante FM a causa di attività sinaptica.

<table border="0" cellpadding="0" cellspacing="0" width="1767">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Pulse generator</td>
			<td>AMPI</td>
			<td>Master-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolated stimulator</td>
			<td>Digitimer</td>
			<td>DS3</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td>This is used for assessing the cell morphology at low magnification.</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TiE</td>
			<td>This is used for high-resolution fluorescence and transmitted light imaging, with minimal focus drift.</td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Nikon</td>
			<td></td>
			<td>Water-immersion lens is recommended. Oil-immersion lens is usable unless an imaged structure is deep from the coverslip surface (e.g. &#62;10 &#956;m).</td>
		</tr>
		<tr>
			<td>Filter cube</td>
			<td>Nikon</td>
			<td>77032509</td>
			<td>490/20-nm ex, 510-nm dclp, 520-nm-LP em for FM1-43</td>
		</tr>
		<tr>
			<td>Filter cube</td>
			<td>Nikon</td>
			<td>77032809</td>
			<td>490/20-nm ex, 510-nm dclp, 650-nm-LP em for FM4-64.&#160;</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor Technology</td>
			<td>iXon EM+ DU-860</td>
			<td>This EMCCD camera is used for high-sensitivity detection of fluorescence.</td>
		</tr>
		<tr>
			<td>Liquid recirculating chiller</td>
			<td>Solid State Cooling Systems</td>
			<td>Oasis 160</td>
			<td>This is used for continuously perfusing the camera with chilled water for maintaining a temperature of -80&#176;C, and thereby reducing noise.</td>
		</tr>
		<tr>
			<td>LED</td>
			<td>CoolLED-Custom Interconnect</td>
			<td>490 nm&#160;</td>
			<td>This light source is used for rapid on/off control of fluorescence excitation.</td>
		</tr>
		<tr>
			<td>Image acquisition software</td>
			<td>Andor Technology</td>
			<td>Solis</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging chamber</td>
			<td>Warner Instruments</td>
			<td>RC-21BRFS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast perfusion system</td>
			<td>Warner Instruments</td>
			<td>SF-77B</td>
			<td></td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris Bioscience</td>
			<td>1045</td>
			<td></td>
		</tr>
		<tr>
			<td>D,L-AP5</td>
			<td>Tocris Bioscience</td>
			<td>0106</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris Bioscience</td>
			<td>1069</td>
			<td>Caution: toxic reagent. Handle with care.</td>
		</tr>
		<tr>
			<td>FM1-43</td>
			<td>Invitrogen</td>
			<td>T35356</td>
			<td></td>
		</tr>
		<tr>
			<td>Aldehyde-fixable FM1-43 (FM1-43FX)</td>
			<td>Invitrogen</td>
			<td>F35355</td>
			<td></td>
		</tr>
		<tr>
			<td>FM4-64</td>
			<td>Invitrogen</td>
			<td>T13320</td>
			<td></td>
		</tr>
		<tr>
			<td>Aldehyde-fixable FM4-64 (FM4-64FX)</td>
			<td>Invitrogen</td>
			<td>F34653</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#8217; balanced salt</td>
			<td>Sigma-Aldrich</td>
			<td>H2387</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>Invitrogen</td>
			<td>51200-038</td>
			<td>This solution does not contain phenol red that will interfere with fluorescence imaging.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Caution: toxic reagent. Handle with care.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
	</tbody>
</table>

examination of synaptic vesicle recycling using fm dyes during evoked, spontaneous, and miniature synaptic activities

Vescicole sinaptiche nelle terminazioni nervose funzionali subiscono esocitosi e endocitosi. Questo riciclo delle vescicole sinaptiche può essere efficacemente analizzata utilizzando coloranti stirilico FM, che rivelano fatturato membrana. Protocolli convenzionali per l&#39;uso di coloranti FM sono stati progettati per analizzare neuroni seguente stimolata (evocati) attività sinaptica. Recentemente, protocolli sono resi disponibili per analizzare i segnali FM che accompagnano attività sinaptica deboli, come eventi sinaptici spontanei o miniatura. L&#39;analisi di queste piccole variazioni di segnali FM richiede che il sistema di imaging è sufficientemente sensibile per rivelare piccole variazioni di intensità, ma che i cambiamenti artefatti di grande ampiezza vengono soppressi. Qui si descrive un protocollo che può essere applicato a evocata, spontanea, e attività sinaptiche in miniatura, e utilizzando colture di neuroni ippocampali come esempio. Questo protocollo incorpora anche un mezzo per determinare il tasso d&#39;photobleaching di coloranti FM, in quanto questo è un significativofonte di artefatti quando di imaging piccole variazioni di intensità.

Come esempio, mostriamo risultati rappresentativi per il decorso decolorante di vescicole sinaptiche (Figura 4). Neuroni dell&#39;ippocampo in coltura sono state colorate con FM4-64 utilizzando l&#39;attività sinaptica spontanea (fase 2.3) e lavato con una soluzione priva di colorante (soluzione di risciacquo 2). La rappresentazione mostra l&#39;andamento iniziale tempo decolorante utilizzando attività spontanea (passo 5.3) (parte iniziale della linea continua, Figura 4A). Segue il de...

Watch this Scientific Journal Video about Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities at JoVE.com

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous and Miniature Synaptic Activities | Neuroscience | Video

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Descriviamo l&#39;uso di coloranti stirilico FM per l&#39;immagine riciclo delle vescicole sinaptiche nei terminali nervosi funzionali. Questo protocollo può essere applicato non solo evocata, ma anche spontanea e attività sinaptiche in miniatura. Il protocollo espande la varietà di eventi sinaptici che possono essere adeguatamente valutati.

Esame delle vescicole sinaptiche Riciclaggio Uso FM coloranti Durante evocati, spontaneo, e miniatura Attività Synaptic

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

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20.9K Views.  University of Iowa Carver College of Medicine.  Descriviamo l'uso di coloranti stirilico FM per l'immagine riciclo delle vescicole sinaptiche nei terminali nervosi funzionali. Questo protocollo può essere applicato non solo evocata, ma anche spontanea e attività sinaptiche in miniatura. Il protocollo espande la varietà di eventi sinaptici che possono essere adeguatamente valutati. 

Video: Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

Studiare Piscine vescicole sinaptiche con fotoconversione di coloranti Styryl

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

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Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Analisi quantitativa di Replenishment pool delle vescicole sinaptiche in colture di neuroni granuli cerebellari utilizzando coloranti FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Diffusione laterale e esocitosi delle proteine ​​di membrana in neuroni in coltura valutata utilizzando Recovery fluorescenza e fluorescenza-loss photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

Derivando La durata di Liquidazione glutammato con una analisi Deconvoluzione delle Correnti Transporter astrociti

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Endocitosi delle vescicole sinaptiche in colture di neuroni ippocampali di misura

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Registrazione di plasticità sinaptica in fettine ippocampali Acute mantenuti in un piccolo volume riciclaggio - aspersione- e sistema di immersione-tipo camera

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Coltura In Vivo-piace murini astrociti utilizzando il protocollo AWESAM veloce, semplice e poco costoso

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

Modello di topo indotta da pentylenetetrazole accensione

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

Combinando optogenetica con microRNA artificiali per caratterizzare gli effetti del Gene atterramento su funzione presinaptica all'interno di circuiti neuronali intatto

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

Un test ottico per riciclo delle vescicole sinaptiche in colture di neuroni che Overexpressing proteine presinaptiche

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...