Les auteurs remercient les membres du laboratoire Harata pour des discussions utiles tout au long de l&#39;exécution de ce travail. Ce travail a été financé par des subventions de l&#39;American Heart Association, la Dystonia Medical Research Foundation, Edward Mallinckrodt, Jr. Foundation, la National Science Foundation et la Fondation Whitehall à NCH

Les auteurs déclarent aucun intérêt financier concurrents.

Nous avons décrit les protocoles de coloration et de décoloration des vésicules synaptiques en réponse à évoqué, l&#39;activité synaptique spontanée et miniature, et pour l&#39;imagerie au cours de la phase de décoloration. En plus des protocoles existants, nous avons inclus un nouveau protocole d&#39;observer la décoloration FM sur la base de l&#39;activité synaptique miniature. L&#39;utilisation de ces protocoles, nous l&#39;avons déjà identifié des anomalies dans les neurones en culture à partir d&#39...

La fonctionnalité des vésicules synaptiques est un déterminant important de la transmission synaptique. Ces vésicules libèrent des neurotransmetteurs quand ils fusionnent avec la membrane plasmique présynaptique (exocytose), et ils seront prêts pour un autre cycle de libération après avoir été régénéré à partir de la membrane plasmique (endocytose) et rechargé avec neurotransmetteur. La recherche sur la dynamique des mécanismes sous-jacents et le recyclage des vésicules synaptiques a été fortement accélérée par l&#39;introduction de colorants styryles FM 1. Ces molécules bipolaires, qui ont chargés positivement groupes de tête hydrophiles et queues hydrophobes (plusieurs colorants dans la figure 1A, stereoview de FM1-43 dans la figure 1B), peuvent réversible entrer et sortir de membranes lipidiques sans les imprégner. Groupes de colorants FM partagent des caractéristiques similaires qui influent sur la plage de la lumière qu&#39;ils émettent. Par exemple, FM2-10, FM1-43, et FM1-84 ont une double liaison entre les deux composés cycliques et shoémission verte w. La différence entre eux est la longueur de la queue hydrophobe, qui détermine son hydrophobie et donc la vitesse de sortie de la membrane (décloisonnement). Dans les cas de FM5 et FM4-95-64, trois doubles liaisons relient les composés cycliques, et ils montrent émission dans le rouge. Ces colorants diffèrent en ce qui concerne leurs parties hydrophiles. Dans tous les colorants FM, l&#39;intensité de fluorescence augmente quand ils sont insérés dans les membranes biologiques, en raison d&#39;une augmentation du rendement quantique dans l&#39;environnement hydrophobe par rapport à l&#39;environnement hydrophile. Ainsi, les variations de l&#39;intensité FM représentent les variations de la valeur de la membrane. Les différentes couleurs (spectres d&#39;émission) et hydrophobicités font la FM teint un outil de recherche polyvalent dans le recyclage des vésicules synaptiques. Sur la base de ces caractéristiques, les colorants FM sont principalement utilisés selon le schéma suivant dans l&#39;analyse de recyclage des vésicules synaptiques (figure 2). Neurons sont baignées dans une solution extracellulaire contenant le colorant FM, ce qui lui permet d&#39;être pris dans des vésicules synaptiques (SVS) car ils forment par endocytose (coloration). Le colorant est ensuite lavé par application d&#39;une solution de colorant extracellulaire libre, ce qui révèle les terminaisons nerveuses fonctionnelles, c&#39;est à dire que ceux subissant une endocytose activement contiendra un groupe de vésicules synaptiques qui sont chargés avec le colorant (figure 2 du bas). Exocytose ultérieur conduit à la perte du colorant FM à l&#39;espace extracellulaire et une perte concomitante de la fluorescence (de décoloration; due à la fois à la décloisonnement à un environnement hydrophile et diffusion en dehors du site de l&#39;exocytose). Par conséquent, les changements dans FM intensité de fluorescence sont des indicateurs de la vésicule synaptique exo-et endocytose. Colorants FM ont été utilisés pour colorer et décolorer les vésicules synaptiques dans divers organismes et préparations 2,3. Les exemples incluent neur mammifèrescultures onal 4-9, des tranches de cerveau de mammifères 10,11, 12,13 jonctions neuromusculaires, les neurones bipolaires de la rétine 14,15 et cellules ciliées de la cochlée 16. En général, dans ces expériences, les colorations et décoloration sont déclenchées par la stimulation largement les neurones (activité évoquée). Récemment, cependant, de la vésicule synaptique recyclage en réponse à la stimulation faible a également été analysée, de même que le recyclage en l&#39;absence d&#39;un stimulus externe (de l&#39;activité synaptique spontanée et miniature) 9,17-19. Activités synaptiques spontanés et miniatures sont définis comme ceux qui se produisent en l&#39;absence de stimuli externes, avec l&#39;ancien impliquant la décharge spontanée de potentiels d&#39;action (figure 3). Ces activités synaptiques faibles sont associés à de faibles changements de signaux FM que celles déclenchées par une vaste stimulation. La mesure exige que les changements dans Déess FMintensité rence reflètent fidèlement l&#39;exocytose des vésicules synaptiques ou endocytose mais les changements ne artéfactuelles en intensité. L&#39;une des causes de l&#39;artefact est la présence d&#39;une coloration non spécifique de la membrane de plasma par des colorants FM. Lavage progressif de cette composante va conduire à un changement progressif dans l&#39;intensité de fluorescence mesurée, qui sera attribué à tort à des activités synaptiques. Ce facteur peut être réduite par des méthodes appropriées (voir Protocole). La cause la plus notable de l&#39;artefact est le photoblanchiment de colorant FM retenu dans les vésicules synaptiques. Les changements liés à l&#39;intensité photoblanchiment-FM doivent être petites par rapport aux biologiques (synaptiques) les changements qui sont évalués. Le développement récent des appareils sensibles, par exemple la caméra dispositif à couplage de charge d&#39;électrons multipliant (EMCCD), permet de réduire photoblanchiment en raccourcissant la durée d&#39;exposition et l&#39;affaiblissement de l&#39;intensité de la lumière utilisée pour exciter le fluorophore. Une autre cause de l&#39;artefact est une dérive in le niveau de focalisation du microscope optique. La dérive de mise au point au cours d&#39;une session de formation d&#39;image peut être causée par des effets mécaniques ou thermiques, et à tort entraîner un changement dans l&#39;intensité de fluorescence mesurée. Nous décrivons ici des protocoles et des équipements qui font qu&#39;il est possible d&#39;utiliser des colorants FM pour analyser recyclage des vésicules synaptiques, même dans le contexte de faible ou pas de stimulation, en particulier, l&#39;activité synaptique miniature. Nous montrons des exemples de la coloration et décoloration de vésicules synaptiques lors d&#39;événements évoqué et spontanées, en utilisant les neurones de rongeurs hippocampe en culture, et l&#39;imagerie de la phase de décoloration. Nous démontrons également comment évaluer le degré de photoblanchiment FM colorant, en l&#39;absence de toute perte de colorant FM en raison des activités synaptiques.

<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

Les vésicules synaptiques dans les terminaisons nerveuses fonctionnelles subissent l&#39;exocytose et l&#39;endocytose. Ce recyclage des vésicules synaptiques peut être efficacement analysé en utilisant des colorants styryle FM, qui révèlent le chiffre d&#39;affaires de la membrane. Des protocoles classiques pour l&#39;utilisation de colorants FM ont été conçus pour l&#39;analyse suivante neurones stimulée (évoqué) l&#39;activité synaptique. Récemment, les protocoles sont devenues disponibles pour l&#39;analyse des signaux FM qui accompagnent les activités synaptiques plus faibles, tels que les événements spontanés ou décoratifs synaptiques. L&#39;analyse de ces petits changements dans les signaux FM nécessite que le système d&#39;imagerie est suffisamment sensible pour détecter de petites variations de l&#39;intensité, pour l&#39;instant que les changements d&#39;artefact de grande amplitude sont supprimées. Nous décrivons ici un protocole qui peut être appliquée à évoqué, spontanée, et les activités synaptiques miniatures, et en utilisant des neurones hippocampiques en culture, par exemple. Ce protocole comporte également un moyen d&#39;évaluer le taux de photoblanchiment des colorants FM, comme il s&#39;agit d&#39;une importantesource d&#39;artefacts lors de l&#39;imagerie de petits changements dans l&#39;intensité.

A titre d&#39;exemple, nous montrons des résultats représentatifs pour le cours du temps de décoloration des vésicules synaptiques (figure 4). Neurones hippocampiques en culture ont été colorées avec FM4-64, en utilisant l&#39;activité synaptique spontanée (étape 2.3) et lavées avec une solution sans colorant (solution de rinçage 2). L&#39;imagerie montre le cours initial de temps de décoloration utilisant activité spontanée (étape 5.3) (partie initiale de la ligne continue, la f...

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

Nous décrivons l&#39;utilisation de colorants styryles FM à l&#39;image synaptique recyclage des vésicules dans les terminaisons nerveuses fonctionnelles. Ce protocole peut être appliqué non seulement à évoquer, mais aussi spontané et activités synaptiques miniatures. Le protocole élargit le choix des événements synaptiques qui peuvent être évaluées de manière efficace.

Examen de la vésicule synaptique recyclage utilisant des colorants FM Pendant évoqués, spontanée, et les activités synaptiques miniatures

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 vues.  University of Iowa Carver College of Medicine.  Nous décrivons l'utilisation de colorants styryles FM à l'image synaptique recyclage des vésicules dans les terminaisons nerveuses fonctionnelles. Ce protocole peut être appliqué non seulement à évoquer, mais aussi spontané et activités synaptiques miniatures. Le protocole élargit le choix des événements synaptiques qui peuvent être évaluées de manière efficace. 

Vidéo: 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.

Etudier Piscines vésicules synaptiques utilisant des colorants styryle photoconversion

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.

Analyse quantitative des vésicules reconstitution Piscine synaptique dans des cultures de neurones granulaires du cervelet utilisant des colorants 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.

Diffusion latérale et l&#39;exocytose des protéines membranaires dans des neurones en culture évaluée à l&#39;aide de récupération de fluorescence et de fluorescence de perte de photoblanchiment

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.

Issu l&#39;évolution temporelle du glutamate Dégagement avec une analyse de déconvolution des courants de Transporteur ASTROCYTIQUES

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.

Mesurer l’endocytose de la vésicule synaptique dans des neurones Hippocampal cultivés

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.

Enregistrement de la plasticité synaptique dans des tranches d’hippocampe aiguës maintenues dans un petit volume recyclage - Perfusion- et système de chambre de Submersion-type

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.

Culture In Vivo-comme les Astrocytes Murine en utilisant le protocole AWESAM rapide, Simple et peu coûteux

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.

Modèle de souris de bois d’allumage induite par le pentylènetétrazole

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.

Alliant Optogenetics artificiel microARN pour caractériser les effets du gène Knockdown fonction présynaptique dans les Circuits de neurones intacts

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.

Une analyse optique pour le recyclage de vésicule synaptique dans les neurones en culture surexprimant protéines présynaptiques

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