Die Autoren danken den Mitgliedern des Harata für die Diskussionen während der Ausführung dieser Arbeit. Diese Arbeit wurde durch Zuschüsse von der American Heart Association, der Dystonie Medical Research Foundation, die Edward Mallinckrodt, Jr. Foundation, der National Science Foundation und der Whitehall-Stiftung finanziert NCH

Die Autoren erklären, keine finanziellen Interessen konkurrieren.

Wir haben Protokolle für Färben und Entfärben synaptischen Vesikeln in Reaktion auf evozierte, spontan und Miniatur synaptische Aktivität und für die Bildgebung bei der Entfärbung Phase beschrieben. Zusätzlich zu den bestehenden Protokollen, haben wir ein neues Protokoll der Beobachtung der FM Entfärben basierend auf Miniatur synaptische Aktivität enthalten. Mit Hilfe dieser Protokolle, bisher haben wir Anomalien in kultivierten Neuronen aus einem Maus-Modell der Bewegungsstörung Dystonie identifiziert. Im Ver...

Die Funktionalität der synaptischen Vesikeln ist eine wichtige Determinante der synaptischen Übertragung. Diese Vesikel Neurotransmitter freisetzen, wenn sie mit der präsynaptischen Plasmamembran (Exozytose) fusionieren, und sie werden, nachdem sie von der Plasmamembran (Endozytose) regeneriert und neu geladen mit Neurotransmitter bereit für einen weiteren Zyklus der Veröffentlichung. Erforschung der Mechanismen und Dynamik der synaptischen Vesikel Recycling Basiswert hat durch die Einführung von Styryl-Farbstoffe FM 1 beschleunigt. Diese amphipathische Moleküle, die (in 1A, stereo von FM1-43 in Figur 1B mehrere Farbstoffe) positiv geladen sind hydrophile Kopfgruppen und hydrophoben Schwänze, reversibel ein-und aussteigen, ohne Lipidmembranen durchdringt sie. Gruppen FM Farbstoffe haben ähnliche Funktionen, die das Spektrum des Lichts, das sie aussenden beeinflussen. Zum Beispiel, FM2-10, FM1-43 und FM1-84 haben eine Doppelbindung zwischen zwei zyklischen Verbindungen und show grüne Emission. Der Unterschied zwischen ihnen ist die Länge der hydrophoben Schwanz, der seine Hydrophobie bestimmt und daher die Rate des Austritts aus der Membran (departitioning). In den Fällen des FM5-95 und FM4-64, drei Doppelbindungen verbinden die zyklische Verbindungen, und sie rote Emission zeigen. Diese Farbstoffe unterscheiden sich in ihrem hydrophilen Teile. In allen FM-Farbstoffe, die Fluoreszenzintensität erhöht, wenn sie in biologischen Membranen eingesetzt, aufgrund einer Erhöhung der Quantenausbeute in der hydrophoben Umgebung relativ zu dem hydrophilen Umgebung. So werden die Veränderungen in der FM-Intensität stellen die Veränderungen in der Membran Umsatz. Die verschiedenen Farben (Emissionsspektren) und Hydrophobien machen das FM färbt ein vielseitiges Forschungsinstrument in der synaptischen Vesikel-Recycling. Basierend auf diesen Merkmalen werden die FM-Farbstoffe meist nach dem folgenden Schema bei der Analyse der synaptischen Vesikel-Recycling (Fig. 2) verwendet. Neurons sind in einer extrazellulären Lösung, die das FM Farbstoff getaucht, so dass es in die synaptischen Vesikel (SV) genommen werden, da sie über Endozytose (Färbung) zu bilden. Der Farbstoff wird dann durch Aufbringen einer farbstofffreien extrazellulären Lösung gewaschen, dies zeigt die funktionelle Nervenenden, dh nur die aktiv Endozytose wird eine Gruppe von synaptischen Vesikeln, die mit dem Farbstoff (Fig. 2 unten) beladen sind, enthalten. Anschließende Exozytose führt zum Verlust des FM-Farbstoff an den extrazellulären Raum und einem gleichzeitigen Verlust der Fluoreszenz (Entfärbung, sowohl wegen der departitioning zu einer hydrophilen Umgebung und Diffusion weg vom Ort der Exozytose). Daher sind die Veränderungen in der Fluoreszenzintensität FM sind Indikatoren für die synaptischen Vesikel Exo-und Endozytose. FM-Farbstoffe werden verwendet, um zu färben und entfärben die synaptischen Vesikeln in verschiedenen Organismen und Zubereitungen 2,3. Beispiele sind Säugetier neuronal Kulturen 4-9, Säugetiergehirnscheiben 10,11, 12,13 neuromuskulären Verbindungen, Netzhaut bipolaren Neuronen 14,15 und Haarzellen der Cochlea 16. Typischerweise in solchen Experimenten werden sowohl Färbung und Entfärbung von extensiv Stimulierung der Neuronen (evozierte Aktivität) ausgelöst. Kürzlich jedoch synaptischen Vesikel-Recycling in Reaktion auf schwache Stimulation wurde auch untersucht, ebenso wie das Recycling in Abwesenheit eines externen Stimulus (spontan und Miniatur synaptische Aktivität) 9,17-19. Spontanen und Miniatur synaptischen Aktivitäten als diejenigen, die in der Abwesenheit von externen Stimuli auftreten definiert, wobei erstere mit der spontanen Zündung von Aktionspotentialen (Fig. 3). Diese schwachen synaptischen Aktivitäten werden mit kleineren Änderungen in der FM-Signale als die von umfangreichen Stimulation ausgelöst verbunden. Die Messung erfordert, daß die Veränderungen in der FM fluoreszierReferenzintensität genau widerspiegeln synaptischen Vesikel Endozytose oder Exozytose aber nicht artifactual Änderungen in der Intensität. Eine Ursache des Artefakts ist die Anwesenheit von nicht-spezifische Färbung der Plasmamembran durch FM-Farbstoffe. Schrittweise Auswaschung dieser Komponente wird zu einer allmählichen Veränderung der Fluoreszenzintensität gemessen, die fälschlicherweise synaptischen Aktivitäten zugeschrieben wird, zu führen. Dieser Faktor kann durch geeignete Methoden reduziert werden (siehe Protokoll). Die wichtigste Ursache des Artefakts ist das Bleichen von FM-Farbstoff in synaptischen Vesikeln erhalten. Die Photobleaching bedingte Veränderungen FM Intensität im Vergleich zu den biologischen (synaptischen) Änderungen, die gemessen werden klein sein. Die jüngste Entwicklung von empfindlichen Kameras, z. B. die Elektronenvervielfachungsladungsgekoppelte Vorrichtung (EM-CCD)-Kamera, ist es möglich, zu minimieren Bleichen durch Verkürzen Belichtungszeit und die Schwächung der Intensität des Lichts verwendet werden, um die Fluorophore anzuregen. Eine weitere Ursache des Artefakts ist eine Drift in die Fokussierung Niveau der Lichtmikroskop. Die Fokusdrift während eines Bildgebungssitzung kann durch mechanische oder thermische Effekte verursacht werden, und fälschlicherweise zu einer Änderung in der gemessenen Fluoreszenzintensität führen. Hier beschreiben wir Protokolle und Geräte, die es möglich, UKW-Farbstoffe verwenden, um synaptische Vesikel-Recycling auch im Rahmen von schwachen oder gar keine Stimulation zu analysieren, insbesondere das Miniatur synaptische Aktivität zu machen. Wir zeigen Beispiele für die Färbung und Entfärbung von Vesikeln während evozierte und spontane synaptische Ereignisse, mit kultivierten Hippocampus-Neuronen Nagetier, und die Abbildung der Entfärbung Phase. Wir zeigen auch, wie der Grad der FM Farbstoff Bleichen zu bewerten, in Abwesenheit von jedem FM-Farbstoffverlust durch synaptische Aktivitäten.

<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

Synaptische Vesikel in Nervenfunktionsterminals unterziehen Exozytose und Endozytose. Diese synaptischen Vesikel-Recycling kann mit FM Styryl-Farbstoffe, die Membran Umsatz offenbaren effektiv analysiert. Herkömmliche Protokolle für die Verwendung von FM-Farbstoffe wurden für die Analyse von Neuronen nach stimulierte (hervorgerufen) synaptische Aktivität entwickelt. Kürzlich Protokolle werden für die Analyse der FM-Signale, die schwächer synaptischen Aktivitäten, wie spontane oder Miniatur synaptischen Ereignisse begleiten verfügbar. Analyse dieser kleine Änderungen in FM-Signale erfordert, dass das Abbildungssystem ist empfindlich genug, um kleine Änderungen in der Intensität zu erfassen, doch ist ein Artefakt Änderungen mit großer Amplitude unterdrückt werden. Hier beschreiben wir ein Protokoll, die angewendet werden, um hervorgerufen, spontan, und Miniatur-synaptischen Aktivitäten, und verwenden Sie kultivierten Hippocampus-Neuronen als Beispiel werden. Dieses Protokoll enthält auch ein Mittel zur Bewertung der Rate der Photobleichung des FM-Farbstoffe, da dies eine erheblicheQuelle von Artefakten bei der Abbildung von kleinen Änderungen in der Intensität.

Als Beispiel zeigen wir repräsentative Ergebnisse für die Entfärbung Zeitverlauf der synaptischen Vesikel (Abbildung 4). Hippokampalen Neuronen wurden mit FM4-64 angefärbt mit der spontanen synaptischen Aktivität (Schritt 2.3) und mit Farbstoff-freie Lösung (Spüllösung 2) gewaschen. Die Abbildungs ​​zeigt die Anfangs Entfärben Zeitverlauf mit Spontanaktivität (Schritt 5.3) (Anfangsteil der durchgehenden Linie, 4A). Dies wird durch die Entfärbung Zeitkurs mit drei Runden d...

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

Wir beschreiben die Verwendung von Styryl FM Farbstoffe Bild synaptischen Vesikel-Recycling in der funktionellen Nervenenden. Dieses Protokoll kann nicht nur beschworen, sondern auch spontan und Miniatur synaptischen Tätigkeiten angewendet werden. Das Protokoll erweitert die Vielzahl von synaptischen Ereignisse, die effektiv ausgewertet werden können.

Die Untersuchung der synaptischen Vesikel Recycling Verwenden des UKW-Farbstoffe Während evozierte, spontan, und Miniatur Synaptic Aktivitäten

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 Aufrufe.  University of Iowa Carver College of Medicine.  Wir beschreiben die Verwendung von Styryl FM Farbstoffe Bild synaptischen Vesikel-Recycling in der funktionellen Nervenenden. Dieses Protokoll kann nicht nur beschworen, sondern auch spontan und Miniatur synaptischen Tätigkeiten angewendet werden. Das Protokoll erweitert die Vielzahl von synaptischen Ereignisse, die effektiv ausgewertet werden können. 

Serie: 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.

Studium synaptischer Vesikel Pools mit Photokonversion Styrylfarbstoffe

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.

Quantitative Analyse der synaptischen Vesikel Pool Replenishment in Cultured Cerebellar Nervenzellen Granulat mit FM Dyes

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.

Laterale Diffusion und Exozytose von Membranproteinen in kultivierten Neuronen Veranlagte mit Fluorescence Recovery-und Fluoreszenz-Verlust 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.

Die Ableitung der zeitliche Verlauf der Glutamate Ausverkauf mit Dekonvolutionsanalyse von Astrozytäre Transporter Currents

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.

Messung der synaptischen Vesikel Endozytose in kultivierten Hippocampal Neuronen

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.

Aufnahme synaptische Plastizität in akuten Hippocampal Scheiben gepflegt in einem kleinvolumigen Recycling - Perfusions- und untertauchen-Typ-Kammer-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 ...

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.

In VivoKultivierung-wie Murine Astrozyten über das schnelle, einfache und kostengünstige AWESAM-Protokoll

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.

Pentylenetetrazole-induzierten anzünden-Maus-Modell

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.

Kombination von Optogenetik mit künstlichen Mikro-RNAs, die Effekte gen Knockdown präsynaptischen Funktion innerhalb intakte neuronale Schaltkreise zu charakterisieren

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.

Ein optischer Test zum synaptischen Vesicle Recycling in kultivierten Neuronen überexprimiert präsynaptischen Proteine

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