Diese Arbeit wurde vom NIH Zuschüsse DA15169 und DA035224 zu HEM finanziert

Alle Tierbehandlung und Gewebe Ernte wurde in Übereinstimmung mit den Richtlinien der University of Massachusetts Medical School Institutional Animal Care Use Committee (IACUC) durchgeführt, nach dem genehmigten Protokoll # A1506 (Melikian, PI). Benötigte Lösungen Künstliche Liquor (ACSF) - Machen Sie täglich frisch 125 mM NaCl, 2,5 mM KCl, 1,2 mM NaH 2 PO 4, 1,2 mM MgCl 2, 2,4 mM CaCl 2, 26 mM NaHCO 3 und 11 mM Glucose Hinweis: Bereiten ACSF als 10x Stammlösung, ohne NaHCO 3 und Glucose. Machen 1x Arbeitslösungen täglich aus dem 10fach, in Ergänzung mit frischen NaHCO 3 und Glucose. Saccharose-ergänzt ACSF (SACSF) - Stellen täglich frisch 250 mM ErfolgRose, 2,5 mM KCl, 1,2 mM NaH 2 PO 4, 1,2 mM MgCl 2, 2,4 mM CaCl 2, 26 mM NaHCO 3 und 11 mM Glucose Hinweis: Bereiten SACSF als 10x Stammlösung, ohne NaHCO 3 und Glucose. Machen 1x Arbeitslösungen täglich aus dem 10fach, in Ergänzung mit frischen NaHCO 3 und Glucose. Sulfo-N-Hydroxysuccinyl-SS-Biotin (Sulfo-NHS-SS-Biotin, Pierce Chemical Company) Auf Lösungen sollten 200 mg / ml in DMSO und sind resistent gegen mehrere Einfrieren / Auftauen. Aliquots werden bei -20 ° C gelagert Die Succinylester schnell in wässriger Lösung hydrolysiert, so Arbeits Lösungen sollten unmittelbar vor dem Aufbringen auf Scheiben hergestellt werden. Scheibe Quench-Lösung ACSF mit 100 mM Glycin supplementiert RIPA Lyse Buffer 10 mM Tris, pH 7,4, 150 mM NaCl, 1,0 mM EDTA, 1% Triton-X-100, 0,1% SDS, 1% Na-Desoxycholat RIPA mit Protease-Inhibitoren (RIPA / PI) - Machen Sie täglich frisch RIPA ergänzt mit 1 &amp;mgr; M Leupeptin, 1 &amp;mgr; M Pepstatin, 1 &amp;mgr; M Aprotinin und 1 mM Phenylmethylsulfonylfluorid. 1. Bereiten Hirnschnitten <ol><li> Stellen Sie frische 1x SACSF und 1x ACSF </li><li> Chill-SACSF auf Eis in einem kleinen Becherglas. Dies wird verwendet, um die frisch geernteten Gehirn der Maus zu halten. </li><li> Tränken Sie das ACSF und SACSF mit Sauerstoff durch Blasenbildung mit 95% / 5% O 2 / CO 2, 20 min auf Eis. </li><li> P30-38-Mäuse sollten für eine optimale Gewebelebensfähigkeit verwendet werden. Sacrifice Tiere durch Genickbruch und Enthauptung, und das Gehirn wird rasch zu entfernen, in vorgekühlte, sauerstoff SACSF. </li><li> Mit Hilfe eines vibrierenden Mikrotom, stellen 300 um Gehirnschnitte in der Region von Interesse. </li><li> Falls gewünscht, können weitere Scheiben vor der Verwertung zerlegt werden, um für bestimmte Hirnregionen oder separaten rechten und linken Hemisphäre als Kontroll-und Versuchsscheiben zu verwenden, bzw. zu bereichern. </li><li> Mit einem feuerpolierten Pasteur Pipette Scheiben Kammern in 24-Well-Platten gesetzt Boden eingreifen. </li><li> Lassen Scheiben für 40 min in Sauerstoff angereichert ACSF erholen, 31 ° C, mit ständigen, sanfte Blubbern. </li></ol> 2. Drug Treatment (falls zutreffend) und Scheibe Biotinylierung <ol><li> Nach der Erholung, waschen Scheiben 3x in vorgewärmten (37 ° C), sauerstoff ACSF sprudelnden ständig mit 95% / 5% O 2 / CO 2. </li><li> In Testverbindungen und Inkubation mit kontinuierlicher Sauerstoffversorgung. <ol><li> Für die Bequemlichkeit, einen 1/10 Volumen 10x konzentrierter Droge, und mischen durch vorsichtiges Umdrehen. </li><li> Schütteln Platten vorsichtig in einem Wasserbad bei der gewünschten Temperatur. </li></ol></li><li> Nach der medikamentösen Behandlung, schnellChill-Scheiben durch Waschen 3x in eiskalter ACSF. </li></ol> 3. Biotinylieren Oberflächenproteine <ol><li> Planen 1,0 mg / ml Sulfo-NHS-SS-Biotin in eiskaltem ACSF unmittelbar vor der Etikettierung. </li><li> In 0,75 ml Sulfo-NHS-SS-Biotin an Scheiben Scheiben und Inkubation auf Eis, 45 min. </li><li> Dreimal waschen Scheiben schnell mit eiskaltem ACSF, dann Inkubation für 10 min in eiskaltem ACSF auf Eis. </li><li> Scheiben waschen dreimal mit eiskaltem Scheibe Quenchpuffer und Inkubation mit 0,75 ml Scheibe Quenchpuffer zwei Mal, 25 min, auf Eis, freie Sulfo-NHS-SS-Biotin zu stillen. </li></ol> 4. Bereiten Gewebelysaten <ol><li> Dreimal waschen Scheiben in eiskalter ACSF und übertragen jede Scheibe zu einem Mikrozentrifugenröhrchen mit einer feuerpolierten Pasteurpipette. </li><li> Pellet vorsichtig Scheibe durch Zentrifugation 200 g, 1 min und sorgfältig absaugen restlichen ACSF. </li><li> In 400 ul eiskaltem RIPA / PI und brechen Gewebe durch Auf-und Abpipettieren einmal durch eine P200 pipette. </li><li> Über dissoziiert Slice / RIPA in ein frisches Röhrchen und Inkubation 30 min, 4 ° C, Drehen, um die Lyse zu vervollständigen. </li><li> Pelletierung von Zelltrümmern durch Zentrifugation, 18.000 x g, 15 min, 4 ° C </li><li> Proteinkonzentrationen bestimmen Lysat unter Verwendung des BCA-Proteinassay mit Rinderserumalbumin (BSA) als Standard. </li></ol> 5. Isolieren biotinylierter Proteine <ol><li> Optimieren Bead / Gesamtprotein-Verhältnis. Hinweis: Einzelne Proteinexpressionsniveaus kann weit in den verschiedenen Hirnregionen unterschiedlich. Wenn nicht alle der biotinylierten Proteinen in einer gegebenen Menge von Gewebelysat erfasst, ist es nicht möglich, alle möglichen Veränderungen in der Oberflächenexpression genau zu erfassen. Daher ist es zwingend notwendig, um empirisch bestimmen die optimale Perlen / Gesamtproteinverhältnis für ein bestimmtes Protein / Hirnregion vor der Einschiffung auf eine neue Scheibe Biotinylierung Studie. <ol><li> Inkubation 25 ul Streptavidin-Agarose-Beads mit zunehmender Menges Gewebelysat (ungefähren Bereich von 25 bis 200 ug) und dann gehen Sie wie folgt für die Bindung, Waschen und Elution Schritten beschrieben. </li><li> Quantifizierung der resultierenden immunreaktiven Banden und wählen Sie eine Perle / Lysat-Verhältnis im linearen Bereich der Bindung, die genaue Quantifizierung von entweder erhöht oder verringert Protein-Oberflächenexpression ermöglichen. </li></ol></li><li> Bereiten Streptavidin-Agarose Beads <ol><li> Bestimmen Sie die Gesamt Wulst Volumen für alle Proben notwendig. Eine ausreichende Schweißnahtvolumen für diesen Betrag plus ein zusätzliches (dh 4 Proben bei 25 ul / Probe = 100 ul Perlen + ein extra = 125 ul Perlen. </li><li> Vortex Streptavidin Wulst Lager und Pipette heraus gewünschte Lautstärke. Bei Verwendung eines P200 Pipette, schneiden Sie das Ende der Spitze zu einer Verstopfung oder Wulst Schäden zu verhindern. </li><li> Waschen Sie Perlen 3-mal in 0,5 bis 1,0 ml RIPA / PI Konservierungsmittel, Verwirbelung nach jedem RIPA hinaus und sammeln Perlen zwischen den Waschgängen durch Zentrifugation 18.000 xg, 1 min zu entfernen, bei Raumtemperaturratur. </li><li> Absaugen Puffer zwischen den Waschgängen mit einem Glas-Pasteur-Pipette auf eine Vakuumflasche angebracht. Ein Kunststoff-P200 Spitze bis zum Ende der Glaspasteurpipette befestigt bietet feinere Kontrolle beim Ansaugen. Es ist nicht unbedingt notwendig, alle Puffer für die ersten zwei Waschungen entfernen, da es Risiken Ansaugen Kügelchen in die Pipette. Nach dem letzten Waschen, zu entfernen, so viel überschüssigen Puffer wie möglich ohne Absaugen der Perlen. </li><li> In RIPA / PI, um Perlen wieder in ihre ursprüngliche Volumen zu bringen, und Abpipettieren zu resuspendieren. Vermeiden Verwirbelung an dieser Stelle, als Perlen werden an der Rohrwand haften. </li><li> Aliquot Perlen in Mikrozentrifugenröhrchen mit einem P200 mit der Spitze abgeschnitten. Pipette nach oben und unten mehrmals zwischen der Probenahme zu gewährleisten, Perlen bleiben gleichmäßig suspendiert und unter den Röhrchen verteilt. </li></ol></li><li> Bind biotinylierten Proteine ​​Streptavidin-Perlen <ol><li> Verteilen Zelllysate auf die Rohre, die die Agarose-Perlen. Für Experimente whe<html> re mehrere Proben verglichen werden, stellen Sie sicher, um die gleiche Menge an Protein für jede Probe für genaue Vergleiche zu verwenden. </li><li> Fügen Sie zusätzliche RIPA / PI, um Proben zu einem minimalen Volumen 200 ul zu bringen. Dies gewährleistet, dass die Proben während der Inkubation ausreichend zu mischen und auch vereinheitlicht die Proteinkonzentrationen in Proben. </li><li> Die Röhrchen in ein Rohr Rotator und über Nacht mischen bei 4 ° C </li><li> In getrennten Röhrchen, verzichtet werden ein Äquivalent des gesamten Lysats Volumen für jede Probe verwendet. Alternativ, wenn die Probenmengen hoch sind, ein Bruchteil des gesamten Lysats Volumen anstelle reserviert werden, um die maximale Ladevolumen auf SDS-PAGE-Gelen unterzubringen. Diese werden verwendet, um die Oberflächenexpression auf die Gesamtproteinmenge zu normalisieren. </li><li> Fügen Sie entweder das gleiche Volumen 2x oder 1/5 Vol. 6x SDS-PAGE-Probenpuffer und Inkubation entweder bei 4 ° C, parallel zu Wulst Proben oder bei -20 ° C bis Proben analysiert. </li></ol></li></ol>ove_title &quot;&gt; 6. Elute und Analysieren von Proben <ol><li> Beads durch Zentrifugation pelle 18.000 × g, 2 min bei Raumtemperatur. </li><li> Überstand wird abgesaugt und Waschperlen dreimal mit 0,75 ml RIPA. Um Wulst Verlust zu minimieren, lassen einen kleinen Kopf Volumen Puffer oben Perlen zwischen Wäschen. Nach dem letzten Waschen, zu entfernen, so viel wie möglich RIPA, ohne dass der Wulst Pellets. </li><li> Eluiere biotinylierten Proteine ​​aus Streptavidin-Kügelchen durch die Reduzierung der Disulfidbrücke. In 25 ul 2x SDS-PAGE-Probenpuffer reduziert, Vortex gut und drehen Proben 30 min, Raumtemperatur. Anmerkung: Viele Membranproteine ​​haben eine hohe Tendenz zu aggregieren, wenn sie in SDS-PAGE-Probenpuffer gekocht, deren elektrophoretische Mobilität stark beeinträchtigen. Wenn dies der Fall für das Protein untersucht wird, zu vermeiden Erhitzen von Proben und stattdessen eluieren bei Raumtemperatur langsam. Wenn kochendem absolut notwendig ist (zB wenn ein anderes Protein parallel beurteilt erfordert Sieden), samplE-Puffer mit 2 M Harnstoff (Endkonzentration) ergänzt werden, um die Aggregation zu minimieren. Während bei der Verringerung Aggregation in einigen Fällen Harnstoff oft Kompromisse Band Auftritte. </li><li> Analysieren Sie die Proben durch Immunoblot. </li><li> Auftauen Gesamt Lysatproben und drehen parallel Wulst Proben, 30 min, Raumtemperatur. </li><li> Getrennte Proteine ​​auf SDS-PAGE-Gelen. </li><li> Identifizieren Protein (e) von Interesse durch Immunoblotting. <ol><li> Seien Sie sicher, dass die Bänder im linearen Bereich der Detektion für die richtige Quantifizierung detektiert. </li><li> Um sicherzustellen, daß das Biotinylierungsreagenz hat keinen Zugang zu intrazellulären Proteinen durch beschädigte / gefährdeten Zellen gewonnen. Dies wird am besten durch Immunoblotting parallel zu einem intrazellulären Protein spezifisch für den Zelltyp untersucht erreicht. </li><li> Quantifizieren Band Dichten und berechnen relative Proteinoberflächendichte in Prozent des Gesamtproteinexpressionsniveau. </li></ol></li></ol>

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Die Autoren haben keine finanziellen Interessen, dass Konflikte mit dieser Arbeit.

Trotz langjährige Wissen, dass endozytischen Handel kritisch Auswirkungen synaptischen Signal im Gehirn, hat es schwierig, quantitativ Veränderungen in der Proteinoberflächen-Expression in adulten Neuronen messen bewährt. In dieser Arbeit, einen zuverlässigen Ansatz zur Oberflächenprotein ex vivo in akuten Hirnschnitten beschriften berichten wir. Hirnschnittpräparate haben eine langjährige Geschichte der Nutzen für elektrophysiologischen Ableitungen, wie sie synaptischen Verbindungen und die Lebensfäh...

Endozytischen Handel ist ein allgegenwärtiges zellulären Mechanismus, der die Feinabstimmung des Plasmamembran Präsentation einer Vielzahl von integralen Membranproteinen. Endozytose liefert lebenswichtige Nährstoffe, um die intrazelluläre Milieu 1 und desensibilisiert-Rezeptor-Signal als Reaktion auf 2-Rezeptor-Aktivierung. Endocytotischen Recycling zurück in die Plasmamembran erhöhen zusätzlich zelluläre Signalgebung durch Erhöhung der Proteinexpressionsniveaus an der Zelloberfläche 3. Darüber hinaus sind Membrantransport Störungen in zahlreichen Krankheiten und pathologischen Bedingungen 4,5 verwickelt, und betonte die Notwendigkeit, die molekularen Mechanismen, die Protein endozytischen Handel regieren zu untersuchen. Während viele Proteine ​​nutzen klassische Clathrin-abhängigen Internalisierung Mechanismen, Anzeichen dafür in den vergangenen Jahren zeigt, dass mehrere Clathrin-unabhängige Mechanismen endozytischen regeln die endozytischen Potential einer wachsenden Palette vonProteine ​​6,7. So hat die Notwendigkeit, endozytischen Mechanismen für Menschenhandel in physiologisch relevanten Systemen zu untersuchen stark gewachsen. Im Gehirn hat endozytischen Handel von Rezeptoren, Ionenkanäle und Neurotransmitter-Transporter eine primäre Rolle bei der Gründung der synaptischen Plastizität 8-11 und der Reaktion auf Drogen 12-15, letztlich Auswirkungen auf die neuronale Erregbarkeit und synaptischen Antworten. Bis heute hat die Mehrheit der neuronalen Handel Studien entweder heterologen Expressionssystemen oder kultivierten primären Nervenzellen verlassen können, von denen weder zuverlässig reflektieren Mechanismen eine Rolle spielen bei erwachsenen Neuronen. Hier ein Ansatz, der Biotinylierung Oberfläche verwendet, um quantitativ Oberflächenproteinspiegel in akuten Hirnschnitten von erwachsenen Nagern abgeleitet messen berichten wir. Mit diesem Ansatz können Daten, die zeigen, dass die Maus striatale Dopamin-Transporter schnell verinnerlicht in re präsentieren wirReaktion auf Phorbolester-vermittelte Protein-Kinase C (PKC).

<table border="0" cellpadding="0" cellspacing="0" width="682">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">sulfo NHS-SS-biotin</td>
			<td style="width:83px;">Pierce</td>
			<td style="width:119px;">21331</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Streptavidin agarose</td>
			<td style="width:83px;">Pierce</td>
			<td>20347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">IgG-free, Protease-free Bovine serum albumin</td>
			<td style="width:83px;">Sigma</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Vibrating microtome sectioner</td>
			<td style="width:83px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking water bath</td>
			<td style="width:83px;">various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Milli-cell mesh-bottomed inserts (8&#181;m pore size)</td>
			<td style="width:83px;">Millipore</td>
			<td style="width:119px;">PI8P 012 50</td>
			<td>These can be washed by hand and re-used</td>
		</tr>
	</tbody>
</table>

brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons

Geregelte endozytischen Handel ist der zentrale Mechanismus erleichtert eine Vielzahl von neuromodulatorischen Veranstaltungen, die von dynamischen Steuerung Rezeptor-Ionenkanal und Transporter Zelloberfläche Präsentation auf einem Minuten-Zeitskala. Es gibt eine breite Vielfalt von Mechanismen, die endozytischen Handel einzelner Proteine ​​steuern. Studien zur Untersuchung der molekularen Grundlagen von Menschenhandel wurden in erster Linie auf die Oberfläche Biotinylierung verlassen, um quantitativ Veränderungen in der Membranproteinoberfläche messen Ausdruck als Reaktion auf exogene Reize und Gen-Manipulation. Allerdings wurde dieser Ansatz vor allem auf kultivierten Zellen, die nicht treu die physiologisch relevanten Mechanismen im Spiel bei erwachsenen Neuronen spiegeln kann begrenzt. Darüber hinaus kann kultivierten Zell Ansätze regionsspezifische Unterschiede in Handel Mechanismen unterschätzen. Hier beschreiben wir einen Ansatz, der Zelloberfläche Biotinylierung der akuten Hirnschnittpräparat erstreckt. Wirzeigen, dass diese Methode bietet eine High-Fidelity-Ansatz zur schnellen Veränderungen in der Membranproteinoberfläche Ebenen bei erwachsenen Nervenzellen messen. Dieser Ansatz dürfte eine breite Dienstprogramm auf dem Gebiet der neuronalen endozytischen Handel zu haben.

Die neuronale Dopamintransporter wird in Reaktion auf PKC-Aktivierung in Zelllinien 16-20 internalisiert. Obwohl viele Berichte zeigen PKC-induzierte DAT Oberflächenverluste in einer Vielzahl von Zelllinien und Expressionssystemen ist es schwierig gewesen, diese Feststellung in kultivierten dopaminergen Neuronen 21-23 bestätigen. Wir verwendeten Maus Striatum Scheiben direkt zu testen, ob DAT verinnerlicht als Reaktion auf PKC-Aktivierung in der Erwachsenen dopaminergen Neuronen. Folgende Schnitt...

Watch this Scientific Journal Video about Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons at JoVE.com

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronale Membrantransport steuert dynamisch die Plasmamembran Protein Verfügbarkeit und wirkt sich deutlich auf die Neurotransmission. Bis heute hat es sich herausfordern, um neuronale endozytischen Handel bei erwachsenen Nervenzellen messen. Hier beschreiben wir eine hochwirksame, quantitative Methode, um schnelle Veränderungen in der Oberflächenproteinexpression ex vivo in akuten Hirnschnitten messen.

Hirnschnitt Biotinylierung: Eine<em&gt; Ex Vivo</em&gt; Ansatz zur Region spezifische Plasma Membrane Protein Handel Erwachsene Neuronen messen

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Research

JoVE Journal

Neuroscience

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

12.8K Aufrufe.  University of Massachusetts Medical School.  Neuronale Membrantransport steuert dynamisch die Plasmamembran Protein Verfügbarkeit und wirkt sich deutlich auf die Neurotransmission. Bis heute hat es sich herausfordern, um neuronale endozytischen Handel bei erwachsenen Nervenzellen messen. Hier beschreiben wir eine hochwirksame, quantitative Methode, um schnelle Veränderungen in der Oberflächenproteinexpression ex vivo in akuten Hirnschnitten messen. 

Serie: Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

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 ...

Forschung

Neurowissenschaften

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Imaging Receptor Insertion in die Plasmamembran pHluorin-Tag in der Grundschule Cultured Mäuseneuronen

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

Mit Hilfe eines α-Bungarotoxin Binding Site Tag an GABA A-Rezeptor-Membranlokalisierung und Handel Studieren

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Slice It Hot: Akute erwachsenen Gehirn Slicing in physiologischen Temperatur

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Ein<em&gt; Ex vivo</em&gt; Laser-induzierte Rückenmarksverletzungen Modell zu Mechanismen der axonalen Degeneration Beurteilen Sie in Echtzeit

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Ex Vivo optogenetische Dissection of Fear Schaltkreise in Hirnschnitten

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Live-Bilder von GLUT4 Protein Menschenhandel Maus primäre hypothalamische Neurone mit Dekonvolution Mikroskopie

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Ein alternativer Ansatz zur primären Ereignissen in Neurodegeneration mit Ex Vivo Ratte Gehirnscheiben studieren

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex-Vivo Calcium Imaging zur Visualisierung von Gehirn Reaktionen auf endokrine Signalisierung bei Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Einsatz von Infrarot-Fluoreszenz und hochauflösendem Scannen zur Messung des Protein-Expression im Rodentrains

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...