Diese Arbeit wurde vom NIH National Institute of Neurological Disorders and Stroke (NINDS) (NS049442) unterstützt.&gt;

Resuspendieren β-Amyloid-Peptid Bevor Sie beginnen müssen bereit: <ul><li> Aß 1-42 (gefriergetrocknetes Pulver) </li><li> 1,1,1,3,3,3-Hexafluor-2-propanol (HFIP) </li><li> Low-Bindung Polypropylen Mikrozentrifugenröhrchen (1,5 ml) </li><li> Gasdichte Hamilton-Spritze mit Teflon stürzen zu stoppen (250 ul) </li><li> Dimethylsulfoxid (DMSO) </li></ul><ol><li> Lassen lyophilisiert Aß 1-42 bei Raumtemperatur für 30 Minuten ins Gleichgewicht zu Kondensation beim Öffnen der Peptid-Fläschchen zu vermeiden. </li><li> Unter der Abzugshaube, resuspendieren Aß-Peptid 1-42 in eiskaltem HFIP zu einer 1 mM Lösung und Vortex die Lösung für ein paar Sekunden zu erhalten. </li><li> Mit einem Glas Gasdichte Hamilton-Spritze mit Teflon-Stecker, schnell teilen die Aß 1-42 / HFIP-Lösung in drei gleich große Polypropylen Ampullen und dichten die Gefäße. </li><li> Die Fläschchen werden dann für 2 Stunden inkubiert, um für Aß Monomerisierung ermöglichen. Dann öffnen Sie die Fläschchen und konzentrieren die Aß 1-42 / HFIP Lösung unter Vakuum mit Hilfe eines SpeedVac Zentrifuge (800 g, Raumtemperatur), bis eine klare Peptid-Film ist an der Unterseite der Fläschchen beobachtet. Prüfen Sie sorgfältig die Temperatur im Inneren des SpeedVac Zentrifuge Peptidabbau vermeiden (max 25 ° C). </li><li> Verschließen Sie die Fläschchen und speichern Sie die Aliquots bei -80 ° C. Aß Film kann für 6 Monate gespeichert werden. </li><li> Re-suspend ein Film, indem DMSO zu einer Konzentration von bis zu erhalten, um 5 mM Aß 1-42. Mit Ultraschall im Wasserbad für 10 Minuten, um sicherzustellen, komplette Resuspension. </li><li> Aliquot dieser 5mM Aß 1-42 Lösung in Polypropylen Ampullen. Seal alle Fläschchen und lagern Sie sie bei -20 ° C. Aliquots sollten nur einmal aufgetaut werden. Hüten Sie sich Kondenswasser im Inneren der Tiefkühltruhe. Peptide in Lösung zersetzen sich sehr leicht. </li></ol> Oligomerisierung Bevor Sie beginnen müssen bereit: <ul><li> Aß 1-42 / DMSO (5mm) </li><li> Sterile Phosphat-Puffer </li><li> Low-Bindung Polypropylen Mikrozentrifugenröhrchen (1,5 ml) </li><li> Low-Bindung Polypropylenzentrifugenröhrchen (50 ml) </li></ul><ol><li> Verdünnen Sie die erhaltenen 5mM Aß 1-42 / DMSO Aliquot mit sterilem Phosphatpuffer (bis zu 100 ul). </li><li> Vortex für 30 Sekunden und anschließend 12 Stunden lang bei 4 ° C </li></ol> Hippokampalen Slice Behandlung Bei der Behandlung von Schnitten des Hippocampus, ist es wichtig, nicht-spezifische Peptid Haftung auf Bechern, Infusionsschlauch Flächen und die Aufnahme Kammer zu vermeiden. Verwenden Sie bevorzugt low-Bindung Polypropylen Rohre und Behälter. Vermeiden Sie die Verwendung von Glas-oder Kunststoff-generic-ware. Perfusion Medien sollten Serum-oder Albumin-frei. Aß Perfusion Bevor Sie beginnen müssen bereit: <ul><li> Oligomerisiert Aß 1-42 </li><li> Recording-Puffer (in mM: 124 NaCl, 4,4 KCl, 1 Na 2 HPO 4, 25 NaHCO 3, 2 CaCl 2, 2 MgCl 2 und 10 Glukose) </li><li> Elektrophysiologische Aufzeichnung Set-up mit Schnittstelle Kammer </li><li> Quer Schnitten des Hippocampus in Sauerstoff Aufzeichnungspuffer für mindestens 90 Minuten nach der Präparation inkubiert (T = 29 ° C; konstant Perfusionsrate = 2 ml / Minute) </li></ul><ol><li> Unmittelbar vor dem Experiment, verdünnen oligomerisiert Aß 1-42 Aliquot auf den entsprechenden Arbeitsgruppen Konzentration (200 nM oder höher) in einem Polypropylen-Rohr mit pre-Sauerstoff Aufzeichnungspuffer. </li><li> Versorgen die Aß 1-42 Lösung für 20 Minuten, um die Scheiben vor der Induktion der synaptischen Plastizität. </li></ol> Induktion von Plastizität und Follow-up <ol><li> Typischerweise ist der am weitesten verbreitete Modell der synaptischen Plastizität der LTP. Es kann durch tetanische Stimulation eines bestimmten Synapsen im Hippocampus induziert werden. Wir Datensatzfeld Antworten von Synapsen zwischen den Shaffers Sicherheiten Projektionen und CA1 pyramidalen Neuronen in der Schicht radiatum. Unsere tetanische Stimulation besteht aus einem Theta-Burst Stimulation, die 3-Züge um 15 Sekunden getrennt sind. Jeder Zug besteht aus 10 Ausbrüche bei 5 Hz, wo jeder Burst bringt 5 Impulse bei 100 Hz. Übernehmen Sie die tetanische Stimulation unmittelbar nach der Perfusion der oligomerisiert Aß abgeschlossen ist. </li><li> Unmittelbar nach der tetanischen Stimulation wieder auf Perfusion mit normaler Aufnahme-Puffer. Aufrechterhaltung einer konstanten Perfusionsrate, Aufnahme Kammertemperatur und ordnungsgemäße Sauerstoffversorgung während der gesamten Dauer des Experiments. </li></ol> Repräsentative Ergebnisse und mögliche Probleme Die Aß oligomerisiert nach dem klassischen Protokoll Stine et coll. 1 erzeugt Aß Monomeren und eine Vielzahl von Oligomeren unterschiedlicher Größes (Dimer, Trimer, etc.) Die Perfusion des oligomerisiert Aß 1-42 führt zu einer verminderten LTP in Aß-behandelten Scheiben im Vergleich zur Kontrollgruppe Scheiben schneiden. Unter unseren experimentellen Bedingungen, wo 3-5 Monate alten C57BL/6J männlichen Mäusen verwendet werden, LTP in 200 nM Aß-behandelten Scheiben auf durchschnittlich 150% der Ausgangswerte in zwei Stunden nach dem tetanischen Stimulation, während die Kontrolle Scheiben LTP-Werte im Durchschnitt 200-250% der baseline2. In einigen Fällen kann die Behandlung mit Aß nicht zu hippocampalen LTP zu reduzieren. Mehrere kritische Fehler, die auftreten könnten, umfassen ein extremes Filmtrocknung während Aß-Konzentration und unvollständiger Oligomerisierung. Aß Perfusion in Schnitten des Hippocampus könnte ein weiterer Anlass zur Sorge wegen der physikalisch-chemischen Eigenschaften des Aß-Peptid in Lösung, die anfällig für unspezifische Haftung auf Kunststoffen ist. Troubleshooting von diesen Fragen wird im Folgenden erörtert.

<ol>
	<li>Stine, W. B., Dahlgren, K. N., Krafft, G. A., LaDu, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+conditions+for+amyloid-beta+peptide+oligomerization+and+fibrillogenesis.">In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis.</a> J Biol Chem. 278, 11612-11622 (2003).</li><li>Vitolo, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+-peptide+inhibition+of+the+PKA/CREB+pathway+and+long-term+potentiation:+reversibility+by+drugs+that+enhance+cAMP+signaling.">Amyloid beta -peptide inhibition of the PKA/CREB pathway and long-term potentiation: reversibility by drugs that enhance cAMP signaling.</a> Proc Natl Acad Sci U S A. 99, 13217-13221 (2002).</li><li>Puzzo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picomolar+amyloid-beta+positively+modulates+synaptic+plasticity+and+memory+in+hippocampus.">Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus.</a> J Neurosci. 28, 14537-14545 (2008).</li><li>Lewczuk, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sample+collection+tubes+on+cerebrospinal+fluid+concentrations+of+tau+proteins+and+amyloid+beta+peptides.">Effect of sample collection tubes on cerebrospinal fluid concentrations of tau proteins and amyloid beta peptides.</a> Clin Chem. 52, 332-334 (2006).</li><li>Masliah, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+synaptic+dysfunction+in+Alzheimer's+disease.">Mechanisms of synaptic dysfunction in Alzheimer's disease.</a> Histol Histopathol. 10, 509-519 (1995).</li><li>Cullen, W. K., Suh, Y. H., Anwyl, R., Rowan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Block+of+LTP+in+rat+hippocampus+in+vivo+by+beta-amyloid+precursor+protein+fragments.">Block of LTP in rat hippocampus in vivo by beta-amyloid precursor protein fragments.</a> Neuroreport. 8, 3213-3217 (1997).</li><li>Itoh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairments+of+long-term+potentiation+in+hippocampal+slices+of+beta-amyloid-infused+rats.">Impairments of long-term potentiation in hippocampal slices of beta-amyloid-infused rats.</a> Eur J Pharmacol. 382, 167-175 (1999).</li><li>Walsh, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+secreted+oligomers+of+amyloid+beta+protein+potently+inhibit+hippocampal+long-term+potentiation+in+vivo.">Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo.</a> Nature. 416, 535-539 (2002).</li><li>Lambert, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nonfibrillar+ligands+derived+from+Abeta1-42+are+potent+central+nervous+system+neurotoxins.">nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins.</a> Proc Natl Acad Sci U S A. 95, 6448-6453 (1998).</li><li>Gong, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+hydrolase+Uch-L1+rescues+beta-amyloid-induced+decreases+in+synaptic+function+and+contextual+memory.">Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual memory.</a> Cell. 126, 775-788 (2006).</li><li>Trinchese, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+calpains+improves+memory+and+synaptic+transmission+in+a+mouse+model+of+Alzheimer+disease.">Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.</a> J Clin Invest. 118, 2796-2807 (2008).</li></ol>

Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften her von der Columbia University IACUC gesetzt durchgeführt.

Wie oben beschrieben, können verschiedene Probleme in der Herstellung von oligomeren Aß in unbeeinträchtigt LTP führen. Eine Möglichkeit zu prüfen, ob Aß Abbau oder der Mangel an Aß Oligomerisierung stattgefunden haben könnte ist, die Aß Vorbereitung mit Tris-Tricin PAGE / Western Blot-Analyse und analytische Ultrazentrifugation (noch nicht in diesem Protokoll beschrieben) zu bewerten. Als Western Blot Proben unter non-denaturing/non-reducing Bedingungen vorbereitet sind, sollte eine erfolgreiche Vorbereitung ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>A&beta; 1-42 (lyophilized) </td>
<td>&nbsp;</td>
<td>American Peptide Co.</td>
<td>62-0-80</td>
<td>&nbsp;</td>
<tr>
<td>1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>52517</td>
<td>&nbsp;</td>
<tr>
<td>Dimethylsulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Biotium</td>
<td>90082</td>
<td>&nbsp;</td>
<tr>
<td>50mL Polypropylene Centrifuge Tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>05-538-67</td>
<td>&nbsp;</td>
<tr>
<td>1.5ml MaxyClear microtubes Maximum recovery</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>MCT-150-L-C</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate-Buffered Saline pH 7.4</td>
<td>&nbsp;</td>
<td>Invitrogen-Gibco</td>
<td>10010-023</td>
<td>&nbsp;</td>
<tr>
<td>GASTIGHT Syringes 250 &mu;L</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>1725</td>
<td>&nbsp;</td>
<tr>
<td>Bath sonicator</td>
<td>&nbsp;</td>
<td>Branson</td>
<td>3510</td>
<td>&nbsp;</td>
<tr>
<td>Savant SpeedVac Concentrator System</td>
<td>&nbsp;</td>
<td>Various distributors</td>
<td>SC 110A</td>
<td>&nbsp;</td>		</tbody>
		</table>
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preparation of oligomeric &beta;-amyloid1-42 and induction of synaptic plasticity impairment on hippocampal slices

Wertminderung von synaptischen Verbindungen geeignet ist, die subtilen amnesic Veränderungen in den frühen Stadien der Alzheimer-Krankheit (AD) zugrunde liegen. β-Amyloid (Aß), ein Peptid, in hohen Mengen in AD produziert, ist bekannt, dass Langzeit-Potenzierung (LTP), ein zelluläres Korrelat von Lernen und Gedächtnis zu reduzieren. In der Tat ist LTP Beeinträchtigung durch Aß verursacht eine nützliche experimentelle Paradigma zur Untersuchung synaptischer Funktionsstörungen in AD-Modellen und zum Screening von Drogen in der Lage zu mildern oder Zurücksetzen wie synaptic Beeinträchtigungen. Studien haben gezeigt, dass Aß die LTP Unterbrechung produziert bevorzugt über ihr oligomerer Form. Hier bieten wir Ihnen ein detailliertes Protokoll für beeinträchtigen LTP durch Perfusion von oligomerisierten synthetischen Aβ1-42-Peptid auf akuten Schnitten des Hippocampus. In diesem Video, skizzieren wir eine Schritt-für-Schritt-Verfahren zur Herstellung von oligomeren Aß<sub&gt; 1-42</sub&gt;. Dann folgen wir ein individuelles Experiment, bei dem LTP im Hippocampus Scheiben ausgesetzt oligomerisiert Aß reduziert wird<sub&gt; 1-42</sub&gt;, Um Scheiben in einem Kontrollexperiment, wo kein Aß im Vergleich<sub&gt; 1-42</sub&gt; Exposition stattgefunden hatte.

Watch this Scientific Journal Video about Preparation of Oligomeric Î²-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices at JoVE.com

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Ein Merkmal der Alzheimer-Krankheit ist die Erhöhung der Aß<sub&gt; 1-42</sub&gt; Peptid. Hier bieten wir ein Protokoll zur Herstellung von synthetischen Aß<sub&gt; 1-42</sub&gt; Oligomere, die Hippocampus Langzeit-Potenzierung beeinträchtigt, ein zelluläres Korrelat der Erinnerung. Dieses Verfahren ist nützlich für die Untersuchung von Mechanismen der Aß-induzierte Pathologie und Wirkstoff-Screening.

Herstellung von oligomeren β-Amyloid 1-42 Und Induktion der synaptischen Plastizität Impairment auf Schnitten des Hippocampus

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

preparation-oligomeric-amyloid1-42-induction-synaptic-plasticity

Research

JoVE Journal

Neuroscience

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

23.0K Aufrufe.  Columbia University. Ein Merkmal der Alzheimer-Krankheit ist die Erhöhung der Aß 1-42 Peptid. Hier bieten wir ein Protokoll zur Herstellung von synthetischen Aß 1-42 Oligomere, die Hippocampus Langzeit-Potenzierung beeinträchtigt, ein zelluläres Korrelat der Erinnerung. Dieses Verfahren ist nützlich für die Untersuchung von Mechanismen der Aß-induzierte Pathologie und Wirkstoff-Screening.

Serie: Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

Vorbereitung der akuten Schnitten des Hippocampus von Ratten und transgenen Mäusen für das Studium der Synaptic Veränderungen während des Alterns und Amyloid-Pathologie

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Forschung

Neurowissenschaften

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation von Neuronen aus Nager Hirnschnitten zu Study Synaptic Transmission and Image präsynaptischen

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

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 ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Post-Einbettung Immunogoldmarkierung der synaptischen Proteine ​​in Hippocampusschnitt Kulturen

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Verbesserte Zubereitung und Konservierung von Hippocampus Maus Slices für eine sehr stabile und reproduzierbare Erfassung der Langzeit-Potenzierung

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Stereotaktische Infusion von oligomeren Amyloid-beta in die Maus-Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

Untersuchung von Synaptic Tagging / Erfassung und Lang Capture mit akuten Schnitten des Hippocampus von Nagetiere

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

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 ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

Bewertung der Synapse Dichte im Hippokampus Nagetier Gehirnscheiben

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...