Name: preparation of oligomeric &beta;-amyloid1-42 and induction of synaptic plasticity impairment on hippocampal slices
Uploaded: 2010-07-14T17:30:00
Description: Watch this Scientific Journal Video about Preparation of Oligomeric ÃŽÂ²-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices at JoVE.com

Detta arbete stöddes av NIH National Institute of neurologiska sjukdomar och stroke (NINDS) (NS049442).&gt;

Resuspending β-amyloid peptiden Innan du börjar ha klart: <ul><li> A-beta 1-42 (frystorkat pulver) </li><li> 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) </li><li> Låg-bindande polypropylen mikrocentrifugrör (1,5 ml) </li><li> Gastäta Hamilton spruta med Teflon störtar stop (250 l) </li><li> Dimetylsulfoxid (DMSO) </li></ul><ol><li> Låt frystorkat A-beta 1-42 utjämna till rumstemperatur i 30 minuter för att undvika kondens när du öppnar peptiden flaskan. </li><...

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	<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>

Som beskrivits ovan kan flera problem i beredningen av oligomeric A-beta leder felfri LTP. Ett sätt att utvärdera om A-beta nedbrytning eller brist på A-beta oligomerisering kan ha uppstått är att utvärdera A-beta-preparatet hjälp av tris-Tricine SIDA / Western blotting analys och analytisk ultracentrifugering (som inte beskrivs i detta protokoll). När Western blotting provföremålen i non-denaturing/non-reducing villkor, bör en lyckad förberedelse generera en Western blotting signatur med banden som motsvara...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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preparation of oligomeric &beta;-amyloid1-42 and induction of synaptic plasticity impairment on hippocampal slices

Nedskrivningar av synapsförbindelser sannolikt ligger bakom de subtila amnesic förändringar som skett i ett tidigt skede av Alzheimers sjukdom (AD). β-amyloid (A-beta), en peptid som produceras i stora mängder i AD, är känt för att minska långsiktig potentiering (LTP), en cellulär korrelera för inlärning och minne. Faktum är LTP orsakats av A-beta ett användbart experimentell paradigm för att studera synaptisk dysfunktioner i AD modeller och för screening läkemedel som kan mildra eller återgå sådant synaptic nedskrivningar. Studier har visat att A-beta producerar LTP störningar företrädesvis via sin oligomeric form. Här har vi en detaljerad protokoll för att försämra LTP av perfusion av oligomerized syntetiska Aβ1-42 peptid på akut hippocampus skivor. I denna video, disposition vi en steg-för-steg förfarande för beredning av oligomeric A-beta<sub&gt; 1-42</sub&gt;. Sedan följer vi ett individuellt experiment där LTP minskar i hippocampus skivor utsätts för oligomerized A-beta<sub&gt; 1-42</sub&gt; Jämfört med skivor i ett kontrollrum experiment där ingen A-beta<sub&gt; 1-42</sub&gt; Exponering hade inträffat.

Watch this Scientific Journal Video about Preparation of Oligomeric ÃŽÂ²-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices at JoVE.com

Preparation of Oligomeric ÃƒÅ½Ã‚Â²-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Ett inslag i Alzheimers sjukdom är den förhöjning av A-beta<sub&gt; 1-42</sub&gt; Peptid. Här ger vi ett protokoll för att förbereda syntetiska A-beta<sub&gt; 1-42</sub&gt; Oligomerer, vilket försämrar hippocampus långsiktig potentiering, ett cellulärt korrelera minne. Detta förfarande är användbart för att undersöka mekanismerna för A-beta-inducerad patologisk förändring och drog screening.

Beredning av Oligomer β-amyloid 1-42 Och induktion av synaptisk plasticitet Nedskrivning av hippocampus Slices

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

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Neuroscience

Preparation of Oligomeric &beta;-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

Beredning av akut hippocampus Skivor från råttor och transgena möss för studier av Synaptic Förändringar under åldrandet och Amyloid patologi

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

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

Vibrodissociation av nervceller från Slices Gnagare Brain att studera synaptisk transmission och bild Presynaptiska Terminaler

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

Lateral Diffusion och exocytos av membranproteiner i odlade nervceller utvärderas med hjälp av fluorescens återhämtning och fluorescens-förlust fotoblekning

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

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

Förbättrad bearbetning och konservering av hippocampus Mouse skivor för en mycket stabil och reproducerbar Inspelning av långsiktig potentiering

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

Stereotaxic Infusion av oligomera amyloid-beta i musen 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

Utredning av Synaptic märkning / Capture och Cross-capture använder akut hippocampus skivor från Gnagare

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

Mäta synaptiskt vesikelprotein endocytos i odlade nervceller i hippocampus

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

Inspelning synaptisk plasticitet i akut Hippocampus skivor underhålls i en små volymer återvinning - Perfusion- och nedsänkning-typ kammaresystem

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

Utvärdering av synaps densitet i hippocampus gnagare hjärnan skivor

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

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole-inducerad Kindling musmodell

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