This work was supported by NIH National Institute of Neurological Disorders and Stroke (NINDS) (NS049442).>

Resuspending &beta;-amyloid peptide
Before getting started have ready:
<ul>
	<li>A&beta;1-42 (lyophilized powder)</li>
	<li>1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP)</li>
	<li>Low-binding polypropylene microcentrifuge tubes (1.5 ml)</li>
	<li>GasTight Hamilton syringe with Teflon plunge stop (250 &mu;l)</li>
	<li>Dimethylsulfoxide (DMSO)</li>
</ul>
<ol>
	<li>Allow lyophilized A&beta;1-42 to equilibrate at room temperature for 30 minutes to avoid condensation upon opening the peptide vial.</li>
	<li>Under the fume hood, re-suspend A&beta;1-42 peptide in ice cold HFIP to obtain a 1 mM solution and vortex the solution for a few seconds.</li>
	<li>Using a glass GasTight Hamilton syringe with Teflon plug, quickly divide the A&beta;1-42/HFIP solution equally into three polypropylene vials and seal the vials.</li>
	<li>The vials are then incubated for 2 hours to allow for A&beta; monomerization. Next, open the vials and concentrate the A&beta;1-42/HFIP solution under vacuum by using a SpeedVac centrifuge (800 g, room temperature) until a clear peptide film is observed at the bottom of the vials. Check carefully the temperature inside the SpeedVac centrifuge to avoid peptide degradation (max 25&deg;C).</li>
	<li>Seal the vials and store the aliquots at -80&deg;C. A&beta; film can be stored for 6 months.</li>
	<li>Re-suspend one film by adding DMSO to obtain a concentration up to 5 mM A&beta;1-42. Sonicate in the water bath for 10 minutes to ensure complete re-suspension.</li>
	<li>Aliquot this 5mM A&beta;1-42 solution into polypropylene vials. Seal all the vials and store them at -20&deg;C. Aliquots should be thawed only once. Beware of water condensation inside the freezer. Peptides in solution degrade very easily.</li>
</ol>
Oligomerization
Before getting started have ready:
<ul>
	<li>A&beta;1-42/DMSO (5mM)</li>
	<li>Sterile phosphate buffer</li>
	<li>Low-binding polypropylene microcentrifuge tubes (1.5 ml)</li>
	<li>Low-binding polypropylene centrifuge tubes (50 ml)</li>
</ul>
<ol>
	<li>Dilute the obtained 5mM A&beta;1-42/DMSO aliquot with sterile phosphate buffer (up to 100 &mu;l).</li>
	<li>Vortex for 30 seconds and then incubate for 12 hours at 4&deg;C</li>
</ol>
Hippocampal slice treatment
When treating hippocampal slices, it is important to avoid non-specific peptide adhesion onto beakers, perfusion tubing surfaces, and the recording chamber. Use preferentially low-binding polypropylene tubing and containers. Avoid the use of glassware or generic plastic-ware. Perfusion media should be serum- or albumin-free.
A&beta; perfusion
Before getting started have ready:
<ul>
	<li>Oligomerized A&beta;1-42</li>
	<li>Recording buffer (in mM: 124 NaCl, 4.4 KCl, 1 Na2HPO4, 25 NaHCO3, 2 CaCl2, 2 MgCl2, and 10 glucose)</li>
	<li>Electrophysiological recording set-up with interface chamber</li>
	<li>Transverse hippocampal slices incubated in oxygenated recording buffer for at least 90 minutes post-dissection (T = 29&deg; C; constant perfusion rate = 2 ml/minute)</li>
</ul>
<ol>
	<li>Immediately prior to the experiment, dilute the oligomerized A&beta;1-42 aliquot to the appropriate working concentration (200 nM or higher) in a polypropylene tube with pre-oxygenated recording buffer.</li>
	<li>Perfuse the A&beta;1-42 solution for 20 minutes to the slices before the induction of synaptic plasticity.</li>
</ol>
Induction of plasticity and follow-up
<ol>
	<li>Typically, the most widely-used model of synaptic plasticity is the LTP. It can be induced through tetanic stimulation of a given synapse in the hippocampus. We record field responses from synapses between the Shaffer's collateral projections and CA1 pyramidal neurons in the stratum radiatum. Our tetanic stimulation consists of a theta burst stimulation which includes 3 trains separated by 15 second intervals. Each train consists of 10 bursts at 5 Hz where each burst entails 5 pulses at 100 Hz. Apply the tetanic stimulation immediately after the perfusion of oligomerized A&beta; has been completed.</li>
	<li>Immediately after the tetanic stimulation switch back to perfusion with normal recording buffer. Maintain a constant perfusion rate, recording chamber temperature and proper oxygenation throughout the duration of the experiment.</li>
</ol>
Representative Results and possible problems
The A&beta; oligomerized according to the classical protocol of Stine et coll.1, generates A&beta; monomers and a variety of oligomers of different sizes (dimer, trimer, etc.) The perfusion of oligomerized A&beta;1-42 leads to decreased LTP in A&beta;-treated slices compared to control slices. Under our experimental conditions, where 3-5 month old C57BL/6J male mice are used, LTP in 200 nM A&beta;-treated slices is on average 150% of the baseline values at two hours after tetanic stimulation, while in control slices LTP values are on average 200-250% of baseline2. In some cases, treatment with A&beta; may fail to reduce hippocampal LTP. Several critical errors that might occur include excessive film drying during A&beta; concentration and incomplete oligomerization. A&beta; perfusion in hippocampal slices might be another source of concern because of the physicochemical properties of the A&beta; peptide in solution, which is prone to non-specific adhesion to plastics. Troubleshooting of these issues is discussed below.

<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>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Columbia University IACUC.

As described above, several problems in the preparation of oligomeric A&beta; may result in unimpaired LTP. One way to evaluate whether A&beta; degradation or the lack of A&beta; oligomerization may have occurred is to evaluate the A&beta; preparation using TRIS-Tricine PAGE/Western Blotting analysis and analytical ultracentrifugation (not described in this protocol). When Western Blotting samples are prepared under non-denaturing/non-reducing conditions, a successful preparation should generate a Western Blotting signa...

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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>
		</div>

preparation of oligomeric &beta;-amyloid1-42 and induction of synaptic plasticity impairment on hippocampal slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

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

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

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

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

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JoVE Journal

Neuroscience

23.0K Views.  Columbia University. One feature of Alzheimer's Disease is the elevation of Aβ1-42 peptide. Here we provide a protocol for preparing synthetic Aβ1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of Aβ-induced pathology and drug screening.