Preparation of Oligomeric β-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Summary

One feature of Alzheimer's Disease is the elevation of Aβ_1-42 peptide. Here we provide a protocol for preparing synthetic Aβ_1-42 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.

Abstract

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). β-amyloid (Aβ), 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 Aβ 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 Aβ produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic Aβ1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric Aβ_1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized Aβ_1-42 compared to slices in a control experiment where no Aβ_1-42 exposure had occurred.

Protocol

Resuspending β-amyloid peptide

Before getting started have ready:

• Aβ_1-42 (lyophilized powder)
• 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP)
• Low-binding polypropylene microcentrifuge tubes (1.5 ml)
• GasTight Hamilton syringe with Teflon plunge stop (250 μl)
• Dimethylsulfoxide (DMSO)

1. Allow lyophilized Aβ_1-42 to equilibrate at room temperature for 30 minutes to avoid condensation upon opening the peptide vial.
2. Under the fume hood, re-suspend Aβ_1-42 peptide in ice cold HFIP to obtain a 1 mM solution and ....

Discussion

As described above, several problems in the preparation of oligomeric Aβ may result in unimpaired LTP. One way to evaluate whether Aβ degradation or the lack of Aβ oligomerization may have occurred is to evaluate the Aβ 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aβ 1-42 (lyophilized)	American Peptide	62-0-80
1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP)	Fluka	52517
Dimethylsulfoxide (DMSO)	Biotium, Inc.	90082
50mL Polypropylene Centrifuge Tubes	Corning	05-538-67
1.5ml MaxyClear microtubes Maximum recovery	Axygen Scientific	MCT-150-L-C
Phosphate-Buffered Saline pH 7.4	Invitrogen	10010-023
GASTIGHT Syringes 250 μL	Hamilton Co	1725
Bath sonicator	Branson	3510
Savant SpeedVac Concentrator System	Various	SC 110A