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 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.
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 Aβ1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized Aβ1-42 compared to slices in a control experiment where no Aβ1-42 exposure had occurred.
Protocol
Resuspending β-amyloid peptide Before getting started have ready: Aβ1-42 (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) Allow lyophilized Aβ1-42 to equilibrate at room temperature for 30 minutes to avoid condensation up…
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH National Institute of Neurological Disorders and Stroke (NINDS) (NS049442).>
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Aβ 1-42 (lyophilized) | American Peptide Co. | 62-0-80 | ||
| 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP) | Fluka | 52517 | ||
| Dimethylsulfoxide (DMSO) | Biotium | 90082 | ||
| 50mL Polypropylene Centrifuge Tubes | Corning | 05-538-67 | ||
| 1.5ml MaxyClear microtubes Maximum recovery | Axygen | MCT-150-L-C | ||
| Phosphate-Buffered Saline pH 7.4 | Invitrogen-Gibco | 10010-023 | ||
| GASTIGHT Syringes 250 μL | Hamilton | 1725 | ||
| Bath sonicator | Branson | 3510 | ||
| Savant SpeedVac Concentrator System | Various distributors | SC 110A |
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Cite This Article
Fa, M., Orozco, I. J., Francis, Y. I., Saeed, F., Gong, Y., Arancio, O. Preparation of Oligomeric β-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices. J. Vis. Exp. (41), e1884, doi:10.3791/1884 (2010).