Induction of hyperphosphorylated tau in living slices of rat hippocampal formation and subsequent detection using an ELISA.
Although hyperphosphorylated tau is an established feature of Alzheimer's Disease, its role in the disease process is poorly understood, partly because of lack of suitable animal models. We describe the use of living slices of rat hippocampal formation to study tau phosphorylation. Using the AT8 antibody in an ELISA, phosphorylated tau was detected in freshly frozen slices and it increased significantly in slices that were incubated in an electrophysiological recording chamber; the amount detected was greatest when the homogenisation buffer contained phosphatase and kinase inhibitors. The phosphorylated tau content of the slices increased significantly after exposure to the phosphatase 1 and 2A inhibitor okadaic acid (OA) - 1.5 microM. Electrophysiological recordings confirmed that slices were alive and that OA had no acute toxic effect. In control slices phosphorylated tau, detected immunohistochemically, was mainly in the somatodendritic compartment of neurones; in OA treated slices, there was an apparent decrease in somatodendritic AT8 staining and an increase in neuropil staining. Our system enables the induction of hyperphosphorylated tau within living slices, in an experimental environment that can be used to study the biological consequences of such a change, and may therefore help further our understanding of the significance of hyperphosphorylated tau in Alzheimer's Disease.
Mudher
A K
AK
University Department of Pharmacology, Oxford, UK. a.mudher@iop.kcl.ac.uk
Woolley
S T
ST
Perry
V H
VH
Greene
J R
JR
D000544:Alzheimer Disease / Q000378:metabolism; D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D001923:Brain Chemistry / Q000187:drug effects / Q000502:physiology; D002021:Buffers; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D018756:GABA Antagonists / Q000494:pharmacology; D006624:Hippocampus / Q000737:chemistry* / Q000201:enzymology*; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D019319:Okadaic Acid / Q000494:pharmacology; D009924:Organ Culture Techniques; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D054645:Protein Phosphatase 1; D051381:Rats; D017208:Rats, Wistar; D016875:tau Proteins / Q000378:metabolism*
D002021:Buffers; D004791:Enzyme Inhibitors; D018756:GABA Antagonists; D007476:Ionophores; D047428:Protein Kinase Inhibitors; D016875:tau Proteins; D019319:Okadaic Acid; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D001640:Bicuculline