Journal: EMBO Reports
Article Title: The Tau/A152T mutation, a risk factor for frontotemporal‐spectrum disorders, leads to NR2B receptor‐mediated excitotoxicity
Figure Lengend Snippet: hTau AT causes elevation of Ca 2+ levels in CA3 hippocampal neurons at resting state and after membrane depolarization through extrasynaptic NMDA receptors Slices (DIV 15) from control mice were loaded with the Ca 2+ ‐sensitive dye Fura‐2AM. Ratiometric images are presented at baseline and after application of high potassium. Note the increase in intracellular Ca 2+ ([Ca 2+ ] i ) after KCl application. Slices (DIV 15) from hTau AT mice were loaded with the Ca 2+ ‐sensitive dye Fura‐2AM. Ratiometric images at baseline and after application of high potassium chloride in area CA3 are depicted. Note the increase in [Ca 2+ ] i (false color legend) under both conditions in hTau AT slices compared to controls. Electrophysiological example traces (control, black; hTau AT , red) of excitatory postsynaptic field potentials (fEPSP) depict examples of depolarizations that are induced by a single high potassium chloride application in stratum pyramidale (s.p.) of area CA3 in slices in which calcium imaging experiments were conducted. The example depolarization evokes averaged calcium influxes into neurons depicted in the calcium imaging graph. The graph shows the mean of [Ca 2+ ] i changes in response to high KCl in stratum radiatum and stratum pyramidale of area CA3 in Fura‐2AM‐loaded hippocampal slices. After depolarization, [Ca 2+ ] i rises up to ˜600 nM in hTau AT slices (red trace, n = 6 slices, prepared from at least three animals), compared to ˜300 nM in control slices (black trace, n = 6 slices; prepared from at least three animals). Note that even under resting conditions, [Ca 2+ ] i is elevated to ˜120 nM due to hTau AT expression. Quantification of the [Ca 2+ ] i from ratiometric images as depicted in (A) and (B) after background subtraction and selection of regions of interest (10 circles of fixed diameter) in stratum radiatum border to stratum pyramidale of area CA3. Under resting conditions, [Ca 2+ ] i was elevated in transgenic slice cultures (˜120 nM, n = 11) compared to controls (˜80 nM, n = 8). Slices were incubated with one of the following channel blockers: nifedipine (NIF, VGCC; Ctrl n = 8; A152T: n = 7), APV (NMDAR; Ctrl n = 7; A152T n = 8), ifenprodil (IFEN, NMDAR; Ctrl n = 8; A152T n = 7), memantine (MEM, NMDAR; Ctrl/A152T n = 6), 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; AMPAR; Ctrl n = 8; A152T n = 7)), tetrodotoxin (TTX, VGNaC; Ctrl n = 4; A152T n = 5), tetanus neurotoxin (TeNT, neurotransmitter release; A152T n = 6) or in Ca 2+ ‐free buffer (Ctrl n = 10; A152T n = 7). For each experiment, slice preparations from at least three different mice were used. One‐way ANOVA followed by Tukey's post hoc test; Ctrl: F (7/51) = 3,533; P = 0.0036; A152T: F (8/55) = 5,230; P . Quantification of the maximum peak increase in intracellular calcium after high KCl stimulation. One‐way ANOVA followed by Tukey's post hoc test Ctrl: F (7/45) = 4,080; P = 0.0015; A152T: F (8/53) = 7,580; P
Article Snippet: For Ca2+ imaging, slices were incubated with the following drugs 120 min before and during Fura‐2AM experiments: 20 μM L‐VGCC inhibitor Nifedipine (Tocris Biosciences, Bristol, UK); 100 μM NMDAR blocker APV (Tocris Biosciences, Bristol, UK); 30 μM AMPAR antagonist CNQX (Tocris Biosciences, US); 20 μM NR2B‐containing NMDA receptor antagonist Ifenprodil (Tocris Biosciences, Bristol, UK); 1 μM sodium channel blocker Tetrodotoxin (TTX) or 8 μM NMDA receptor antagonist memantine.
Techniques: Mouse Assay, Imaging, Expressing, Selection, Transgenic Assay, Incubation