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Fig. 4 | Molecular Neurodegeneration

Fig. 4

From: Soluble oligomeric amyloid-β induces calcium dyshomeostasis that precedes synapse loss in the living mouse brain

Fig. 4

Extracellular calcium is the source of calcium overload in primary neurons expressing human APP. a Tg2576 neurons were incubated with the ratiometric calcium indicator indo-1 AM and then imaged before and after application of NiCl2 to block all voltage gate calcium channels. The ratios of bound vs. unbound indo-1 were calculated, converted to calcium concentration and pseudocolored using the jet color map. Distribution curves of [Ca2+] i (bound/unbound ratio on the lower x-axis and [Ca2+] i on the upper x-axis) are presented in the lower panel showing a wider distribution at baseline, which became more narrow and was shifted to the left after application of NiCl2. The black line indicates the threshold for calcium overload. The percentage of neurons with calcium overload, noted on the graphs before and after treatment, is significantly reduced by NiCl2 from 21.3 to 9.9% (n = 366 neurons before treatment and 321 after treatment in three separate experiments, Fisher exact test, p < 0.0001). b Neurons were transfected with YC3.6 and imaged before and after application of the NMDA receptor antagonist, MK-801. Ratios were converted to calcium levels and pseudocolored as in A. Upper panel shows resting calcium measured in the same neuron before and after inhibition of NMDA receptors. Distribution curves of [Ca2+] i (YFP/CFP on the lower x-axis and [Ca2+] i on the upper x-axis) are shown on the lower panel. Again, the wide distribution of calcium initially observed in transgenic neurons was shifted to the left after treatment with MK-801, dramatically reducing calcium overload from 15.7 to 0.5% (n = 189 neurons in 4 separate experiments, Fisher exact test p < 0.0001). c Summary bar graph for experiments with antagonists for different calcium channel measured with YC3.6 as described in B. The relative change in ratio (ΔR/Ri) after exposure with each inhibitor was calculated for individual cells and presented here as the group mean + SEM (each inhibitor was tested in three independent experiments: vehicles: ddw – n = 243, DMSO – n = 161; inhibitors: Nifedipine (L-type voltage channels) – n = 158, ω-conotoxin (N-type channels) – n = 141, 2-APB (IP3R) – 173, dantrolene (RyR) – 208). The effect of the treatment was tested by comparing each inhibitor to its appropriate vehicle using one way ANOVA with post hoc Dunn’s Multiple comparison test (* p < 0.05, ** p < 0.01, ***p < 0.0001). (Scale bar = 50 μm)

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