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Fig. 1 | Molecular Brain

Fig. 1

From: The phosphorylation status of eukaryotic elongation factor-2 indicates neural activity in the brain

Fig. 1

The eEF2 phosphorylation status is sensitive to synaptic and neuronal activities. ad Administration of excitatory or inhibitory synaptic modulators rapidly modify the p-eEF2/eEF2 ratio in the forebrain and hippocampus. a Representative western blot images of p-eEF2 and eEF2 in the forebrain lysates obtained 1 h after drug or saline administration; α-tubulin was used as a loading control. b The ratios of phosphorylated eEF2 to total eEF2 (p-eEF2/eEF2) in the forebrain lysates obtained from drug-injected mice were normalized to saline-injected controls. Numbers in the parentheses indicate brucine doses (mg/kg) administered intraperitoneally. N = 6 mice for each group. Muscimol: t(10) =  − 24.11, p = 3.42 × 10−10; NBQX: t(10) =  − 3.65, p = 0.0045; Brucine (50): t(10) = 5.10, p = 4.62 × 10−4; Brucine (1): t(10) =  − 8.44, p = 7.30 × 10−6; Student’s t-test. Picrotoxin: U = 3.0, Z =  − 2.40, and p = 0.016 by Mann–Whitney test. c The p-eEF2 and total eEF2 protein levels in the hippocampal lysates obtained 1 h after drug administration. d Summary of the effects of synaptic modulators and blockers on the p-eEF2/eEF2 ratio in the mouse hippocampus. N = 6 mice for each group. Muscimol: U = 0.0, Z =  − 2.88, and p = 0.004; Picrotoxin: U = 1.0, Z =  − 2.72, and p = 0.006; Mann–Whitney test. NBQX: t(10) =  − 5.89, p = 1.52 × 10−4; Brucine (50): t(10) = 4.30, p = 0.0016; Brucine (1): t(10) =  − 4.55, p = 0.0011; Student’s t-test. e Schematic diagram of Cre-dependent expression of mCherry or hM4Di-mCherry in the principal cells in the hippocampus. f Immunohistochemical staining of a coronal brain section showing mCherry expression in the hippocampal principal cells of CaMKIIα-Cre mice unilaterally injected with AAV2-hM4Di-mCherry. The section was co-immunostained with mCherry (red) and the neuronal marker NeuN (green). Scale bar, 1 mm. g Enhanced hippocampal p-eEF2 level (left) and the p-eEF2/eEF2 ratio (right) in the hM4Di-expressing mice compared to mCherry-expressing mice. N = 4 mice for each group. U = 1.0, Z =  − 2.02, and p = 0.043 by Mann–Whitney test. h-j Novel context exploration induces rapid dephosphorylation and subsequent phosphorylation of eEF2 in the hippocampus. Schematic diagram of the experimental design (h). i Representative western blot images of hippocampal p-eEF2 and eEF2 at various timepoints. j The hippocampal p-eEF2/eEF2 ratios in mice that explored a novel context were normalized to those of home cage controls and plotted against exploration time. N = 5–6 mice for each group. 5 min: t(8) = 3.96, p = 0.0042; 15 min: t(8) = 4.067, p = 0.0036; 30 min: t(8) =  − 3.772, p = 0.0054; 60 min: t(10) =  − 10.99, p = 6.66 × 10−7; Student’s t-test. k, l Restraint stress dephosphorylates eEF2 in the hippocampus. The experimental design (k), representative western blots (l, top), and quantification (l, bottom) showing reduced p-eEF2 levels in the hippocampus of mice exposed to restraint stress. N = 5–6 mice for each group. 5 min: t(8) = 2.735, p = 0.0257; 30 min: t(10) = 5.77, p = 1.79 × 10−4; 60 min: t(8) = 4.840, p = 0.0013; Student’s t-test. m The basal levels of p-eEF2 proteins (top) and the p-eEF2/eEF2 ratio (bottom) are reduced in the hippocampus of Xpnpep1−/− mice that lack aminopeptidase p1 (APP1) protein. N = 6 (Xpnpep1+/+) and 5 (Xpnpep1−/−) mice. t(9) = 2.31 and p = 0.047 by Student’s t-test; U = 3.0, Z =  − 2.19, and p = 0.028 by Mann–Whitney test. a, c, g, i, l, and m The blots were cropped, and full blots are presented in the Additional file 1: Fig. S2

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