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. 2019 Jun 10;116(26):13097–13106. doi: 10.1073/pnas.1903203116

Fig. 5.

Fig. 5.

GR-PO4 is required for training-evoked spine maturation. (A) Avidin pulldown of biotinylated proteins from primary neurons at day in vitro 14 electroporated with GR-WT or GR-KI at day in vitro 0, and stimulated with 25 ng/mL BDNF and 1 μM dexamethasone for the indicated time. Each column of the immunoblot (IB) for for GluA1 and p-GluA1 represents an independent sample. (B) Surface GluA1-PO4/GluA1 in cortical neurons expressing GR-WT or GR-KI. Mean ± SEM of n = 11 WT mice and n = 8 KI mice per group; two-way ANOVA: effect of mutant (F1,53 = 6.61, P = 0.016). Pairwise group comparison by unpaired t test (*P < 0.05, #P < 0.001). ns, not significant. (C) In vivo two-photon uncaging of 20 mM MNI-glutamate (90 iterations per laser at 720 nm, 0.7 mW, 0.1 Hz) in Hepes buffer in M1. The arrow indicates photostimulated spine (1) compared with the underlying dendritic shaft (d) and neighboring unstimulated spine (2). (Scale bar, 3 μm). (D) Specificity of glutamate-evoked spine enlargement normalized to baseline spine size in WT mice. Mean ± SEM of n = 10 stimulated (Stim) spines, n = 9 mock-stimulated spines (Mock-stim), and n = 20 neighbor spines (Unstim) in three mice; two-way ANOVA: effect of stimulation (F2,237 = 83.9, P < 0.0001) and time (F6,237 = 6.78, P < 0.0001), post hoc Tukey test (*P < 0.0001). (E) Effect of genotype on glutamate-evoked spine enlargement after 2 d of rotarod training. Mean ± SEM of n = 23 stimulated spines WT, n = 40 stimulated spines KI, n = 18 neighbor spines WT, and n = 20 neighbor spines KI in four mice per group; two-way ANOVA: effect of stimulation (F3,631 = 21.93, P < 0.0001) and time (F6,631 = 2.43, P = 0.024), post hoc Tukey test (#P < 0.0001, *P < 0.0001, †P = 0.009). (F) Effect of RU486 (20 mg/kg i.p. 20 min before each training session) on glutamate-evoked spine enlargement. Mean ± SEM of n = 17 stim+RU486 WT spines, n = 13 stim+RU486 KI spines in three mice; two-way ANOVA: effect of stimulation (F3,605 = 10.13, P < 0.0001) and time (F6,605 = 3.96, P = 0.0007). 2-P, two-photon.