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. 2020 Feb 12;7(1):ENEURO.0303-19.2019. doi: 10.1523/ENEURO.0303-19.2019

Figure 6.

Figure 6.

Ketamine inhibits JNK and prevents spine regression when given prophylactically, whereas JNK inhibition provides more robust rescue even 2 h after CORT. A, We tested the effect of ketamine (10 μM) on JNK activity in 16-d hippocampal neurons using the mRuby2-JNKAR1EV-Clover FRET reporter. FC FRET responses in spine-head ROIs are from 7 separate experiments plotted individually; p values are shown from repeated measures one-way ANOVA with Bonferroni correction. Ketamine inhibited JNK activity, visible by 10 min postaddition. B, The effect was long lasting. Representative FRET ratio images show mRuby2-JNKAR1EV-Clover JNK activity reporter FRET before and 2 h following ketamine. C, Representative images of 16-d hippocampal neurons expressing mCherry-actin (magenta), SEP-GluR2 (green) ± LOV2-JBD. Cells were stimulated with 100 nM corticosterone (CORT) or 10 μM ketamine (KET), as indicated. Blue boxes indicate ROI where 458-nm light was applied (1-s pulses at 3-min intervals) to photoactivate LOV2-JBD. D, Estimated spine volume changes were calculated from multiple images as shown in C, normalized to baseline. Eight cells, four spines per cell were used. E, Quantitative data of cell surface SEP-GluR2 (SEP-Glur2 fluorescence/mCherry-actin fluorescence) are shown from the same cells as in D. Measurements are from eight experiments. F, Dendritic spine volume changes were calculated from cells pretreated with 10 μM ketamine or 10 μM DJNKI-1 (to inhibit JNK) 2 h before addition of CORT (100 nM) for 2 h. G, Cell surface SEP-GluR2 levels (SEP-GluR2 fluorescence/mCherry-actin fluorescence) was measured from the same cells. H, Estimated spine volume changes were from more than or equal to eight experiments; 16-d neurons were treated with CORT (100 nM) for 2 h followed by KET (10 μM) or DJNKI-1 (10 μM). Estimated spine volume was measured and volume at 4 h was expressed relative to control. I, Cell surface GluR2 levels (SEP-GluR2/mCherry-actin) were calculated from the same cells. Mean data ± SEM are shown. Adjusted p values are shown from repeated measures one-way ANOVA with Bonferroni correction.