Fig. 6: Acute stress effects on hippocampal spine dynamics are mediated by MMP9-dependent BDNF maturation ex vivo.

a Experimental timeline of OHSC preparation, treatment and time lapse two-photon imaging of CA1. After 13–15 days in culture (DIC), OHSC are treated for 4-6 h with 1 µM Dex or vehicle and during the last 30 min additionally with 50 nM MMP9i or vehicle. After the treatment has ended, media is harvested for molecular analysis and dendrites of pyramidal neurons are imaged twice (t0 and t30 min) for capturing spine dynamics. Quantification of western blotting for b CTSD, c MMP9, d proBDNF, e mBDNF from harvested OHSC medium. Data shown as mean ± s.e.m. of n = 3 biologically independent samples. One-Way ANOVA with Tukey’s multiple comparisons test was performed. *P < 0.05; ***P < 0.001; ****P < 0.0001. f Representative time lapse images of GFP-expressing dendritic segments in hippocampal region CA1 treated with either vehicle, 1 µM Dex and Dex 1 µM + 50 nM MMP9i (scale bar 5 µm). Orange arrowheads indicate disappearing spines, while blue arrowheads indicate novel spines appearing between t0 and t30. g Quantification and Chi-square analyses of dendritic spines classified into changed and unchanged between t0 and t30. h Quantitative representation of increasing spine densities only in different conditions, expressed as percentage of total spines counted.