Fig 1: Inhibition of endogenous MMP-9 is required for the maturation of dendritic spines in dissociated hippocampal cultures.(A) Results of the DQ-gelatin assay. MMP-9 activity (red) was inhibited by TIMP-1 (blue). The effect of MMP-9 inhibition by TIMP-1 was blocked by incubation with iaMMP-9 (gray). (B) Representative images of live cell imaging of 21 DIV hippocampal neurons that expressed EGFP, revealing changes in dendritic spine morphology (indicated by red arrows) after incubation with cLTP+iaMMP-9. The same set of dendritic spines was analyzed before and after stimulation. (C) Relative changes (mean ± SEM) in the length/width ratio of dendritic spines incubated with cLTP+iaMMP-9. The spines became longer and thinner after 10 min cLTP+iaMMP-9 treatment (nspine = 370; length/width, 0.069 ± 0.026; p < 0.001 and p = 0.004) compared with stimulation without sequestration of the endogenous inhibitor (cLTP only; nspine = 641; length/width, -0.101 ± 0.021) and control (DMSO, vehicle of cLTP; nspine = 379; length/width, -0.025 ± 0.021). After 40 min, the changes in dendritic spine length were persistent. cLTP+iaMMP-9 stimulation increased the length/width ratio (nspine = 370; length/width, 0.120 ± 0.027; p < 0.001 and p = 0.01) compared with cLTP (nspine = 641; length/width, -0.253 ± 0.022) and the control (nspine = 379; length/width, 0.032 ± 0.024). (D) Relative changes (mean ± SEM) in the head width of dendritic spines. Dendritic spine heads after 10 min cLTP were wider (nspine = 641; head width, 0.133 ± 0.018; p < 0.001 and p < 0.001) compared with dendritic spines in cLTP+iaMMP-9 treatment (nspine = 370; head width, -0.019 ± 0.021) and control (nspine = 364; head width, 0.044 ± 0.018). Stimulation for 40 min with cLTP induced enlargement of head width (nspine = 641; head width, 0.091 ± 0.017; p < 0.001 and p < 0.001) compared with cLTP+iaMMP-9 (nspine = 370; head width, -0.027 ± 0.023) and the control (nspine = 364; head width, -0.016 ± 0.020).