Matrix metalloproteinase-9 contributes to kindled seizure development in pentylenetetrazole-treated mice by converting pro-BDNF to mature BDNF in the hippocampus

Abstract

Recurrent seizure activity has been shown to induce a variety of permanent structural changes in the brain. Matrix metalloproteinases (MMPs) function to promote neuronal plasticity, primarily through cleavage of extracellular matrix proteins. Here, we investigated the role of MMP-9 in the development of pentylenetetrazole (PTZ)-induced kindled seizure in mice. Repeated treatment with PTZ (40 mg/kg) produced kindled seizure, which was accompanied by enhanced MMP-9 activity and expression in the hippocampus. No change in MMP-9 activity was observed in the hippocampi of mice with generalized tonic seizure following single administration of PTZ (60 mg/kg). MMP-9 colocalized with the neuronal marker NeuN and the glial marker GFAP in the dentate gyrus of the kindled mouse hippocampus. Coadministration of diazepam or MK-801 with PTZ inhibited the development of kindling and the increased MMP-9 levels in the hippocampus. Marked suppression of kindled seizure progression in response to repeated PTZ treatment was observed in MMP-9((-/-)) mice compared with wild-type mice, an observation that was accompanied by decreased hippocampal levels of mature brain-derived neurotrophic factor. Microinjecting the BDNF scavenger TrkB-Fc into the right ventricle before each PTZ treatment significantly suppressed the development of kindling in wild-type mice, whereas no effect was observed in MMP-9((-/-)) mice. On the other hand, bilateral injections of pro-BDNF into the hippocampal dentate gyrus significantly enhanced kindling in wild-type mice but not MMP-9((-/-)) mice. These findings suggest that MMP-9 is involved in the progression of behavioral phenotypes in kindled mice because of conversion of pro-BDNF to mature BDNF in the hippocampus.

Figure 1.

Seizure and kindling evoked by treating mice once (A) or repeatedly (B) with PTZ. A, Mice received a single injection of PTZ at a dose of 20, 40, or 60 mg/kg. Values are means ± SE (n = 4–6). B, Mice were administered PTZ 11 times at a dose of 40 mg/kg. *p < 0.05 versus control group. Values are means ± SE (n = 4–10). C, Hippocampus-dependent fear memory in control mice treated with saline, mice treated with a single dose of PTZ (60 mg/kg), and kindled mice treated repeatedly with PTZ (40 mg/kg). Context-dependent freezing was measured 24 h after training in a conditioned fear-learning test. Values are means ± SE (n = 8–10). *p < 0.05 versus saline-treated mice. ^#p < 0.05 versus single PTZ dose (60 mg/kg).

Figure 2.

MMP-9 activity in the hippocampi of mice after single (A) or repeated (B, C) doses of PTZ. A, Mice were administered PTZ at a dose of 60 mg/kg and killed 2 h later. Control (Cont) mice were administered saline. MMP-9 standard (MMP-9 Std.) was used as a positive control. Values are means ± SE (n = 4). B, C, Mice were administered PTZ at least 11 times at a dose of 40 mg/kg and killed 2 h (B) or 24 h (C) after the final dose. Values are means ± SE (n = 7 for B; n = 4 for C). D, In situ zymography detected gelatinase (MMP-9) activity in the mouse hippocampus after repeated PTZ treatment. MMP-2^(−/−) mice were administered saline (a–c) or PTZ (d–f) at least 11 times at a dose of 40 mg/kg and killed 2 h after the final dose. Brain sections were incubated with fluorescent gelatin. Cleavage of gelatin by proteinases resulted in increased fluorescence. Scale bar, 100 μm.

Figure 3.

Changes in MMP-9 (A) and MMP-2 (B) protein levels after repeated PTZ treatment in the mouse hippocampus. Mice were administered PTZ at least 11 times at a dose of 40 mg/kg and killed 24 h after the final dose. Control (Cont) mice were administered saline. Values are means ± SE (n = 5–6 for A; n = 4 for B). *p < 0.05 versus control group. C–E, Representative photos of MMP-9 protein expression in mouse hippocampi after repeated treatment with PTZ. Mice were administered saline (Ca, Cc) or PTZ (Cb, Cd) at a dose of 40 mg/kg at least 11 times and killed 2 h after the final treatment. Double immunostaining of the hippocampus for MMP-9 (red, Da, Dd) and NeuN (green, Db, De), or MMP-9 (green, E) and GFAP (red, E) is shown. Scale bars, 100 μm (scale bar in inset, 50 μm).

Figure 4.

Effects of diazepam on kindled seizure (A) and MMP-9 activity (B) evoked by repeated PTZ treatment. A, Mice were administered diazepam at a dose of 1 mg/kg or CMC 30 min before each PTZ dose. Values are means ± SE (n = 6). *p < 0.05 versus CMC + PTZ. B, Mice were killed 2 h after the final PTZ dose. Values are means ± SE (n = 5–12). *p < 0.05 versus cont. #p < 0.05 versus PTZ. Dia, Diazepam.

Figure 5.

Effects of MK-801 on kindled seizure (A) and MMP-9 activity (B) evoked by repeated PTZ treatment. A, Mice were administered MK-801 at a dose of 0.1 mg/kg or saline 30 min before each PTZ dose. Values are means ± SE (n = 5–6). *p < 0.05 versus saline + PTZ. B, Mice were killed 2 h after the final PTZ dose. Values are means ± SE (n = 4). *p < 0.05 versus cont. #p < 0.05 versus PTZ.

Figure 6.

Seizure and kindling in MMP-9^(−/−) mice administered PTZ once (A) or repeatedly (B). A, Mice received a single injection of PTZ at doses of 35, 40, or 60 mg/kg. Values represent means ± SE (n = 5). B, Mice were administered PTZ 11 times at a dose of 35 mg/kg. Values are means ± SE (n = 13). *p < 0.05 versus wild-type mice.

Figure 7.

BDNF mRNA and mature BDNF protein expression in the mouse hippocampus. A–C, Mice were administered PTZ 11 times at a dose of 35 mg/kg and killed 6 h after the final dose. Control animals are saline-treated wild-type mice. Values are means ± SE (n = 5–6). *p < 0.05 versus the saline-treated group. B, D, Representative images of Western blots of samples obtained after 11 or 5 PTZ doses, respectively. Mature BDNF (rBDNF) was used as a positive control. WT: wild-type mice. KO: MMP-9^(−/−) mice. D, E, Mice were administered PTZ five times at a dose of 35 mg/kg and killed 6 h after the final dose. Control animals are saline-treated wild-type mice. Values are means ± SE (n = 5). *p < 0.05 versus saline-treated group. #p < 0.05 versus wild-type mice (PTZ).

Figure 8.

Effects of TrkB-Fc and pro-BDNF on PTZ-induced kindling in wild-type and MMP-9^(−/−) mice. A, B, Mice received microinjections of TrkB-Fc or IgG₁-Fc into the right ventricle 30 min before each PTZ treatment (25 mg/kg). Values are means ± SE (n = 10 for A; n = 8 for B). *p < 0.05 versus IgG₁-Fc-injected wild-type mice. C, D, Mice received microinjections of pro-BDNF or PBS bilaterally into the hippocampal DG 30 min before each PTZ treatment (25 mg/kg). Values are means ± SE (n = 8–10 for A; n = 7 for B). *p < 0.05 versus PBS-injected wild-type mice.