The extracellular protease matrix metalloproteinase-9 is activated by inhibitory avoidance learning and required for long-term memory

Abstract

Matrix metalloproteinases (MMPs) are a family of extracellularly acting proteolytic enzymes with well-recognized roles in plasticity and remodeling of synaptic circuits during brain development and following brain injury. However, it is now becoming increasingly apparent that MMPs also function in normal, nonpathological synaptic plasticity of the kind that may underlie learning and memory. Here, we extend this idea by investigating the role and regulation of MMP-9 in an inhibitory avoidance (IA) learning and memory task. We demonstrate that following IA training, protein levels and proteolytic activity of MMP-9 become elevated in hippocampus by 6 h, peak at 12-24 h, then decline to baseline values by approximately 72 h. When MMP function is abrogated by intrahippocampal infusion of a potent gelatinase (MMP-2 and MMP-9) inhibitor 3.5 h following IA training, a time prior to the onset of training-induced elevation in levels, IA memory retention is significantly diminished when tested 1-3 d later. Animals impaired at 3 d exhibit robust IA memory when retrained, suggesting that such impairment is not likely attributed to toxic or other deleterious effects that permanently disrupt hippocampal function. In anesthetized adult rats, the effective distance over which synaptic plasticity is impaired by a single intrahippocampal infusion of the MMP inhibitor of the kind that blocks IA memory is approximately 1200 microm. Taken together, these data suggest that IA training induces a slowly emerging, but subsequently protracted period of MMP-mediated proteolysis critical for enabling long-lasting synaptic modification that underlies long-term memory consolidation.

Figure 1.

MMP-9 protein levels increase in response to IA training. (A) Representative immunoblot of hippocampal homogenates prepared from unpaired (UP) control rats or from rats killed post-IA training at the times indicated (in hours). Membranes were probed with antibodies that recognize both pro- and active-forms of MMP-9 (pro-9, act-9, respectively), or GAPDH, which was used as a loading control. (B,C) Graphs showing quantification of immunoblots from trained and unpaired animals (n = 4 animals per UP group or per post-training time point, *P < 0.05 in comparison with levels in UP controls). Levels of both pro- (B) and active- (C) forms of MMP-9 rise significantly between 3 and 6 h post-training, peak at 24–48 h, then decline to control levels by 75 h post-training. (D) Graph showing quantification of immunoblots from UP and HC control animals (n = 6 UP, n = 3 HC). There were no differences between these two control groups in levels of pro- or active-MMP-9.

Figure 2.

MMP-9 proteolytic activity increases in response to IA training. (A) Representative in vitro gelatin zymograph showing time course in elevation of MMP-9 proteolytic activity post-IA training in comparison with an unpaired (UP) control rat. The zymograph lanes of the UP control and the 24-h post-training rats are shown at higher magnification in the insets, below. The pro-form of MMP-9 is the middle, thick band (pro-9); the proteolytically active-form of MMP-9 (act-9) is the thin, fainter band immediately subjacent to the pro-band. The pro-form appears in zymographs because of some auto-activation following the partially denaturing conditions of SDS gel electrophoresis; the higher molecular weight activity bands (arrow) may represent unprocessed forms that also undergo some auto-activation (Snoek-van Beurden and Von den Hoff 2005). Asterisks indicate time points (12 and 24 h) of significant elevation in proteolytic activity in comparison with UP control levels. (B) Graph showing quantification of MMP-9 proteolysis (active-MMP-9) following training. MMP-9 activity reaches significantly elevated levels by 12–24 h post-IA training, then declines to baseline values by 48–75 h (*P < 0.05; n = 4 animals per group (UP) or per post-training time point). (C) Graph showing quantification of MMP-9 proteolysis (active-MMP-9) in lysates from home cage (HC) and UP controls. There are no differences between these two control groups in levels of MMP-9 proteolysis.

Figure 3.

Intrahippocampal infusion of MMP-9 inhibitor blocks IA memory. (A) Representative Nissl-stained section through dorsal hippocampus showing the track of the infusion needle used to deliver MMP inhibitor or vehicle. DG, dentate gyrus; CA1, area CA1. (B) Timeline of blocking experiments. Animals were trained at time 0 h, then received bilateral intrahippocampal infusions of Inhibitor II or vehicle at 1.5 and 9.5 h post-training (arrowheads). Retention latencies were then tested at 24 h (test). (C) Graph showing mean IA training and retention latencies of three groups of rats (n = 9 unoperated; n = 5 vehicle-infused; n = 6 MMP inhibitor-infused). Retention latency was assessed at 24 h. The MMP inhibitor-infused rats displayed significantly shorter 24-h retention latencies in comparison with those of the other two groups (*P < 0.004). Within the MMP inhibitor group, there was no significant difference between their training and retention latencies, indicating that they were amnesic when tested for IA memory at 24 h. Data are means + SEM.

Figure 4.

IA memory impairment following MMP-9 inhibition is persistent. (A) Timeline of experiments. The initial training and infusion schedule (arrowheads) was identical to that shown in Fig. 3, but, in these experiments, retention latancies were first assessed at 72 h (test 1). The MMP inhibitor-infused rats were then retrained immediately following the 72-h retention test and retested for IA memory 24 h later (test 2). (B) Graph showing mean IA training and retention latencies of three groups of rats (n = 10 unoperated; n = 9 vehicle-infused; n = 8 MMP inhibitor-infused). The MMP inhibitor-infused rats had significantly shorter 72-h retention latencies in comparison with those of the other two groups (*P < 0.05, **P < 0.01), indicating significantly impaired IA memory. There was no significant difference in Test 1 retention latencies between the unoperated and vehicle-infused control groups (P > 0.2). The MMP inhibitor-infused rats were immediately retrained and tested 24 h later (Test 2). Now, they exhibited retention latencies that were significantly longer than their own group’s Test 1 latency (**P < 0.01), and similar in magnitude to that of unoperated control animals tested at 24 h (P > 0.1). These experiments demonstrate that IA memory impairment at 72 h is not a reflection of toxic or other deleterious effects that permanently disrupt hippocampal function. Data are means + SEM.

Figure 5.

A single, post-training infusion of MMP-9 inhibitor blocks IA memory. (A) Timeline of experiment. The design is identical to that shown in Fig. 1, except that only a single infusion (arrowhead) of vehicle or MMP inhibitor was made at 3.5 h post-training. (B) Graph showing IA training and retention latencies of vehicle-infused rats (n = 7) or MMP inhibitor-infused rats (n = 9). Retention latencies were assessed at 24 h. MMP inhibitor-infused rats displayed significantly shorter entrance latencies in comparison with those of the vehicle-infused group (*P < 0.01). Data are means + SEM.

Figure 6.

Range of effective blockade of synaptic plasticity across dorsal hippocampus following infusion of MMP-9 inhibitor. A combination of pharmacological and electrophysiological methods was applied to urethane-anesthetized adult rats in order to determine the effective distance over which a single intrahippocampal infusion of MMP-9 inhibitor blocks maintenance of LTP of CA3–CA1 synapses. Parameters of infusion were identical to those used to block IA memory. (A) Schematic showing timeline and experimental design. (B) Surface view schematic of rat brain showing position of stimulating (Stim) and recording (Rec) electrodes used to elicit LTP in area CA1, and the positions of single inhibitor infusions into the dorsal hippocampus (black circles). Each animal received only a single infusion. (Left) Representative EPSP traces shown at two such positions: potentiation decayed back to baseline by 90 min post-tetanic stimulation (post-tet stim) when infusion site distances were ≤1200 μm away from the recording site (upper trace), while no effects on LTP were observed when infusion site distances were ≥1400 μm away from the recording site (lower trace). (C) Distances between recording and infusion were grouped into three ranges. LTP was elicited reliably in all experiments (gray “LTP” bar, taken at 30 min post-tetanic stimulation), but decayed back to baseline at sites ≤1200 μm separation distance (black “Decay” bars). Only at sites ≥1400 μm separation was LTP maintained (arrow).