Concomitant deficits in working memory and fear extinction are functionally dissociated from reduced anxiety in metabotropic glutamate receptor 7-deficient mice

Abstract

Metabotropic glutamate receptor 7 (mGluR7), a receptor with a distinct brain distribution and a putative role in anxiety, emotional responding, and spatial working memory, could be an interesting therapeutic target for fear and anxiety disorders. mGluR7-deficient (mGluR7-/-) mice showed essentially normal performance in tests for neuromotor and exploratory activity and passive avoidance learning but prominent anxiolytic behavior in two anxiety tests. They showed a delayed learning curve during the acquisition of the hidden-platform water maze, and three interspersed probe trials indicated that mGluR7-/- mice were slower to acquire spatial information. Working memory in the water maze task and the radial arm maze was impaired in mGluR7-/- mice compared with mGluR7+/+. mGluR7-/- mice also displayed a higher resistance to extinction of fear-elicited response suppression in a conditioned emotional response protocol. In a non-fear-based water maze protocol, mGluR7-/- mice displayed similar delayed extinction. These observed behavioral changes are probably not attributable to changes in AMPA or NMDA receptor function because expression levels of AMPA and NMDA receptors were unaltered. Extinction of conditioned fear is an active and context-dependent form of inhibitory learning and an experimental model for therapeutic fear reduction. It appears to depend on glutamatergic and higher-level brain functions similar to those involved in spatial working memory but functionally dissociated from those that mediate constitutional responses in anxiety tests.

Figure 1.

Elevated plus maze performance and marble-burying task in mGluR7^+/+ (black bars) and mGluR7^−/− (white bars) mice. Total number of arm entries were recorded during a 10 min trial (A) as well as time spent in the open arms (B) and number of crossings into the open versus closed arms (C). mGluR7^−/− mice were generally more active during this test but also showed anxiolytic-like behavior because they spent more time and entered more in the open arms than mGluR7^+/+. The marble-burying task revealed significantly fewer marbles buried by mGluR7^−/−. Values are expressed as mean ± SEM; **p < 0.01, significant difference between mGluR7^+/+ and mGluR7^−/− values.

Figure 2.

Fixed-platform water maze performance in mGluR7^−/− mice and wild-type littermates. All mice learned to find the submerged platform during 13 consecutive trial blocks (A), but, when measuring the mean escape latency, it took mGluR7^−/− (open circles) significantly longer than mGluR7^+/+ (filled squares) to find the hidden platform (n = 26–31 per group), which is only partly attributable to the reduced swim speed in mGluR7^−/− (see inset). In contrast, when measuring the time the animals spent near the wall, both groups quickly abandoned the thigmotactic behavior (B; difference not significant) and searched actively for the hidden platform. Three interspersed probe trials (P1–P3) were performed to evaluate whether the animals had developed spatial preference for the target quadrant (C). Total time in each quadrant (i.e., target, black bar; opposite target, gray bar; adjacent 1 and 2, white bars) is plotted for each probe trial, expressed as mean ± SEM. Whereas mGluR7^+/+ show a clearly established preference for the target quadrant already at probe 2, mGluR7^−/− show a significant preference for target quadrant only at probe 3. **p < 0.01, significant preference for the target quadrant.

Figure 3.

Assessment of working memory in the water maze task in mGluR7^+/+ (filled circles) and mGluR7^−/− (open circles) mice. In daily trial blocks of five swims, mice had to find a hidden platform with daily changing location of the platform. During the cued matching-to-position experiment (first swim indicates platform location for the following 4 swims), mGluR7^+/+ mice found the hidden platform slightly faster than mGluR7^−/− mice (A; p < 0.05). During the uncued experiment (platform location during first swim opposite location of the following 4 swims), both genotypes needed a similar amount of time to find the hidden platform (B). Data are means ± SEM for swim 2–5, averaged by genotype. Savings in escape latency between swim 1 and 2 during cued matching-to-position experiment was increased in mGluR7^+/+ mice, but the difference was not significant (C). The comparison between cued and uncued experiment revealed that mGluR7^+/+ (black bars) display a significantly larger saving when presented with a cue than without (p < 0.01), whereas mGluR7^−/− (white bars) show similar performance. Values are means ± SEM for individual swim 1–swim 2 latencies averaged over 6 trial days.

Figure 4.

Extinction of spatial reference memory in Morris water maze. After both groups had learned the position of the hidden platform by swimming preferentially in the target quadrant during probe trials, 3 extinction days were performed with the platform removed. Two, three, and three trial blocks per day were conducted, each trial block consisting of four swim trials. Already on day 2, both groups quickly abandon the preference for the former target quadrant (A). A detailed analysis of the swim paths revealed a very different search strategy between the two groups. Whereas mGluR7^+/+ (filled squares) quickly stopped searching in the center and started swimming closer to the wall, mGluR7^−/− (open circles) continued to search in the center for the platform and display less thigmotactic behavior (B). That this is part of an active searching strategy is also indicated by the relatively small increase of floating behavior (C) and reduction in swim speed (D).

Figure 5.

Radial arm maze acquisition performance in mGluR7^−/− (open circles) and mGluR7^+/+ (filled circles). Over several weeks, subjects were trained to retrieve pellets from food wells located in the eight arms of the radial maze. Non-visits or repeat visits were counted as errors, and a trial lasted 5 min or until all pellets were retrieved. mGluR7^+/+ mice were faster in learning to retrieve all pellets compared with mGluR7^−/− mice (p = 0.004), which were more variable and inconsistent in their performance. Values are means ± SEM of counted errors.

Figure 6.

Food-rewarded operant conditioning and CER in mGluR7^−/− (open symbols) and wild-type (filled symbols) mice. During the operant sessions (A), food-deprived mice were trained to nose poke for food pellets by positive reinforcement (CRF, FR-5 and FR-10, and VI-30s trials). On establishing a stable response rate, eight CER acquisition trials (B) were followed by eight CER extinction trials (C). Both genotypes displayed similar response suppression during the acquisition trials, but, during extinction, mGluR7^+/+ mice resumed instrumental responding significantly faster than mGluR7^−/− (see Results). Values are means ± SEM.

Figure 7.

Comparison of AMPA and NMDA receptor subunit protein expression in control and mGluR7^−/− brains. A, Nissl-stained adjacent sections were used to identify the different brain regions after immunostaining. Cyclamen and dark blue dots indicate the sampling sites for striatum (8 circles with a diameter of 0.3 mm) and the perirhinal cortex (4 circles with a diameter of 0.3 mm), respectively. B, Schematic diagram illustrating the sampling method used to compare immunoreactivities in different hippocampal regions (for details, see Materials and Methods). The colors represent layers of the hippocampal formation: red, stratum oriens (SO); green, stratum radiatum (SR); yellow, stratum lacunosum-moleculare (SLM); plum, stratum moleculare of dentate gyrus (SM); blue, hilum of dentate gyrus (H); lilac, stratum lucidum of CA3 (SL); pink, striatum (ST); dark blue, perirhinal cortex (PR). Density readings were taken by placing open circular cursors with a diameter of 0.1 mm at the indicated adjacent positions along SO (8), SR (6), SLM (7), SM (12), H (6), and SL (7). C, D, Representative histoblots of corresponding regions of mGluR7^+/+ and mGluR7^−/− brains immunostained with antibodies against GluR1–GluR4 (C) or NR1 (D) iGluR subunit proteins. For illustration proposes, grayscale histoblot images were converted to color gradients using gradient mapping. Scale bars, 1 mm. The bar diagrams indicate the pixel density levels (arbitrary units) in various hippocampal and cortical regions in mGluR7^+/+ (open bars) and mGluR7^−/− (filled bars) animals for the indicated iGluR subunit proteins. Quantitative comparison revealed no significant differences (p > 0.05).