• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC Sep 25, 2009.
Published in final edited form as:
PMCID: PMC2746052
NIHMSID: NIHMS104270

metabotropic glutamate receptor 5 (mGluR5) has a critical role in inhibitory learning

Abstract

The mechanisms that contribute to the extinction of previously acquired memories are not well understood. These processes, often referred to as inhibitory learning, are thought to be parallel learning mechanisms that require a reacquisition of new information, and suppression of previously acquired experiences in order to adapt to novel situations. Using newly generated metabotropic glutamate receptor 5 (mGluR5) knockout mice we investigated the role of mGluR5 in the acquisition and reversal of an associative conditioned task and a spatial reference task. We found that acquisition of fear conditioning is partially impaired in mice lacking mGluR5. More markedly, we found that extinction of both contextual and auditory fear was completely abolished in mGluR5 knockout mice. In the Morris Water Maze test (MWM), mGluR5 knockout mice exhibited mild deficits in the rate of acquisition of the regular water maze task, but again had significant deficits in the reversal task, despite overall spatial memory being intact. Taken together these results demonstrate that mGluR5 is critical to the function of neural circuits that are required for inhibitory learning mechanisms, and suggest that targeting metabotropic receptors may be useful in treating psychiatric disorders in which aversive memories are inappropriately retained.

Keywords: metabotropic glutamate receptor, mGluR5, fear extinction, Morris Water Maze, inhibitory learning, knockout

Introduction

Metabotropic glutamate receptors (mGluRs) modulate neural activity via their linkage to various intracellular cascades (Nakanishi, 1992). The eight individual mammalian mGluRs can be subdivided into 3 groups based on their sequence homologies and physiological activities. mGluR5 belongs to the Group I mGluRs and is coupled to inositol phosphate/Ca2+ signal transduction pathway (Abe et al., 1992). mGluR5 has been demonstrated to have important roles in several forms of synaptic plasticity (Lu et al., 1997; Jia et al., 1998; Huber et al., 2000; Bikbaev et al., 2008) and learning behaviors (Lu et al., 1997; Chiamulera et al., 2001; Balschun and Wetzel, 2002), and has been suggested as a potential therapeutic target in several neurological disorders (Brody et al., 2004; Slassi et al., 2005; Marino and Conn, 2006; Dolen et al., 2007). mGluR5 is expressed throughout the central nervous system including in the hippocampus and lateral nucleus of the amygdala (Abe et al., 1992), which are both structures central to learning and memory mechanisms. Previous studies have demonstrated a role for mGluR5 in two very different forms of learning associated with the hippocampus and the amygdala. Systemic injection (Schulz et al., 2001) or local amygdala perfusion (Rodrigues et al., 2002) of mGluR5 antagonists can disrupt the acquisition of the fear response, and contextual fear conditioning is impaired in mice deficient for mGluR5 (Lu et al., 1997). Similarly mGluR5 knockout mice show deficits in the acquisition of hippocampal dependent learning (Lu et al., 1997). Thus, while there is significant evidence that mGluR5 is involved in the acquisition of new memories and synaptic plasticity mechanisms, the role of mGluR5 in one important aspect of learning is unknown: the reversal or extinction of a previously acquired task, an important adaptive process that corrects for an altered environmental or situation (Bouton, 1993). These learning mechanisms are important to re-tasking, and are particularly relevant to anxiety disorders in humans such as phobias and posttraumatic stress disorder (Barad, 2005). The mechanisms and molecules involved in these adaptive learning processes, which are often termed inhibitory learning (Bouton and Bolles, 1979; Bouton, 1993; Barad, 2005; Myers and Davis, 2007), are not fully understood, although both ionotropic and metabotropic glutamate receptors have been implicated (Walker et al., 2002; Callaerts-Vegh et al., 2006; Kim et al., 2007; Fendt et al., 2008).

In this study, we tested whether mGluR5 plays a role in inhibitory learning processes using newly generated mGluR5 knockout mice (Supplementary Fig. 1). Towards this end, we tested mGluR5 knockout mice in the acquisition and reversal of an associative conditioned task and a spatial reference task. In the classic Pavlovian fear conditioning test, we found that mGluR5 null mice were impaired in the acquisition of fear conditioning, confirming a role for this receptor in neural circuits required for this form of learning (Lu et al., 1997; Rodrigues et al., 2002). Even more strikingly we observed a complete deficit in the ability of mGluR5 knockout mice to extinguish the fear association in both a tone-cued or context-cued test. In the MWM test, mGluR5 knockout mice exhibited mild deficits in the rate of acquisition of the regular water maze task. However, when the task was reversed and mice were compelled to learn a new location for the escape platform, mGluR5 knockout mice performed poorly in this novel situation. Taken together these findings demonstrate a significant role for mGluR5 in adaptive processes that underlie inhibitory learning.

Materials and Methods

All experiments were approved by the Institutional Animal Care and Use Committees of the Salk Institute for Biological Studies.

Generation of mGluR5 knockout mice

Standard gene targeting techniques were used to generate mutant floxed mGluR5 mice (Supplementary Fig. 1). Genomic DNA for mGluR5 was cloned from a phage library of 129 SVJ mouse genomic DNA fragments (Stratagene, La Jolla, CA). A cassette containing a neomycin resistance (neo) gene, flanked by loxP sites, under the control of the phosphoglycerol kinase (PGK) promoter was introduced into the intron 720 bp downstream of exon 7 (Supplementary Fig. 1a). R1 embryonic stem cells (Nagy et al., 1993) were electroporated with the linearized targeting construct, maintained under G418 positive selection, and screened by Southern blot analysis for homologous recombination (Supplementary Fig. 1b). Chimeric animals produced by injection of these cells into C57BL/6 blastocysts were bred with C57BL/6 mice, and germ-line transmission of the mutation was assessed by PCR and Southern blot. The neo cassette was removed in mice by crossing with transgenic mice with Cre recombinase under control of the protamine promoter (O'Gorman et al., 1997) to produce the floxed mice mGluR5loxP/loxP (Supplementary Fig. 1c-e). Finally mGluR5loxP/loxP mice homozygous for the conditional allele were crossed to protamine-cre mice to generate the mGluR5 knockout mice (mGluR5del/del).

Same sex littermates were housed 2-5 per cage, and maintained at 22°C, with a 12hr light/dark cycle. mGluR5loxP/loxP and mGluR5del/del mice used in this study were produced by heterozygous breeding of mGluR5loxP/del, or heterozygous breeding of mGluR5loxP/+ and mGluR5del/+. Age (2-4 month old) and gender matched littermates were used for behavioral studies.

Fear conditioning

An automated video tracking system was used to monitor mice in the fear conditioning paradigm (Med Associates Inc). Samples were collected at the rate of 30 frames/s. A freezing event was registered only when activity was below motion threshold (20 arbitrary units) for at least 0.5s. Mice were handled daily for 1 week before the test and were transferred to a holding place adjacent to the testing room for 20 min to 1 h on the day of test. The training on d0 lasted for 6 minutes. Each mouse was subjected to either training paradigm A, that consisted of 3 minutes of baseline monitoring, followed by 3 pairs of 20s tone (85db, 2900Hz) co-terminated with 1s footshock (0.7 mA) given at 1-min intervals (Fig. 1a), or paradigm B, which was similar to the paradigm A except that the 1s footshock was presented without the tone (Fig. 1b).

Figure 1
Deficits in fear acquisition in mice lacking mGluR5

For contextual memory testing each mouse was returned to the same chamber with the exact contextual settings, but without tone and footshock and monitored for 6 min. Chambers were cleaned with isopropanol between each set of mice (Fig. 2a, d1 and d15). For the extinction of contextual memory the same procedure was repeated daily for 10 consecutive days (Fig. 3).

Figure 2
Effects of deleting mGluR5 on for both contextual and tone-cued fear conditioning
Figure 3
Contextual fear extinction was abolished in mGluR5-deficient mice

For testing auditory fear (Fig. 2a, d2 and d16; Fig. 4), the conditioning chamber was altered by covering the grid floor and the three sides with patterned plastic boards, and the top with colored paper. New visual cues were provided on the inside wall of the insulating box and on the walls of the testing room. The conditioning chamber was also scented with Windex and vanilla solution. Windex was used for cleaning after each testing. The testing lasted 6 minute for each session. For the experiments presented in Fig. 2 (d2 and d16), each mouse was placed into the chamber for 3 minutes and three sets of 20s tones were presented at 1-min intervals. For the extinction of auditory fear conditioning each mouse was put into the chamber for 3 minutes before the 3-min tone was presented. The same procedure was repeated daily for 16 consecutive days (Fig. 4).

Figure 4
Extinction of auditory fear conditioning was abolished in mGluR5-deficient mice

Morris water maze

The Morris Water Maze experiments were conducted as previously described with some modifications (van Praag et al., 2005; Zhang et al., 2008). A white plastic water tank of 120 cm diameter was filled with water at room temperature. The water was made opaque with white non-toxic Crayola washable paint. A transparent platform (8 cm ×13 cm) was submerged 1cm below the surface of opaque water. An automated video tracking system (Ethovision; Noldus Information Technology) was used to record the swim path, velocity and time taken to reach the platform (latency) or the time spent in each zone. The water maze procedure consisted of three phases: (1) visible platform training; (2) hidden platform training and probe test 1; and (3) reversed platform and probe test 2.

Mice were first trained to find the visible platform for 3 days (3 trials per day). A thin black plastic brick (8cm × 13cm × 1.5cm) was placed above the transparent platform and a flag was installed 10-15 cm above the surface, allowing mice to visualize the location of the platform. Mice were released from the Southwest (SW) quadrant for all trials. The platform was rotated from the Northeast (NE) to Northwest (NW) to Southeast (SE) quadrant for each trial. Upon release, each mouse was allowed a maximum of 60s to find the visible platform. Mice failed to find platform within 60s were placed onto the platform. The mouse was allowed to remain on the platform for 15s after each trial.

Hidden platform training was conducted one day after the completion of the visible platform training. Mice were trained for 7 days (3 trials per day) to find the submerged platform at a fixed position (center of NE quadrant) without any visible local cues. Distal cues in the testing room, such as a computer desk and patterned cardboard on a white wall, were provided as spatial references. Each trial lasted either until the mouse found the platform, or for 60s. Starting points were changed every trial. Mice were allowed to rest on the platform for 15s after each trial. The first probe trial was administered 24 h after the last trial of the hidden platform training. During the probe trial the mouse was allowed to swim for 60 s without the platform in the tank.

For the reverse platform training, the hidden platform was moved from the NE quadrant to the center of the SW quadrant without changing any distal visual cues. Mice were then trained to find this new platform location for 4 days (3 trials per day). Day 1 of reverse training was conducted 2-3 hrs following the first probe test. Starting points were changed every trial. The second probe test was carried out 7 days after the final training.

Statistical analysis

Statistical analyses were conducted with Graphpad Prism (San Diego, CA). For the multiple trial experiments including fear extinction tests and MWM tests, Two-way Repeated-Measures ANOVA was conducted to assess the effects of both genotype and sessions/trials. Bonferroni post hoc tests were conducted to compare genotype effects at individual session/trial (Fig. 3b; Fig. 5a-b, Fig. 6a-c). Repeated measures one-way ANOVA were also conducted to assess the effects of training blocks/trials within the same genotype group. Dunnett's Multiple Comparison post-test was also performed to compare the means at individual time points to the control (first trial/block) (Fig. 6a-b). Differences between two means presented in Fig. 2d-e were assessed with t-tests. Post-test for linear trend was performed to determine if there is an increasing/deceasing trend in fear response during fear extinction tests (Fig. 3 and Fig. 4). Data are presented as mean ± S.E.M. Differences were considered significant if p < 0.05.

Figure 5
Deleting mGluR5 impaired performance in the MWM
Figure 6
Ablation of mGluR5 caused a significant deficit in performance of the reversal task of the MWM

Results

In order to test whether mGluR5-deficient mice demonstrate memory or learning impairments we performed two learning and memory tasks: Pavlovian fear conditioning and the Morris Water Maze (MWM) test. Pavlovian fear conditioning is a task in which a fear response to a neutral conditioned stimulus (CS) is learned when the CS is repeatedly paired with an aversive unconditioned stimulus (US) (LeDoux, 2000). In these experiments the CS was either a context specific environment or a loud tone (85db, 2900Hz) that were coupled to the US (footshock, 0.7 mA). To determine whether ablation of mGluR5 affects acute acquisition of fear, animals were presented with three pairings of the CS and US (Fig. 1a, paradigm A). mGluR5loxP/loxP mice displayed immediate postshock freezing that progressively increased after repeated presentations of CS-US. mGluR5del/del mice froze after footshocks, but the increment in freezing behavior was significantly lower in comparison with the control group upon repeated trials (Fig. 1a). mGluR5del/del mice were also impaired in a second training paradigm in which mice were given three footshocks without the presentation of the tone (Fig. 1b, paradigm B). Thus, acquisition of fear is impaired in mice lacking mGluR5. This impairment is unlikely to be caused by reduced pain sensitivity to electric foot shock because mGluR5del/del mice showed the same pain response as mGluR5loxP/loxP mice (Fig. 1c).

Next we tested whether mGluR5del/del mice retained contextual cued and auditory fear memories. In the first context test performed 24 hr after training (d1) in paradigm A, mGluR5del/del mice showed a significant reduction in the context-cued freezing time (Fig. 2b, in d1). The fractions of context-cued freezing were 0.49 ± 0.04 and 0.29 ± 0.05 for mGluR5loxP/loxP mice and mGluR5del/del mice respectively (Fig. 2d) (p=0.0047, t-test). However, the ratio of freezing during acquisition (d0) to d1 was comparable between the two groups, (0.47 ± 0.12 and 0.38 ± 0.11 for mGluR5loxP/loxP and mGluR5del/del respectively; p>0.05, t-test), suggesting that even though mGluR5del/del mice are impaired in the initial fear acquisition, they retain the ability to express the once memorized fear response. In contrast to this context specific deficit, we found that 24 hrs later (d2) when mice were subjected to the CS tone-cued test, both groups showed similar pre-tone freezing (Fig. 2c, 2e; 0-3 min in d2) and post-tone freezing (Fig. 2c, 2e; 4-6 min in d2). Therefore mGluR5del/del mice are able to remember the association between the CS and US two days after training. In order to determine the retention of these memories at later time points, mice were tested again after two weeks (d15 & d16). Contextual fear (d15) was slightly reduced in mGluR5loxP/loxP mice (paired t-test, p=0.044) but was slightly in mGluR5inasecreddel/ldel mice (paired t-test, p>0.05) (Fig. 2b. 2d, d1 vs. d15). In the second CS tone-cued test conducted in d16, both groups displayed similar post-tone freezing (Fig. 2c, 2e in d16). Noticeably however, pre-tone freezing (0-3 min) was reduced in mGluR5loxP/loxP mice but was elevated in mGluR5del/del mice, although these differences were not significant (Fig. 2e, d2 vs. d16). Pre-tone freezing in the second auditory fear memory test was 2.5 times greater in mGluR5del/del mice than the control group (Fig. 2e, d16) (p=0.0028 t-test), suggesting that mGluR5del/del mice became less discriminative to the fear environment compared to the control group after training.

A previously learned aversive association can be suppressed or extinguished by repeated presentation of the CS in the absence of the US. With this training the animal learns that the CS no longer predicts the US such that the fear response is “inhibited”. The most widely accepted theory for the neural basis of extinction is that it is a parallel process distinct from fear acquisition and is often termed inhibitory learning (Bouton and Bolles, 1979; Bouton, 1993; Barad, 2005; Myers and Davis, 2007). This is a distinct mechanism from when extinction is performed very shortly after acquisition of the fear association, when it may be a reversal or unlearning process (Myers et al., 2006). In order to determine whether inhibitory learning requires mGluR5 we performed fear extinction studies. In the first experiment, mice were trained with paradigm B in which they learn to associate the footshock with a context. Mice were then returned to the same context everyday without footshock for 10 consecutive days (Fig. 3a). Freezing in mGluR5loxP/loxP mice decreased during the extinction training, as revealed by one-way ANOVA (F9,210 = 5.644, P<0.0001) (Fig. 3b). Dunnett's multiple comparison test revealed a significant difference between day 1 and all subsequent days. In addition, the post-test for linear trend revealed a significant decreasing trend in freezing during the period of extinction training in mGluR5loxpP/loxP mice (slope=-1.438, p<0.0001). In contrast to this, fear extinction in mGluR5del/del mice was completely abolished (Fig. 3a). One-way ANOVA comparison did not detect any effects of extinction training for mGluR5del/del mice (F9,184=0.7882, P>0.05) (Fig. 3b). In fact, mGluR5del/del mice exhibited a slightly increased amount of freezing on day 10 (d10) (51 − 5.2 %) than on the first day (d1) (37 ± 3.9 %), even though the difference did not reach significance (by Dunnett's post-tests analysis).The post-test for linear trend revealed a significant increasing trend in freezing for mGluR5del/del mice (slope=0.5644, p=0.0036). These experiments are the first to demonstrate a substantial role for mGluR5 in contextual fear extinction.

Next, in order to determine if a similar deficit might be observed in extinction of auditory fear, we performed a separate series of experiments. Mice were first trained in paradigm A (tone-cued test), and were then tested in a different context without footshock but with tone presentation for 16 consecutive days. There was a clear difference between mGluR5loxP/loxP and mGluR5del/del animals in the extinction of both pre-tone and post-tone fear (Fig. 4a). In order to better assess fear behaviors, freezing was analyzed in four categories: total freezing (0-6 min, Fig. 4b), pre-tone freezing (0-3 min, Fig. 4d), post-tone freezing (4-6 min, Fig. 4e) and tone-cued freezing (post-tone freezing minus pre-tone freezing, Fig. 4c). Although initially mGluR5del/del mice showed less post-tone freezing than control mice (Fig 4e), this freezing behavior did not diminish during the course of repeated daily exposure to the tone. The control group showed a clear reduction in post-tone freezing which was significant between days 9 and 16 of extinction training (Fig. 4e). Both groups displayed similar amount of pre-tone freezing between days 1 and 9 of training (Fig. 4d). However as training progressed there was a steady reduction of pre-tone freezing in mGluR5loxP/loxP mice whereas mGluR5del/del mice continued to display the same degree, or increased, pre-tone freezing during the late stages of extinction training (Fig. 4d). To further examine the freezing response as a more direct result of the tone cue, we subtracted pre-tone freezing from post-tone freezing and presented it as tone-cued fear in Fig. 4c. At the onset (d1-d5), tone-cued freezing was lower in mGluR5del/del mice as compared to the control group which displayed the most robust freezing on the second and third day (Fig. 4c). However, tonecued freezing was progressively reduced in mGluR5loxP/loxP group, while it was maintained at a similar level throughout the test in mGluR5del/del mice. Near completion of the test (d9-d16), tone-cued freezing was no longer different between the two genotypes groups (Fig. 4c). Overall, there was a significant trend in the decrease of the fear curve in mGluR5loxP/loxP mice (slope=-0.29, p<0.0031), but not in mGluR5del/del mice (slope= 0.10, p=0.23) (Fig. 4c). Finally, comparing total freezing revealed the opposite trends between mGluR5loxP/loxP mice and the mGluR5del/del mice: no difference in the early part of the test, and significant differences later (Fig. 4b). Together these data demonstrate that mice lacking mGluR5 were not able to extinguish tone-cued fear (Fig.4c) and became less able to discriminate the fear environment (Fig. 4d) during the course of extinction training. Notably, even the extinction of pre-tone freezing in the control group was much slower compared to the extinction of context-cued freezing shown in Fig. 3; although it might be expected that pre-tone freezing should stay at relatively low levels when the mice were exposed to a neutral context. A likely explanation for this is that during the extinction training, the mice learned to associate the neutral environment to tone, which they had previously associated to the US (footshocks). In support of this idea, extinction of pre-tone freezing correlated very well with the extinction of post-tone freezing for both genotype groups, suggesting that the original neutral environment had become another CS for fear.

We also conducted a control experiment to examine if presentation of the 85 db 2900 Hz tone alone, without footshock, could cause freezing in mice. We found that an 85 db tone lasting for 3 minutes induced little freezing in mice (less than 3 % in both groups, see supplementary Fig. 3). Therefore the influence of this freezing in our experiments is small, and unlikely to confound our conclusions.

In the next series of experiments we determined if mGluR5del/del mice demonstrated a deficit in spatial learning in the standard MWM test. A previous study had found that mGluR5 null mice had a robust deficit in performance in this test (Lu et al., 1997). In contrast we found only a mild impairment in performance of mGluR5del/del mice in the MWM. During the first 3 experimental days, mice were trained to find a visible platform, and both groups performed equally on this task, suggesting that mutant and control mice could visualize the platform and swim equally effectively (Fig. 5a, b; left). During the next seven experimental days, mice were trained to find a hidden platform (3 trials/day) (Fig. 5a, b; middle). Although both groups acquired the task during training, mGluR5del/del mice consistently showed longer escape latencies and path lengths over the training blocks. These disparities were not caused by differences in swim speed. In fact, mGluR5del/del mice swam slightly faster during hidden platform training (Supplementary Fig. 2). We next performed a probe trial in which the platform was removed from the learned location. The first probe test conducted 24-h after the initial training demonstrated that both groups remembered the location of the platform (Fig. 5c, d). mGluR5loxP/loxP slightly outperformed mGluR5del/del by spending more time in the target quadrant (NE) and the platform zone (Fig. 5c). However, the genotype-by-quadrant interaction was not significant. mGluR5loxP/loxP also made slightly more entries to the target quadrant and the platform zone than mGluR5del/del (Fig. 5d), but again, the genotype-by-quadrant interaction was not significant.

In the final test we performed a reversal of the hidden platform in the MWM. Immediately following probe test 1, the platform was moved to the opposite quadrant location (SW) and mice were trained for four days (3 trials per day) in this reversed setting. mGluR5del/del mice showed significantly longer escape times (Fig. 5a, right) and path lengths (Fig. 5b, right) over the training blocks. When data were analyzed by trials, the group differences were more evident in early training, with significant differences between the genotypes occurring in both latency and distance as early as the second and third trial (Fig. 6a, b). While mGluR5del/del mice continued to return to the previous target location, the control cohort more rapidly found the platform in the new location (Fig. 6d, representative tracks taken from trial 2). Throughout this training, mGluR5del/del mice entered the NE (previous target location) more often then the control group (Fig. 6c). Meanwhile, the number of entries into the SE and NW (no platform zone) quadrants were much lower for mGluR5del/del mice, and were comparable between mGluR5del/del and mGluR5loxP/loxP mice (Fig. 6c). These results demonstrate that a persistent search in the previous target zone resulted in a significant delay for mGluR5del/del mice to find the new target location. The second probe test in which the platform was again removed was conducted after a further 7 days and revealed no differences in performance between groups (Fig. 6e, f). Thus, spatial memory was intact in mice lacking mGluR5, whereas mice had a clear deficit in the reversal task. These experiments further support a role of mGluR5 in inhibitory learning.

Discussion

Previous work has highlighted a role for mGluR5 in learning and memory, and in various forms of synaptic plasticity. In the present study we generated a novel strain of mGluR5 mutant mice to test the role of mGluR5 in two common learning paradigms, fear conditioning and the MWM. We found that mGluR5 plays a role in the initial steps of memory acquisition rather than memory storage and retrieval. mGluR5-deficient mice were defective in the acquisition of fear conditioning. However, they retained the ability to express the once-memorized fear response (Fig. 2). Similarly, mGluR5-deficient mice displayed mild impairments in the acquisition of the standard of version of the MWM, but were able to remember the location of the hidden platform during the probe test (Fig. 5). Taken together these findings suggest that mGluR5 mutant mice are defective in the acquisition, rather than the retention and retrieval of the memories. These findings are consistent with prior studies which have demonstrated a role for mGluR5 in the acquisition of fear and spatial memories (Schulz et al., 2001; Rodrigues et al., 2002).

Our study is not the first to generate mGluR5 knockout mice. The mice used here were generated as conditional allele mutations to eventually allow ablation of mGluR5 in a spatial and temporally restricted pattern. In a prior study a conventional global knockout allele of mGluR5 was generated (Lu et al., 1997). In behavioral test these mice were impaired in contextual fear conditioning and acquisition of the standard MWM task in agreement with our finding with mGluR5del/del mice. In contrast to the prior study, in our experiments we found only mild impairments in the acquisition of spatial learning in the MWM whereas Lu et al, had reported more robust deficits in this form of learning. Notably, in both studies similar deficits were observed in acquisition during the early trial blocks. Knockout mice displayed progressive learning during these early trial blocks, although their escape latencies were longer than the control groups. The main discrepancy between our findings and the prior study arise in the late training blocks. In the previous study, mGluR5 knockout mice were unable to improve their performance further in the MWM task in late trial blocks. Their escape latencies leveled at 35-40 sec throughout the rest of the training (Figure 5 in Lu et. al., 1997). In our experiments, our knockout mice had a steady increase in learning throughout all the training blocks. At the end of training, knockout mice showed similar escape latencies as control group (Fig. 5). While it is unclear how these discrepancies might arise they may be due to differences in experimental protocols or strain differences in the mice used in each study.

The primary novel finding in our study is that mGluR5-deficient mice are impaired in the extinction or reversal of learning. We used two inhibitory learning paradigms to assess a role for mGluR5 in these adaptive learning processes. In the first we found that mGluR5-deficient mice were completely unable to extinguish a previously acquired fear response (Fig. 3--4).4). In the second we found that mGluR5-deficient mice were impaired in their ability to re-learn a reversed platform location after they had previously learned the task (Fig. 5--66).

The genetic components that contribute to fear extinction are not fully understood although a number of molecules have been implicated (Falls et al., 1992; Lin et al., 2001; Lu et al., 2001; Cain et al., 2002; Marsicano et al., 2002; Lin et al., 2003; Cain et al., 2004; Wang et al., 2004; Chen et al., 2005; Ponnusamy et al., 2005; Callaerts-Vegh et al., 2006; Chhatwal et al., 2006; Davis et al., 2006; Kamprath et al., 2006; Burgos-Robles et al., 2007; Kim et al., 2007; Sananbenesi et al., 2007; Sotres-Bayon et al., 2007; Fendt et al., 2008; Hefner et al., 2008)(for a review see (Myers and Davis, 2007)). Among these are two members of metabotropic glutamate receptor family: mGluR1(Kim et al., 2007) and mGluR7 (Callaerts-Vegh et al., 2006; Fendt et al., 2008). Thus, with the results of our study, it appears that mGluRs play a central role in fear extinction.

We also found a previously unknown role for mGluR5 in the reversal task of the water maze. Interestingly, we found that mGluR5 knockout mice were more impaired in the reversal spatial learning task, than in the initial acquisition of spatial learning task of the regular water maze. Thus mGluR5 knockout mice provide yet another example that acquisition of standard spatial learning, and reversal spatial learning can be dissociated by genetic or pharmacological manipulation (Hawasli et al., 2007; Duffy et al., 2008).

The fundamental concept of inhibitory leaning, which is determined by the animal learning to suppress a prior learned response is broadly used to describe fear extinction studies (Bouton, 1993; Barad, 2005; Myers and Davis, 2007). The same concept of inhibitory learning has also been applied to reversal of spatial learning (Lattal and Abel, 2001; Varvel et al., 2005; Rossato et al., 2006; Duffy et al., 2008; Labrie et al., 2009). In the reversal task, mice learn that navigating to an area where they had previously learned a platform location is no longer an effective escape strategy. Consequently, mice able to perform this task return to this region with decreasing frequency during the training. Thus although fear extinction and reversal spatial learning are quite separate and distinct tasks they are linked as inhibitory learning tasks which require an active suppression of previously learned associations (Lattal and Abel, 2001; Barad, 2005; Myers and Davis, 2007). Our finding that mice lacking mGluR5 are impaired in both the fear extinction and reversal spatial learning, suggest that these two inhibitory learning paradigms may share common mechanistic properties which involve signaling through mGluR5. There are not many other examples of signaling molecules that have been demonstrated to have similar clear roles in multiple forms of inhibitory learning. However, one such molecule is the CB1 receptor (Marsicano et al., 2002; Varvel et al., 2005). CB1 knockout mice have significant deficits in fear extinction (Marsicano et al., 2002) and in the reversed MWM task (Varvel et al., 2005) similar to the mGluR5-deficient mice. Interestingly, mGluR5 and CB1 receptors are linked together by their roles in synaptic plasticity. mGluR5 activation is required for endocannabinoid-mediated long-term depression of GABA-ergic synapses (eCB-LTD) in the hippocampus (Chevaleyre and Castillo, 2003) and depolarization-induced suppression of inhibition (DSI) in the basolateral amygdala (Zhu and Lovinger, 2005). In particular, the mechanisms of DSI in the basolateral amygdala require mGluR5-mediated release of endocannabinoids and activation of presynaptic CB1 receptors. Therefore it is interesting to speculate that a potential cellular basis for inhibitory learning might be the depression of inhibitory synapses in these brain regions. The findings of our study demonstrate a clear role for mGluR5 in fear extinction and reversal of a spatial task, suggesting that both forms of learning could be linked. However a definitive shared molecular link between these two forms of inhibitory learning will require further experimental clarification.

Neural circuits in several different brain regions play a role in fear extinction and reversal tasks. These include the basolateral nucleus of the amygdala (Falls et al., 1992; Walker et al., 2002), the prefrontal cortex (Quirk et al., 2000; Milad and Quirk, 2002; Burgos-Robles et al., 2007) and the hippocampus (Sananbenesi et al., 2007). mGluR5 is expressed in all these structures, and therefore whether or not mGluR5 signaling in all of these regions, or in specific locales are required for inhibitory learning, remains an open question. In future studies, the novel mice described here will enable us to ablate mGluR5 in specific brain regions in order to directly address this question, and will provide more detailed information of which circuits are involved in inhibitory learning. Reversal or extinction of previously acquired memories allows animals to adapt to a novel environment or situation. These mechanisms are perturbed in several neuropsychiatric disorders in which traumatic memories persist. Therefore mGluR5 provides a potential target for therapeutic intervention in processes of maladaptive learning.

Supplementary Material

Supp1

Acknowledgements

We thank Jelena Radulovic for comments on the manuscript. This study was supported by grants from the NIH/NINDS (5R01NS058894 to AC) and NIH/NINDS (NS28709 to SFH)

References

  • Abe T, Sugihara H, Nawa H, Shigemoto R, Mizuno N, Nakanishi S. Molecular characterization of a novel metabotropic glutamate receptor mGluR5 coupled to inositol phosphate/Ca2+ signal transduction. J Biol Chem. 1992;267:13361–13368. [PubMed]
  • Balschun D, Wetzel W. Inhibition of mGluR5 blocks hippocampal LTP in vivo and spatial learning in rats. Pharmacol Biochem Behav. 2002;73:375–380. [PubMed]
  • Barad M. Fear extinction in rodents: basic insight to clinical promise. Curr Opin Neurobiol. 2005;15:710–715. [PubMed]
  • Bikbaev A, Neyman S, Ngomba RT, Conn J, Nicoletti F, Manahan-Vaughan D. MGluR5 mediates the interaction between late-LTP, network activity, and learning. PLoS ONE. 2008;3:e2155. [PMC free article] [PubMed]
  • Bouton ME. Context, time, and memory retrieval in the interference paradigms of Pavlovian learning. Psychol Bull. 1993;114:80–99. [PubMed]
  • Bouton ME, Bolles RC. Role of conditioned contextual stimuli in reinstatement of extinguished fear. J Exp Psychol Anim Behav Process. 1979;5:368–378. [PubMed]
  • Brody SA, Dulawa SC, Conquet F, Geyer MA. Assessment of a prepulse inhibition deficit in a mutant mouse lacking mGlu5 receptors. Mol Psychiatry. 2004;9:35–41. [PubMed]
  • Burgos-Robles A, Vidal-Gonzalez I, Santini E, Quirk GJ. Consolidation of fear extinction requires NMDA receptor-dependent bursting in the ventromedial prefrontal cortex. Neuron. 2007;53:871–880. [PubMed]
  • Cain CK, Blouin AM, Barad M. L-type voltage-gated calcium channels are required for extinction, but not for acquisition or expression, of conditional fear in mice. J Neurosci. 2002;22:9113–9121. [PubMed]
  • Cain CK, Blouin AM, Barad M. Adrenergic transmission facilitates extinction of conditional fear in mice. Learn Mem. 2004;11:179–187. [PMC free article] [PubMed]
  • Callaerts-Vegh Z, Beckers T, Ball SM, Baeyens F, Callaerts PF, Cryan JF, Molnar E, D'Hooge R. Concomitant deficits in working memory and fear extinction are functionally dissociated from reduced anxiety in metabotropic glutamate receptor 7-deficient mice. J Neurosci. 2006;26:6573–6582. [PubMed]
  • Chen X, Garelick MG, Wang H, Lil V, Athos J, Storm DR. PI3 kinase signaling is required for retrieval and extinction of contextual memory. Nat Neurosci. 2005;8:925–931. [PubMed]
  • Chevaleyre V, Castillo PE. Heterosynaptic LTD of hippocampal GABAergic synapses: a novel role of endocannabinoids in regulating excitability. Neuron. 2003;38:461–472. [PubMed]
  • Chhatwal JP, Stanek-Rattiner L, Davis M, Ressler KJ. Amygdala BDNF signaling is required for consolidation but not encoding of extinction. Nat Neurosci. 2006;9:870–872. [PMC free article] [PubMed]
  • Chiamulera C, Epping-Jordan MP, Zocchi A, Marcon C, Cottiny C, Tacconi S, Corsi M, Orzi F, Conquet F. Reinforcing and locomotor stimulant effects of cocaine are absent in mGluR5 null mutant mice. Nat Neurosci. 2001;4:873–874. [PubMed]
  • Davis M, Ressler K, Rothbaum BO, Richardson R. Effects of D-cycloserine on extinction: translation from preclinical to clinical work. Biol Psychiatry. 2006;60:369–375. [PubMed]
  • Dolen G, Osterweil E, Rao BS, Smith GB, Auerbach BD, Chattarji S, Bear MF. Correction of fragile X syndrome in mice. Neuron. 2007;56:955–962. [PMC free article] [PubMed]
  • Duffy S, Labrie V, Roder JC. D-serine augments NMDA-NR2B receptor-dependent hippocampal long-term depression and spatial reversal learning. Neuropsychopharmacology. 2008;33:1004–1018. [PubMed]
  • Falls WA, Miserendino MJ, Davis M. Extinction of fear-potentiated startle: blockade by infusion of an NMDA antagonist into the amygdala. J Neurosci. 1992;12:854–863. [PubMed]
  • Fendt M, Schmid S, Thakker DR, Jacobson LH, Yamamoto R, Mitsukawa K, Maier R, Natt F, Husken D, Kelly PH, McAllister KH, Hoyer D, van der Putten H, Cryan JF, Flor PJ. mGluR7 facilitates extinction of aversive memories and controls amygdala plasticity. Mol Psychiatry. 2008;13:970–979. [PubMed]
  • Hawasli AH, Benavides DR, Nguyen C, Kansy JW, Hayashi K, Chambon P, Greengard P, Powell CM, Cooper DC, Bibb JA. Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation. Nat Neurosci. 2007;10:880–886. [PMC free article] [PubMed]
  • Hefner K, Whittle N, Juhasz J, Norcross M, Karlsson RM, Saksida LM, Bussey TJ, Singewald N, Holmes A. Impaired fear extinction learning and corticoamygdala circuit abnormalities in a common genetic mouse strain. J Neurosci. 2008;28:8074–8085. [PMC free article] [PubMed]
  • Huber KM, Kayser MS, Bear MF. Role for rapid dendritic protein synthesis in hippocampal mGluR-dependent long-term depression. Science. 2000;288:1254–1257. [PubMed]
  • Jia Z, Lu Y, Henderson J, Taverna F, Romano C, Abramow-Newerly W, Wojtowicz JM, Roder J. Selective abolition of the NMDA component of long-term potentiation in mice lacking mGluR5. Learn Mem. 1998;5:331–343. [PMC free article] [PubMed]
  • Kamprath K, Marsicano G, Tang J, Monory K, Bisogno T, Di Marzo V, Lutz B, Wotjak CT. Cannabinoid CB1 receptor mediates fear extinction via habituation-like processes. J Neurosci. 2006;26:6677–6686. [PubMed]
  • Kim J, Lee S, Park H, Song B, Hong I, Geum D, Shin K, Choi S. Blockade of amygdala metabotropic glutamate receptor subtype 1 impairs fear extinction. Biochem Biophys Res Commun. 2007;355:188–193. [PubMed]
  • Labrie V, Duffy S, Wang W, Barger SW, Baker GB, Roder JC. Genetic inactivation of D-amino acid oxidase enhances extinction and reversal learning in mice. Learn Mem. 2009;16:28–37. [PMC free article] [PubMed]
  • Lattal KM, Abel T. Different requirements for protein synthesis in acquisition and extinction of spatial preferences and context-evoked fear. J Neurosci. 2001;21:5773–5780. [PubMed]
  • LeDoux JE. Emotion circuits in the brain. Annu Rev Neurosci. 2000;23:155–184. [PubMed]
  • Lin CH, Yeh SH, Leu TH, Chang WC, Wang ST, Gean PW. Identification of calcineurin as a key signal in the extinction of fear memory. J Neurosci. 2003;23:1574–1579. [PubMed]
  • Lin CH, Yeh SH, Lin CH, Lu KT, Leu TH, Chang WC, Gean PW. A role for the PI-3 kinase signaling pathway in fear conditioning and synaptic plasticity in the amygdala. Neuron. 2001;31:841–851. [PubMed]
  • Lu KT, Walker DL, Davis M. Mitogen-activated protein kinase cascade in the basolateral nucleus of amygdala is involved in extinction of fear-potentiated startle. J Neurosci. 2001;21:RC162. [PubMed]
  • Lu YM, Jia Z, Janus C, Henderson JT, Gerlai R, Wojtowicz JM, Roder JC. Mice lacking metabotropic glutamate receptor 5 show impaired learning and reduced CA1 long-term potentiation (LTP) but normal CA3 LTP. J Neurosci. 1997;17:5196–5205. [PubMed]
  • Marino MJ, Conn PJ. Glutamate-based therapeutic approaches: allosteric modulators of metabotropic glutamate receptors. Curr Opin Pharmacol. 2006;6:98–102. [PubMed]
  • Marsicano G, Wotjak CT, Azad SC, Bisogno T, Rammes G, Cascio MG, Hermann H, Tang J, Hofmann C, Zieglgansberger W, Di Marzo V, Lutz B. The endogenous cannabinoid system controls extinction of aversive memories. Nature. 2002;418:530–534. [PubMed]
  • Milad MR, Quirk GJ. Neurons in medial prefrontal cortex signal memory for fear extinction. Nature. 2002;420:70–74. [PubMed]
  • Myers KM, Davis M. Mechanisms of fear extinction. Mol Psychiatry. 2007;12:120–150. [PubMed]
  • Myers KM, Ressler KJ, Davis M. Different mechanisms of fear extinction dependent on length of time since fear acquisition. Learn Mem. 2006;13:216–223. [PMC free article] [PubMed]
  • Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci U S A. 1993;90:8424–8428. [PMC free article] [PubMed]
  • Nakanishi S. Molecular diversity of glutamate receptors and implications for brain function. Science. 1992;258:597–603. [PubMed]
  • O'Gorman S, Dagenais NA, Qian M, Marchuk Y. Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells. Proc Natl Acad Sci U S A. 1997;94:14602–14607. [PMC free article] [PubMed]
  • Ponnusamy R, Nissim HA, Barad M. Systemic blockade of D2-like dopamine receptors facilitates extinction of conditioned fear in mice. Learn Mem. 2005;12:399–406. [PMC free article] [PubMed]
  • Quirk GJ, Russo GK, Barron JL, Lebron K. The role of ventromedial prefrontal cortex in the recovery of extinguished fear. J Neurosci. 2000;20:6225–6231. [PubMed]
  • Rodrigues SM, Bauer EP, Farb CR, Schafe GE, LeDoux JE. The group I metabotropic glutamate receptor mGluR5 is required for fear memory formation and long-term potentiation in the lateral amygdala. J Neurosci. 2002;22:5219–5229. [PubMed]
  • Rossato JI, Bevilaqua LR, Medina JH, Izquierdo I, Cammarota M. Retrieval induces hippocampal-dependent reconsolidation of spatial memory. Learn Mem. 2006;13:431–440. [PMC free article] [PubMed]
  • Sananbenesi F, Fischer A, Wang X, Schrick C, Neve R, Radulovic J, Tsai LH. A hippocampal Cdk5 pathway regulates extinction of contextual fear. Nat Neurosci. 2007;10:1012–1019. [PMC free article] [PubMed]
  • Schulz B, Fendt M, Gasparini F, Lingenhohl K, Kuhn R, Koch M. The metabotropic glutamate receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) blocks fear conditioning in rats. Neuropharmacology. 2001;41:1–7. [PubMed]
  • Slassi A, Isaac M, Edwards L, Minidis A, Wensbo D, Mattsson J, Nilsson K, Raboisson P, McLeod D, Stormann TM, Hammerland LG, Johnson E. Recent advances in non-competitive mGlu5 receptor antagonists and their potential therapeutic applications. Curr Top Med Chem. 2005;5:897–911. [PubMed]
  • Sotres-Bayon F, Bush DE, LeDoux JE. Acquisition of fear extinction requires activation of NR2B-containing NMDA receptors in the lateral amygdala. Neuropsychopharmacology. 2007;32:1929–1940. [PubMed]
  • van Praag H, Shubert T, Zhao C, Gage FH. Exercise enhances learning and hippocampal neurogenesis in aged mice. J Neurosci. 2005;25:8680–8685. [PMC free article] [PubMed]
  • Varvel SA, Anum EA, Lichtman AH. Disruption of CB(1) receptor signaling impairs extinction of spatial memory in mice. Psychopharmacology (Berl) 2005;179:863–872. [PubMed]
  • Walker DL, Ressler KJ, Lu KT, Davis M. Facilitation of conditioned fear extinction by systemic administration or intra-amygdala infusions of D-cycloserine as assessed with fear-potentiated startle in rats. J Neurosci. 2002;22:2343–2351. [PubMed]
  • Wang H, Ferguson GD, Pineda VV, Cundiff PE, Storm DR. Overexpression of type-1 adenylyl cyclase in mouse forebrain enhances recognition memory and LTP. Nat Neurosci. 2004;7:635–642. [PubMed]
  • Zhang CL, Zou Y, He W, Gage FH, Evans RM. A role for adult TLX-positive neural stem cells in learning and behaviour. Nature. 2008;451:1004–1007. [PubMed]
  • Zhu PJ, Lovinger DM. Retrograde endocannabinoid signaling in a postsynaptic neuron/synaptic bouton preparation from basolateral amygdala. J Neurosci. 2005;25:6199–6207. [PMC free article] [PubMed]
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...