Article Figures & Data
Figures
- Figure 1.
Deficits in fear acquisition in mice lacking mGluR5. a , Fear acquisition by three footshocks paired with tones. mGluR5del/del mice (n = 28) froze significantly less than mGluR5loxP/loxP(n = 27) (F (1,106) = 13.14, p = 0.0006 for genotype; F (2,106) = 33.93, p < 0.0001 for training–genotype interaction; Bonferroni post-tests for genotype, p < 0.05 at 4–5 min, p < 0.0001 at 5–6 min). b , Fear acquisition by three footshocks. There was less freezing in mGluR5del/del mice (n = 20) than mGluR5loxP/loxPmice (n = 25) (F (1,86) = 6.31, p = 0.0158 for genotype; F (2,86) = 14.48, p < 0.0001 for training–genotype interaction; Bonferroni post-tests for genotype, p < 0.0001 at 5–6 min). c , Acute pain threshold to footshock was not altered in mGluR5 null mice. We measured the minimal currents required to elicit four stereotyped reactions against footshock: flinch, move, vocalization, and jump. mGluR5del/del mice (n = 8) and mGluR5loxP/loxP mice (n = 13) showed the same pain sensitivity to an increasing electric footshock (t test, p > 0.05 for each reactions between genotypes). Data are presented as SEM (**p < 0.01,***p < 0.001 for Bonferroni post-tests).
- Figure 2.
Effects of deleting mGluR5 on for both contextual and tone-cued fear conditioning. a , The experimental paradigm. Mice were trained at d0 by three footshocks paired with tones in context 1 (marked by gray box), retuned to the same context at d1 and d15 for 6 min without tone presentation. At d2 and d16, mice were put into different context (marked by light blue circle) for 6 min, with three pairs of 20 s tone presentations of 1 min intervals starting at 3 min. b , After fear acquisition at d0, context-cued tests were conducted at d1 and d15, respectively. Freezing was scored at 1 min intervals (mGluR5del/del, n = 17; mGluR5loxP/loxP, n = 18). c , Auditory fear conditioning tests were conducted at d2 and d16, respectively. Freezing was scored at 1 min intervals (mGluR5del/del, n = 17; mGluR5loxP/loxP, n = 18). Time periods for tone presentations were highlighted with blue bars. d , Freezing in ( b ) was scored at 6 min intervals. mGluR5del/del mice exhibited <50% postshock freezing (d0) compared with mGluR5loxP/loxP mice (p = 0.0104, t test). In the first context test (d1) performed 24 h after training, mGluR5del/del mice showed a 40% reduction in the context-cued freezing time (p = 0.0047, t test). The ratios of postshock freezing in d0 versus contextual freezing in d1 were comparable between the two groups (0.465 ± 0.1154 and 0.3830 ± 0.1123 for mGluR5loxP/loxP and mGluR5del/del respectively; p > 0.05, t test). Contextual fear in the second test (d15) was slightly reduced in mGluR5loxP/loxP mice (d1 vs d15; paired t test, p = 0.047), but was slightly increased in mGluR5del/ldel mice (d1 vs d15; paired t test, p > 0.05). e , Freezing in ( c ) was scored at 3 min intervals (for pretone fear and post-tone fear, respectively). Pretone freezing in the second auditory fear conditioning test (d16) was 2.5 times greater in mGluR5del/del mice than the control group (p = 0.0028 t test). A significant difference was not detected between other genotype comparisons. Pretone freezing was reduced in mGluR5loxP/loxP mice but was elevated in mGluR5del/del mice (d2 vs d16), but the differences were not significant. Data are presented as SEM (*p < 0.05, **p < 0.01, for t tests).
- Figure 3.
Contextual fear extinction was abolished in mGluR5-deficient mice. a , Extinction of contextual fear. Mice [mGluR5del/del, n = 20 (d1–d9), n = 14 (d1–d10); mGluR5loxP/loxP, n = 25 (d1–d7), n = 15, (d1–d10)] were trained in d0 with three footshocks and returned to the same context (marked by gray box) up to 10 consecutive days for 6 min each day. Freezing was analyzed at 1 min intervals. During extinction training (d1–d10), mGLuR5loxP/loxP mice generally showed bell-shaped time–response with highest freezing occurring at 3–4 min, whereas mGluR5del/del mice showed a trend of increase freezing toward the end of 6 min training session. b , Freezing in a was scored at 6 min intervals. mGluR5del/del mice displayed no extinction over the trials (nonmatching two-way ANOVA, F (1,394) = 21.26, p < 0.0001 for genotype; F9, 394 = 4.22, p < 0.0001 for day–genotype interaction; Bonferroni post-tests for genotype, *p < 0.05 at d1 and d6). For the mGluR5loxP/loxP group, one-way ANOVA detected significant effect of training (F (9,210) = 6.644, p < 0.0001). Post-test further detected significant trend of fear extinction (slope = −1.44, p < 0.0001). For the mGluR5del/del group, effect of training was not significant by one-way ANOVA (F (9,184) = 0.61, p > 0.05). However, post-test for liner trend detected a significant positive trend (slope = 0.57, p = 0.036). Data are presented as SEM (*p < 0.05, for Bonferroni post-tests; ### p < 0.001 for two-way ANOVA). (Linear slope is denoted by “m”; + p < 0.05 and +++ p < 0.001 for ANOVA post-test for linear trend). ns, Not significant.
- Figure 4.
Extinction of auditory fear conditioning was abolished in mGluR5-deficient mice. a , Extinction of auditory fear conditioning. Mice were trained with three footshocks paired with tones at d0 in one context (gray square) and subsequently returned to a different context (blue circles) for 16 consecutive days. Extinction training on each day lasted 6 min, with one tone of 3 min presented during the final 3 min. Freezing was scored at 1 min intervals to compare within-session fear expression profiles between the two genotype groups (mGluR5del/del n = 14; mGluR5loxP/loxP n = 16). Time periods for the tone presentations were highlighted with blue bars (each represents 3 min). Gray square marked for the context for fear acquisition. Blue circles indicate the neutral context where extinction trainings were conducted. b , Total freezing in each day analyzed in 6 min block. Initially, mGluR5del/del mice (n = 14) showed slightly less freezing (F (1,140) = 3.86, p = 0.0513, d1–d5), but this freezing behavior was not diminished during the course of repeated tone presentations. In contrast, mGluR5loxP/loxP mice (n = 16) showed extinction of auditory fear conditioning upon repeated daily exposure, resulting in less freezing in the second half of testing (F (1, 224) = 32.64, p < 0.001, d9–d16). Overall, day–genotype interaction was significant (F (15,420) = 7.52, p < 0.0001). Post-test detected significant trend of fear extinction in mGluR5loxP/loxP mice (slope = −0.70, p < 0.0001) and significant trend of increase in fear in mGluR5del/del mice (slope = 0.44, p < 0.0001). c , Extinction of tone-cued freezing. At the onset, tone-cued freezing was less in mGluR5del/del mice (F (1,140) = 29.57, p < 0.0001, d1–d15). Near the completion of the test, tone-cued freezing amounts were similar between the two groups (F (1,224) = 2.16, p = 0.14, d9–d16). Two-way ANOVA showed a significant day–genotype interaction from d1 to d16 (F (15,420) = 2.80, p = 0.0004). Post-test for linear trend detected significant trend of decrease of fear in mGluR5loxP/loxP mice (slope = −0.29, p < 0.0031) but not in mGluR5del/del mice (slope = 0.11, p = 0.23). d , Pretone freezing analyzed in 3 min block. Although both groups displayed similar amounts of freezing in the early and middle sessions of training (d1–d9), mGluR5del/del mice froze more in the late sessions (d9–d16) (F1, 224 = 40.15, p < 0.0001). Two-way ANOVA showed a significant day–genotype interaction from d1 to d16 (F (15,420) = 4.13, p < 0.0001). Post-test for linear trend detected significant trend of fear reduction in mGluR5loxP/loxP mice (slope = −0.55, p < 0.0001) and a significant trend of fear increase in mGluR5del/del mice (slope = 0.38, p < 0.0001). e , Post-tone freezing analyzed in 3 min block. Initially, post-tone freezing was less in mGluR5del/del mice (F (1,140) = 12.87, p = 0.0005, d1–d15). However, mGluR5del/del mice froze more in the end (F (1,224) = 20.2, p < 0.0001, d9–d16). Two-way ANOVA showed a significant day–genotype interaction from d1 to d16 (F (15,420) = 7.50, p < 0.0001). A significant trend of fear reduction in mGluR5del/del mice (m = −0.83, p < 0.0001) and a significant trend of fear increase in mGluR5del/del mice (m = 0.49, p < 0.0001) were detected. Data are presented as SEM (### p < 0.001 for two-way ANOVA). (Linear slope is denoted by “m”; ++ p < 0.01 and +++ p < 0.001 for ANOVA post-test for linear trend). ns, Not significant.
- Figure 5.
Deleting mGluR5 impaired performance in the MWM. The water maze procedure consisted of three phases: (1) visible platform training, d1–d3; (2) hidden platform training and probe test 1, d4–d11; and (3) reversed platform and probe test 2, d11–d21. The experimental paradigm is illustrated at the bottom of the figure. a , b , Escape times ( a ) and path lengths ( b ) taken to reach the platform. Escape times and path lengths were analyzed by training block (3 trials per day). In the visible platform test, both groups learned the task, as indicated by incremental reductions in escape times (F (2,72) = 82.31, p < 0.0001) and distances (F (2,72) = 53.91, p < 0.0001) during training. There was no significant difference in either escape times (F (1,72) = 0.36, p = 0.5499) or path lengths (F (1,72) = 0.07, p = 0.7951) between genotype groups (mGluR5del/del, n = 19; mGluR5loxP/loxP n = 19) in the visible platform test (left block). Mice were then trained to find a hidden platform during the next seven consecutive days (3 trials per day). mGluR5del/del mice consistently showed a longer escape latencies and path lengths (middle block) over the training blocks. Repeated-measure two-way ANOVA showed a significant effect of genotype (F (1,216) = 15.49, p = 0.0004) and day (F (6,216) = 21.59, p < 0.0001) but not of the genotype–day interaction (F (6, 216) = 0.83 p = 0.5494) on escape latency. Bonferroni post-tests detected significant differences at hidden day 4, **p < 0.01. Similarly, there were significant effects of genotype (F (1,216) = 23.99, p < 0.0001) and day (F (6,216) = 26.44, p < 0.0001) but not genotype–day interaction (F (6,216) = 1.17, p = 0.3236) on escape length (Bonferroni post-tests for genotype, *p < 0.05 hidden day 3, **p < 0.01, hidden 4). A probe test was conducted 24 h after the completion of regular platform training ( c , d ). Immediately after the first probe test, the platform was removed to the opposite quadrant in the pool and mice were trained at three trials per day for another 4 d in this reversed setting. Although both groups acquired the task after 4 d training, mGluR5del/del mice demonstrated longer escape time (Fig. 5 a, right block) (F (3,108) = 42.38, p < 0.0001 for day; F (1,108) = 45.68, p < 0.0001 for genotype; F (3,108) = 0.80, p = 0.4978 for day–genotype interaction; Bonferroni post-tests, p < 0.001 for reverse day 1 and day 3, p < 0.01 for reverse day 4), and path lengths (Fig. 5 b, right block) (F (3,108) = 36.59, p < 0.0001 for day; F (1,108) = 35.52, p < 0.0001 for genotype; F (3,108) = 0.90, p = 0.4418 for day–genotype interaction; Bonferroni post-tests, p < 0.001 for reverse day 1, day 2, and day 3, p > 0.05 for reverse day 4). c , d , Performances in the first probe test. The first probe test was conducted 24 h after the completion of regular platform training. Analysis of the time spent in the four quadrants ( c ) revealed a significant effect of quadrant (F (4, 144) = 86.20, p < 0.0001). mGluR5loxP/loxP mice spent slightly more time in the target quadrant (NE) and platform zone than mGluR5del/del. However, the genotype–quadrant interaction was not significant (F (4, 144) = 2.24, p = 0.0678). Further analysis of times entered in each testing zone ( d ) also revealed a significant effect of quadrant (F (4, 144) = 125.02, p < 0.0001). Entries to NE quadrant and platform zone were slightly higher in mGluR5loxP/loxP mice compared with mGluR5del/del mice, but the genotype–quadrant interaction was not significant (F (4, 144) = 1.33, p = 0.2615). Data are presented as SEM (*p < 0.05, **p < 0.01,***p < 0.001 for Bonferroni posttests; ### p < 0.001 for two-way ANOVA).
- Figure 6.
Ablation of mGluR5 caused a significant deficit in performance of the reversal task of the MWM. a , b , Trial-by-trial analysis of escape times ( a ) and path lengths ( b ) taken to reach the hidden platform at a reversed position. Two-way ANOVA analysis of escape duration showed significant effect for genotype group (F (1,396) = 45.68, p < 0.0001), trial (F (11,396) = 18.39, p < 0.0001) and trial–genotype interaction (F (11,396) = 1.99, p = 0.0284). Dunnett's multiple comparison test comparing to trial #1 showed that the improvement in mGluR5loxP/loxP mice reached significant difference at trial #3 (+++ p < 0.001), The improvement in mGluR5del/del group became significant on fifth trial (+ p < 0.05). Two-way ANOVA analysis of path lengths also showed significant effect for genotype group (F (1,396) = 35.52, p < 0.0001), trial (F (11, 395) = 15.85, p < 0.0001) and trial–genotype interaction (F (11,396) = 1.95, p = 0.0322). Dunnett's multiple comparison test showed that the improvement reached significant difference on trial #3 (+++ p < 0.001) for mGluR5del/del mice and fifth trial (+ p < 0.05) for mGluR5loxP/loxP mice, respectively. Bonferroni posttests used to compare individual points between genotype groups detected significant differences in escape latency and distance as early as trial 2 and trial 3 (mGluR5del/del, n = 19; mGluR5loxP/loxP, n = 19). c , Throughout training, mGluR5del/del mice entered the NE quadrant (previous target location) more often then the control group (F (1,180) = 11.70, p = 0.0016, for genotype, trial 0–5; F (5,180) = 3.88, p = 0.0023, for genotype–trial interaction, trial 0–5) (left). Meanwhile, entries to SE quadrant (trial 0–5, genotype effect: F (1,180) = 2.90, p = 0.097, genotype–trial interaction: F (5,180) = 0.45, p = 0.8164) and NW quadrant (F (1,180) = 4.29, p = 0.0455 for genotype; F (5,180) = 1.44, p = 0.2116 for genotype–trial interaction) were much lower for the mGluR5del/del mice, and were more comparable to the control group. Trial 0 represents frequencies entered in each testing zone during probe test 1 from the same group of animals (mGluR5del/del, n = 19; mGluR5loxP/loxP, n = 19). Due to an unknown reason, mGluR5del/del mice performed poorly in trial 8. They had much longer escape latency (Fig. 6 a) and distance (Fig. 6 b) than the control group. Their longer search in trial 8 also resulted in much higher number of entries into all quadrants compared with the control group. d , Representative trajectories taken from trial 2, demonstrating that mGluR5del/del mice continued to return to the previous target location while control mice more rapidly found the new location. e , f , Second probe test performed 7 d after reversed platform training. Both groups remembered the last target location (SW) (F (4,144) = 59.61, p < 0.0001 for time spent in the different quadrants; F (4,144) = 52.54, p < 0.0001 for number of entries). There was no significant genotype–quadrant interaction for time spent (F (4,144) = 0.29, p = 0.8815) ( e ) and entry numbers (F (4, 144) = 0.50, p = 0.7369) ( f ). Data are presented as SEM (*p < 0.05, **p < 0.01, ***p < 0.001 for Bonferroni posttests; ## p < 0.01, ### p < 0.001 for two-way ANOVA; + p < 0.05, +++ p < 0.001 for Dunnett's multiple comparison test).
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