Knockdown of Prodynorphin Gene Prevents Cognitive Decline, Reduces Anxiety, and Rescues Loss of Group 1 Metabotropic Glutamate Receptor Function in Aging

Group 1 mGluR expression is specifically increased in old Pdyn^−/− mice hippocampus. A, Hippocampal mGluR1α protein level was significantly increased in old Pdyn^−/− mice compared with WT mice. B, Hippocampal mGlu5 receptor level was significantly increased in old Pdyn^−/− mice compared with WT mice, particularly in the CA1 region as shown by immunohistochemistry (C). C, Western blot values represent mean ± SEM of five separate experiments (n = 6 for each age group and genotype), and data are expressed as percentage of control (6-month WT mice). Scale bar, 200 μm. *p < 0.05 (two-way ANOVA with Bonferroni post test). **p < 0.01 (two-way ANOVA with Bonferroni post test).

Group 1 mGluR function is increased in old Pdyn^−/− mice hippocampus. A, After 30 min of baseline recording, DHPG, a specific Group 1 mGluR agonist, was applied for 10 min to slices from either old WT or Pdyn^−/− mice. Only Pdyn^−/− mice expressed significant LTD in the hippocampal CA1 region at 60 min after DHPG application. B, Percentage depression of fEPSP slope at 60 min after DHPG application in old Pdyn^−/− slices was significantly higher than WT slices. C, mGluR-LTD function is similar in young WT and Pdyn^−/− mice. D, Percentage depression of fEPSP slope at 60 min after DHPG application was not significantly different between young WT and Pdyn^−/− mice. Recordings represent mean ± SEM of six separate experiments (n = 6 slices from 3 or 4 mice per genotype). *p < 0.05 (unpaired two-tailed t test for LTD magnitude).

No aging-related spatial memory impairment in old Pdyn^−/− mice. A, All mice learned to swim to the visible platform to escape water after three trials of 60 s regardless of age and genotype (delay of 30 min between each trial). A flag was positioned on the platform as a visual clue. B, Hidden platform MWM learning acquisition curves of the young (6 months), middle-aged (10–14 months), and old (18–25 months) WT versus Pdyn^−/− mice (3 trials/d). C, After 2 d of rest, the platform was moved to the opposite quadrant to assess inhibitory learning acquisition (3 trials/d). Data represent mean ± SEM for all animals of each group (n = 10–18 for each group and genotype) trained in two different cohorts. *p < 0.05 (two-way ANOVA followed by Bonferroni post test). **p < 0.01 (two-way ANOVA followed by Bonferroni post test). ***p < 0.001 (two-way ANOVA followed by Bonferroni post test).

Old Pdyn^−/− mice display intact spatial memory in MWM probe tests. A, Average platform crossings during probe tests conducted 1 h after the last training trial of normal and reversal spatial learning in old WT and Pdyn^−/− mice. B, Average swim speed was similar for age-matched WT and Pdyn^−/− mice. Data represent mean ± SEM for all animals of each group (n = 10–18 for each group and genotype) trained in 2 different cohorts. *p < 0.05 (two-way ANOVA followed by Bonferroni post test). **p < 0.01 (two-way ANOVA followed by Bonferroni post test).

Spatial memory is rescued in old WT mice by treatments with mGluR5-positive allosteric modulator CDPPB or κ-opioid receptor antagonist norBNI, whereas MPEP, a selective antagonist of mGluR5, impaired old Pdyn^−/− mice cognition. A, All old mice (18–25 months) learned to swim to the visible platform to escape water after three trials of 60 s on day 0. B, Mice were injected intraperitoneally 20 min before testing on day 1 with CDPPB, norBNI, or DMSO (vehicle) for old WT mice group and MPEP or DMSO (vehicle) for old Pdyn^−/− group. Drugs did not affect mice cued test performance. C, Learning acquisition of the hidden platform position after pharmacological treatments compared with untreated old WT and Pdyn^−/− mice (3 trials/d). D, On day 8, the platform was moved to the opposite quadrant to assess inhibitory learning acquisition (3 trials/d). E, Average platform crossings during probe tests conducted 1 h after the last training trial of normal and reversal spatial learning. Data represent mean ± SEM for all animals of each group (n = 5/genotype for vehicle, n = 8/drug for injected mice, and n = 10–16/genotype for control untreated mice). For CDPPB or MPEP treatment: *p < 0.05 (two-way ANOVA followed by Bonferroni post tests performed between treated groups and untreated controls); **p < 0.01 (two-way ANOVA followed by Bonferroni post tests performed between treated groups and untreated controls); ***p < 0.001 (two-way ANOVA followed by Bonferroni post tests performed between treated groups and untreated controls). After injection with norBNI: #p < 0.05 (two-way ANOVA followed by Bonferroni post tests performed between treated groups and untreated controls); ##p < 0.01 (two-way ANOVA followed by Bonferroni post tests performed between treated groups and untreated controls); ###p < 0.001 (two-way ANOVA followed by Bonferroni post tests performed between treated groups and untreated controls).

Immediate early gene Homer 1a and Arc/Arg 3.1 expression is correlated with MWM performances. A, Homer 1a protein level was significantly higher in old Pdyn^−/− mice homogenates compared with WT animals. B, Homer 1a expression level was significantly correlated to old mice MWM performances during probe tests. C, Homer 1a double immunostaining with the neuronal marker MAP2 confirmed higher expression of this IEG in old Pdyn^−/− mice CA1 hippocampus region. D, Arc protein level decreased with aging in WT mice, whereas it remained unaltered in Pdyn^−/− mice. E, Like Homer 1a, Arc expression level was significantly correlated with old mice MWM performances. F, Arc staining was slightly enhanced in the CA1 area of Pdyn^−/− mice hippocampus compared with WT mice. Western blot values represent mean ± SEM from three separate experiments (n = 6 for each age group and genotype), and data are expressed as percentage of 6-mo WT mice. *p < 0.05 (two-way ANOVA). Correlations were determined with the Pearson test. Scale bar, 50 μm. Arrowheads indicate IEG-positive cells.

Recognition memory is unaffected despite aging in Pdyn^−/− mice. A, Whereas old WT mice became memory-impaired at 18–25 months of age, old Pdyn^−/− mice spent significantly more time interacting with the novel objects on average and after a 60 min delay. No difference was observed for young and middle-aged Pdyn^−/− mice compared with WT animals of the same age. B, Pdyn^−/− mice displayed higher exploratory behavior than WT mice. C, Representative paths for young, middle-aged, and old mice in the novel object recognition arena (novel object top right in white). D, Treatment with norBNI (#) or CDPPB (*) improved recognition memory of old WT mice by increasing time treated mice spent on novel objects. E, Exploratory behavior was unaffected by drugs injections. Data represent mean ± SEM for all animals of each group (n = 7–17 for each age group and genotype for untreated mice, n = 5/genotype for vehicle, n = 8/drug for injected mice) trained in three different cohorts. Statistical significance was evaluated with two-way ANOVA followed by Bonferroni post tests performed between genotypes (A,B) or treated groups and untreated controls (D) and one-way ANOVA for total distance traveled after pharmacological treatments (E). *p < 0.05. **p < 0.01. #p < 0.05.

Anxiety-related behaviors are reduced in middle-aged and old Pdyn^−/− mice. A, Middle-aged and old Pdyn^−/− mice traveled a longer distance in the EPM open arms compared with WT mice of the same age, suggesting lower anxiety. B, Young and old Pdyn^−/− mice traveled a significantly longer overall distance in the EPM arena compared with WT mice of the same age, suggesting lower anxiety and higher explorative behavior in a bright environment. C, Representative paths for young, middle-aged, and old WT and Pdyn^−/− mice. Black represents closed arms; gray represents open arms. D, MPEP treatment significantly decreased the distance traveled in EPM open arms for old Pdyn^−/− mice. E, Old WT mice injected with norBNI showed higher explorative behavior compared with old untreated WT mice. Data represent mean ± SEM for all animals of each group (n = 7–15 for each age group and genotype and 5–8 for injected old mice groups) trained in three different cohorts. Two-way ANOVA followed by Bonferroni post tests was performed between genotypes and age groups, and one-way ANOVA between treated groups and controls. *p < 0.05. **p < 0.01. #p < 0.05.