Anxiety and spontaneous activity
Anxiety-like behavior in the elevated plus maze was indexed by the animals' reluctance to venture into the open arms relative to the enclosed arms. This was expressed by the percent time spent in the open arms, or the proportion of arms entries into the open arms (). Separate 2 × 2 (age × genotype) ANOVAs of the two measures did not yield any significant effect [F's<1]. Thus, neither genotype nor age significantly modified the expression of anxiety. Similarly, separate analysis of the total distance travelled in the maze was analysed, which also did not yield any significant effects [F's<1] (see ), thus confirming that the test was not confounded by possible group differences in spontaneous locomotor activity.
Table 1 Anxiety-like behavior in the elevated plus maze (EPM) as indexed by (i) percentage of entries made into the open arms, and (ii) percentage of time spent in the open arms. Spontaneous locomotor activity was indexed by total distance travelled in the elevated (more ...)
Locomotor activity was further evaluated in the open field for a longer period of time sufficient for the assessment of locomotor habituation effect. Habituation was evident from the monotonic reduction of activity over the course of the 60 min period – an effect that was equivalently seen in all groups (see ). The overall level of activity was highly comparable across genotype as well as age groups. A 2 × 2 × 6 (age × genotype × 10-min bins) ANOVA of distance travelled only yielded a significant effect of bins [F(5,190)=101.3, p<0.001].
Spontaneous locomotor activity in the open field (OF) shown as total distance travelled per 10 min bins. All values refer to mean±SEM.
Potential difference in general anxiety or baseline locomotor activity might confound the measure of conditioned fear in the form of freezing in the subsequent conditioned freezing experiment. These were excluded by these two separate tests.
Here, we focused on the conditioned freezing paradigm since this test revealed the most robust promnesic effect in adult mutants (Yee et al., 2006
). On the day of conditioning, the level of freezing generally increased over successive periods of inter-trial intervals (ITIs, ). A similar increase from the first to the second CS presentation in each CU-US pairing was observed in the adult but not the aged mice (). Separate split-plot ANOVAs of freezing recorded during ITIs and during CS presentations yielded only a main effect of ITI periods [F(2,76)=55.07, p<0.001].
The acquisition of conditioned freezing to the tactile CS on day 1 (A) and context freezing on day 2 (B) are depicted. All values refer to mean±SEM.
Conditioned freezing to the context was tested 24h later, and the levels of freezing obtained did not reveal any effect of genotype [F(1,38)=1.78, p=0.20]. However, there was an increase in freezing level over the course of the 8-min test that was apparent only in the adult but not aged mice (). A 2 × 2 × 2 (genotype × age × half-session) ANOVA of percent freezing revealed a significant age by half-session interaction [F(1,38)=4.39, p<0.05].
Next, conditioned freezing to the CS was assessed in two extinction tests conducted across consecutive days in a neutral context. Each test began with a 2-min pre-CS period allowing the evaluation of baseline freezing levels. Analysis of Pre-CS freezing by a 2 × 2 × 2 (age × genotype × days) ANOVA did not reveal any clear significant difference between the two ages [F(1,38)=1.66, p=0.20] or genotypes [F(1,38)=4.07, p=0.05]. Next, the tactile CS was presented for 8 min. The overall freezing response to the CS was stronger in the mutant mice compared with the moderate freezing levels seen in the controls (). However, the time course of the freezing response to the CS markedly differed between adult and aged mutant subjects (). Adult mutants reacted to the initiation of the CS with a pronounced freezing response which then quickly subsided in the course of the 8 min test period as a result of within-session extinction () – a profile that was similarly seen on both test days. In contrast, the temporal profile of the aged mutants' freezing response remained relatively stable over time () – suggesting weaker extinction within a test session compared with adult mutants. The contrasting profile is more readily discernable in , which shows the within-session profile averaged across the two consecutive test days. While within-session extinction appeared weak in aged mutants, between-day extinction was still evident in all groups.
Figure 3 Freezing to the vibration CS in the first and second CS test sessions (conducted 48 h and 72 h after conditioning, respectively) is depicted in two perspectives: (a) comparison of age of each phenotype, and (b) comparison of genotypes at each age. They (more ...)
A 2 × 2 × 2 × 4 (genotype × age × days × 2-min bins) ANOVA of percent freezing revealed a significant effect of genotype [F(1,38)=7.06, p<0.05], supporting the overall presence of enhanced conditioned freezing to the CS in the mutants regardless of age. Additionally, the presence of a significant bins [F(3,114)=7.00, p<0.001] and days effects [F(1,38)=21.35, p<0.001] reflected the overall presence of both within- and between-day extinction, respectively. Lastly, evidence for age-dependent phenotypes was revealed when within-session freezing over bins was examined, as supported by the presence of a significant genotype × age × bins interaction [F(3,114)=3.85, p<0.05] (). Orthogonal contrast analysis indicated that this interaction was predominantly attributable to the linear component of bins [F(1,38)=5.10 p<0.05], which accounted for 80% of the variance explained by the three-way interaction. We therefore calculated the linear component for each group for additional post-hoc comparisons. A downward sloping trend was detectable in all groups, and their mean (±SEM) magnitudes are as follows, adult control = 0.63±0.98, adult mutant = 4.17±0.89, aged control = 0.99±0.82, aged mutant-=0.38± 0.98. The significantly higher value for the adult mutant [maximum p=0.01] than the other three groups (), which did not differ from each other, suggested that the linear rate of extinction within-session was the highest in the adult mutants.
Spontaneous recovery of conditioned CS freezing was evaluated by a 2 × 2 × 2 × 4 (genotype × age × days × 2-min bins) ANOVA that allowed a direct comparison of the animals' freezing response exhibited on the CS tests conducted before and after the 7-day retention period. As shown in , the CR recovered to a somewhat higher level, and mutant mice regardless of age, again, exhibited a stronger response than controls. The analysis yielded a significant effect of days suggestive of a spontaneous recovery effect [F(1,38)=8.36, p<0.01] as well as a significant genotype effect [F(1, 38)=8.02, p<0.01] (). Although aged mutant mice once again appeared to exhibit a weaker within-session extinction profile on this test, there was no evidence for a significant genotype by bins interaction [F<1], which might be due to the lack of a strong overall within-session extinction effect in this test, as suggested by the lack of a statistically significant bins effect [F(3,114)=1.63, p>0.05].
Figure 4 Freezing to the CS 7 days after the last CS-test (A) and overall freezing for the 3 CS-test (B) are depicted. Percent time freezing is indexed as a function of 2-min bins including the 2 minutes of pre-CS. Sp refer to the CS 7 days after the last CS-test. (more ...)
Visualization of apoptosis by Caspase-3 immunohistochemistry
The possible effect of forebrain neuronal GlyT1 knockout on apoptotic cell death was assessed by caspase-3 immunostaining. Caspase-3 is a cysteine protease and a key degradative enzyme involved in apoptosis (Rami, Jansen, Giesser, & Winckler, 2003
). Quantification of caspase-3-ir cells did not reveal any difference between groups. The mean numbers of cells per unit volume (±SEM) were: control: adult=7.67 ± 2.7, aged=7.55 ± 2.36; mutant: adult=4.42 ± 2.46, aged=8.95 ± 3.09. A 2 × 2 (age × genotype) ANOVA of the number of the caspase-3-ir cells in the granular cell layer of the DG in the dHPC revealed neither a significant main effect of genotype and age nor its interaction.
NR1 and NR2B immunohistochemistry
None of the measurements revealed any genotype effect (see ). Separate 2 × 2 (age × genotype) ANOVAs (per marker per region) only yielded a main effect of age in the analysis of CA3 NR2B immunodensity [F(1,35)=7.74, p<0.05]. Representative photographs taken from each group are illustrated in .
Table 2 Summary of the relative optical densities of NMDA receptor subunit 1 (NR1) and NMDA receptor subunit 2B (NR2B) in the subregions of the dHPC (CA1/2 and CA3), Amygdala (BLA and CeA) and Prefrontal cortex (Cg1, PrL and IL) of adult and aged mutant and control (more ...) Visualization of immature neurons by doublecortin immunohistochemistry
Evaluation of the stereological estimates by a non-parametric ANOVA revealed a clear reduction in the number of DCX-positive cells in the aged animals as expected [F(1,34)=107.07; p<0.05] in comparison with the adults (). Importantly, a significant genotype effect was also detected [F(1,34)=4.47; p<0.05] suggesting an increase in DCX-positive cells in the mutant mice. This effect did not appear to be significantly affected in senescence [Genotype × Age interaction: F(1,34)=0.36, p=0.55] in spite of the substantial age-related decrease.
Figure 6 Representative photomicrographs of doublecortin-positive (DCX) immunostaining (scale bar = 200 μm). Separate box-plots showing the stereological estimates of DCX-positive cells per unit volume in the dentate gyrus granule cell layers. Each box (more ...)