Pde8b is expressed in an anatomically restricted pattern in the mouse brain
To investigate the distribution of either Pde8a or Pde8b
gene expression in the mouse brain, we utilized mouse lines in which Pde8a or Pde8b
are inactivated by replacement of either exon 17 (Pde8a
) or exons 14 and 15 (Pde8b
) with a lacZ
cDNA () (Tsai et al. 2011
; Vasta et al. 2006
). Expression of the exogenous nls-lacZ
allows visualization of β-galactosidase (β-gal) activity under the control of the endogenous Pde8a or Pde8b
promoter (Tsai et al. 2011
; Vasta et al. 2006
). Analysis of β-gal activity on sagittal brain sections from PDE8A KO mice revealed that β-gal expression is largely undetectable in the brains of these mice (). In contrast to PDE8A KO mice, we observed β-gal activity in PDE8B KO mice, with the highest levels of activity in the dentate gyrus and CA1 regions of the hippocampus, the ventral striatum, and the Purkinje cell layer of the cerebellum (). These results consistent with observed in situ
hybridization analysis reported by the Allen Institute for Brain Science (http://mouse.brain-map.org
Pde8b, but not Pde8A expression is enriched in specific brain regions
Inactivation of Pde8b leads to enhancement of select behaviors
We have previously demonstrated that replacement of exons 14 and 15 of Pde8b
, which encodes the catalytic domain of PDE8B, with a cDNA encoding lacz
is sufficient to disrupt functional PDE8B activity (Tsai et al. 2011
). To determine the behavioral significance of Pde8b
expression in the brain, we analyzed PDE8B KO mice and WT littermates in a suite of behavioral tasks. We observed that numerous basic behaviors are unaltered by Pde8b
inactivation (). However, we did observe selective enhancement of context-dependent fear, spatial memory, performance in an instrumental conditioning task, and motor coordination (summarized below) indicating that some hippocampal-, striatal-, and cerebellar-dependent behaviors are enhanced following Pde8b
inactivation. These findings are consistent with several of the well-known functions of cAMP signaling in these brain regions.
Summary of behavioral analysis of PDE8B KO mice
Contextual and spatial memory is enhanced in PDE8B KO mice
appears most highly expressed in the dentate granule cell layer and CA1 pyramidal cell layers of the hippocampus (). These hippocampal subdivisions have been demonstrated to be critical in contextual memory recall and spatial pattern separation (Bourtchuladze et al. 1994
; Rolls & Kesner 2006
). Therefore, we assessed whether Pde8B inactivation would alter contextual fear or spatial memory using either contextual fear conditioning paradigm or Morris water maze.
To determine if genetic inactivation of Pde8b
alters contextual fear conditioning, we assayed freezing behavior in WT control and PDE8B KO mice, as described (Athos et al. 2002
). Briefly, mice were conditioned with a single foot shock (0.7 mA, 2 sec) in a novel context and assayed for context-induced freezing 24 hours later in the same context. PDE8B KO mice exhibited an increase in freezing time compared to WT littermate controls (; t-test, t21
=2.24, p<0.05). Increased freezing behavior was not due to altered sensitivity to foot shock (), nor to mobility (data not shown). To determine whether enhanced freezing in PDE8B KO mice is context specific, we monitored freezing behavior in separate cohorts of control and PDE8B KO mice that were conditioned in a context distinct from the testing apparatus. There was no difference between the two groups (WT, 17.58 ± 1.68 vs. KO, 21.61 ± 3.59; t-test, t20
Contextual and spatial memoty is enhanced in PDE8B KO relative to WT mice
To address the specificity of the enhancement in contextual fear conditioning in PDE8B KO mice, we utilized a delayed cue fear-conditioning task which is less dependent on the hippocampus and more dependent upon the amygdala (Phillips & LeDoux 1992
). Mice were conditioned with a single pairing of a 30 second auditory cue (90–100 dB) that co-terminated with foot shock (0.7 mA, 2 sec). Cue-evoked freezing was assessed in a context independent of the conditioning chamber 24 hours later. Learning in this task was indistinguishable between PDE8B KO and WT control mice (; t-test, t20
Next, we investigated whether functional PDE8B activity might be important in spatial memory using a Morris water maze. Mice were given three training trials per day for 2–3 days to learn the location of a hidden platform. Mice were tested 24 hours following the last conditioning day with the hidden platform removed to assess memory. During training there was no difference between PDE8B KO and control mice in escape latencies, or swim speed ( and data not shown; acquisition, two-way repeated measures ANOVA: genotype effect, F1,23=1.27, p=0.27; effect of trial, F5,115=18.31, p<0.001).
During the probe test, PDE8B KO mice made significantly more entries into the target area (NE quadrant) than controls (; two-way repeated measures ANOVA, genotype X quadrant interaction, F3,69=3.28, p<0.05). Increased frequency of entry into the target area was associated with a trend towards increased time spent in the target quadrant; though this measure did not reach statistical significance (; two-way repeated measures ANOVA: genotype X quadrant interaction, F3,69=1.52, p=0.22). No differences in the latency to first cross (WT, 22.93 ± 6.59 vs. KO, 19.44 ± 4.42, t-test: t23=0.44, p=0.67) or frequency of crossings (WT, 3.92 ± 0.81 vs. KO, 5.08 ± 0.43, t-test: t23=1.29, p=0.10) of previous platform location were observed.
We next performed a remote memory test on cohorts of mice distinct from those described above. Mice were trained to find the hidden platform as above, except all mice received 4 days of conditioning. Analysis of acquisition revealed a significant effect of trial (; two-way repeated measures ANOVA: trial, F11,198=22.01, p<0.001), no genotype effect was detected (two-way repeated measures ANOVA: trial, F1,18=2.29, p=0.14). Similar to the probe test performed 1 day following conditioning described above, a probe test 10 days following conditioning revealed a significant genotype x quadrant interaction on the frequency of quadrant entries (, two-way repeated measures ANOVA: genotype x quadrant, F3,57=4.22, p<0.01). Again, this increased frequency was associated with a trend towards increased time spent in the target quadrant by PDE8B KO mice (; two-way repeated measures ANOVA: genotype X quadrant interaction, F3,57=2.5, p<0.07). No differences in the latency to first cross (WT, 68.20 ± 17.45 vs. KO, 42.39 ± 11.07, t-test: t19=1.27, p=0.10) or frequency of crossings (WT, 4.30 ± 1.42 vs. KO, 7.27 ± 1.76, t-test: t19=1.30, p=0.10) of the previous platform location were observed.
Both PDE8B KO and control mice appeared to take longer to first cross the previous platform location in the remote memory test compared to the 1 day probe test (WT 1 day, 22.93 ± 6.59 vs. WT remote, 68.20 ± 17.45; KO 1 day, 19.44 ± 4.42 vs. KO remote, 42.39 ± 11.07). Statistical analysis across all groups revealed a significant difference in control mice during the remote memory test compared to the 1 day test, this was not significant in PDE8B KO mice (one-way ANOVA: F3 =4.62, p<0.01; Bonferroni’s Multiple Comparisons test, WT remote vs. WT 1 day, p<0.05 and WT remote vs. KO 1 day, p<0.01).
Appetitive instrumental behavior is enhanced in PDE8B KO mice
Analysis of β-gal activity in PDE8B KO mice demonstrated a high level of enrichment of Pde8b expression in the ventral striatum. Because PDEs are important for hydrolysis of cAMP generated in response to dopamine D1 receptors in the striatum and this signaling is important for reward learning and motivated behavior, we hypothesized that appetitive instrumental learning, motivation, or both might be altered in PDE8B KO mice. First, we tested PDE8B KO and WT control mice in a fixed ratio (FR) schedule in which a single lever press resulted in delivery of a reward pellet (FR1). Two levers were activated at the start of each trial signaled by the illumination of the chamber house light. Following a successful trial the house light was turned off, the pellet was dispensed, and the levers where inactivated; six seconds later the house lights were re-illuminated and the levers were re-activated. Both active and inactive lever presses where recorded over 50 trials or for 1 hour, whichever came first. Total lever presses per day did not differ significantly between groups (; two-way repeated measures ANOVA: genotype X day interaction, F6,144=0.74, p=0.62); however, there was a trend towards an overall effect of genotype (two-way repeated measures ANOVA: genotype F1,167=3.62, p=0.059).
PDE8B KO mice have better performance in a simple instrumental conditioning task than WT mice
To determine the precision of lever pressing behavior we scored non-rewarded lever presses across days. Analysis of non-rewarded lever presses revealed an overall genotype effect (; two-way repeated measures ANOVA: genotype, F1,167=5.55, p=0.02), with PDE8B KO mice making more non-rewarded presses, particularly during the first days of training; however, no significant genotype X day interaction was detected (two-way repeated measures ANOVA: genotype X day interaction, F6,144=2.01, p=0.06).
Following the fourth day of training all mice were completing 50 lever presses within the 1 hour time window; moreover, total lever presses and total non-rewarded lever presses were equivalent indicating both groups were equally precise in their performance (, days 5–7). Intriguingly, PDE8B KO were faster at completing the task at this later stage compared to control mice (, days 5–7). To elucidate further the extent to which PDE8B KO mice are performing better than control mice we analyzed cumulative reward acquisition on days 5–7. PDE8B KO mice performed significantly better than controls on all three days (; two-way repeated measures ANOVA: day 5, genotype X time, F29,725=3.47, p<0.0001; day 6, genotype X time, F29,725=8.86, p<0.0001; day 7, genotype X time, F29,725=5.22, p<0.0001).
To assess whether increased performance in instrumental conditioning by the PDE8B KO mice reflects an enhancement of motivated behavior, we tested PDE8B KO and control mice in a progressive ratio task. Break point analysis, as assessed by the total lever processes in the last trial completed, did not differ between PDE8B KO and control mice (). Finally, to determine whether differences in performance between PDE8B KO and WT mice are due to differences in instrumental learning, we performed a two-lever discrimination task. Mice were given block-training sessions for 6 days to associate a cue light above one of two levers with the active lever. Block trials of 10 light-lever pairings were given on each lever. On day seven mice were assayed for two-lever discrimination based on their ability to discriminate which of the two levers were active; active levers were assigned pseudo randomly. No difference was observed between control and PDE8B KO mice, with both groups performing at or above 80% accuracy ().
Age-induced decay of motor performance is attenuated in PDE8B KO mice
Expression of Pde8b
in the cerebellum, together with previous observations linking Pde8b
to ADSD, a progressive motor degenerative disorder, suggests that Pde8b
inactivation may be associated with altered motor coordination (Appenzeller et al. 2010
; Perez-Torres et al. 2003
). To investigate this further, we monitored latency to fall from a rotating rod using an escalating rotarod task in both young () and aged () adult mice. Analysis of young adult mice revealed a significant effect of training (two-way repeated measures ANOVA: trial, F8,96
=8.00, p<0.001), but not genotype (two-way repeated measures ANOVA: genotype, F1,12
=4.16, p=0.06). In contrast, Pde8b
inactivation had a significantly increased latency to fall in aged adult mice (two-way repeated measures ANOVA: genotype, F1,8
=17.91, p=0.003). Analysis of average latency to fall across all groups revealed a significant decrease in performance in aged WT mice compared to young adult mice that was not observed in PDE8B KO mice (, ANOVA, F3
= 37.42, followed by Bonferroni’s Multiple Comparisons Test: WT vs WT aged p<0.01). Additionally, this analysis revealed a significant difference in young adults between WT and PDEB KO mice (Bonferroni’s Multiple Comparisons Test: WT vs PDE8B KO p<0.05).
Age-induced decline in rotarod performance is abrogated in PDE8B KO mice compared to WT mice
Anxiety-like behavior is increased in PDE8B KO mice
Improved memory in contextual fear conditioning and a spatial learning task, along with improved performance in instrumental conditioning and motor coordination suggests that suppression of PDE8B activity overall improves cognitive and motor abilities. Given that alterations in the accumulation of cAMP are linked to numerous psychiatric disorders and the desire to identify PDEs for targeted therapeutic treatment of mood disorders (Xu et al., 2011
), we asked whether genetic inactivation of Pde8b
is associated with changes in affective state. To address this we monitored anxiety-like behavior using two standard laboratory methods, open field and elevated plus maze. Due to the difficulty in detecting increased anxiety levels in these tests under normal lighting conditions, we utilized low-light testing conditions which result in significantly higher percentages of time spent in the center of the open field arena and the open arms of the elevated plus maze (Bruchas et al. 2009
). PDE8B KO mice spent significantly less time in the center of the arena than WT controls (; t-test, t9
=2.41, p<0.05). Similarly, PDE8B KO mice spent significantly more time in the closed arms of elevated plus maze than the open arms (), contrary to what was observed in WT control mice (PDE8B KO; closed vs. open arms, t-test, t8
PDE8B KO mice have increased basal anxiety levels compared to WT mice, but do not display alterations in sensory-motor gating as assessed by acoustic startle and PPI
Alterations in cAMP levels are associated with perturbations of sensory motor gating, such as those measured by prepulse inhibition (PPI) of the acoustic startle reflex, which is a frequent endophenotype of psychiatric disorders such as schizophrenia (Clapcote et al. 2007
). To determine whether Pde8b
inactivation alters PPI, we first monitored acoustic startle responses (ASR) across multiple startle pulse intensities to determine whether baseline differences in ASR exist between PDE8B KO and WT control mice. ASR curves between the two groups were indistinguishable (, two-way repeated measures ANOVA: genotype effect, F1,10
=0.03, p=0.86). To monitor PPI, we measured acoustic startle at 120 dB in the presence or absence of 70, 75, and 80 dB prepulses. No significant differences in PPI between PDE8B KO and WT control mice were detected (, two-way repeated measures ANOVA: genotype effect, F1,12