A negative allosteric modulator of PDE4D enhances learning after traumatic brain injury

Abstract

Traumatic brain injury (TBI) significantly decreases cyclic AMP (cAMP) signaling which produces long-term synaptic plasticity deficits and chronic learning and memory impairments. Phosphodiesterase 4 (PDE4) is a major family of cAMP hydrolyzing enzymes in the brain and of the four PDE4 subtypes, PDE4D in particular has been found to be involved in memory formation. Although most PDE4 inhibitors target all PDE4 subtypes, PDE4D can be targeted with a selective, negative allosteric modulator, D159687. In this study, we hypothesized that treating animals with D159687 could reverse the cognitive deficits caused by TBI. To test this hypothesis, adult male Sprague Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury. After 3 months of recovery, animals were treated with D159687 (0.3 mg/kg, intraperitoneally) at 30 min prior to cue and contextual fear conditioning, acquisition in the water maze or during a spatial working memory task. Treatment with D159687 had no significant effect on these behavioral tasks in non-injured, sham animals, but did reverse the learning and memory deficits in chronic TBI animals. Assessment of hippocampal slices at 3 months post-TBI revealed that D159687 reversed both the depression in basal synaptic transmission in area CA1 as well as the late-phase of long-term potentiation. These results demonstrate that a negative allosteric modulator of PDE4D may be a potential therapeutic to improve chronic cognitive dysfunction following TBI.

Keywords: Cognition; Fluid Percussion Injury; Hippocampus; Memory; Phosphodiesterase; Traumatic brain injury.

Fig. 1

D159687 treatment did not alter cognitive functioning or hippocampal synaptic plasticity in the non-injured, sham animals. (A) Timeline of behavioral testing. Animals received vehicle or D159687 (0.3 mg/kg, i.p., arrows) at 30 min prior to training for fear conditioning (Fear cond), water maze acquisition, spatial working memory (Work mem) and shock threshold assessment (Shock thresh). Retention in the water maze and fear conditioning (FC ret) were assessed without drug treatment. D159687 treatment had no significant effects in non-injured, sham animals on (B) contextual and cued fear conditioning, (C) water maze acquisition, (D) quadrant preference during the water maze probe trial, (E) escape latency to match the location in a spatial working memory task, (F) hippocampal fEPSP I-O curves in area CA1, (G) hippocampal LTP and (H) fEPSP responses between 170–180 min after LTP induction. Mean±SEM, Sham+Vehicle n=6, Sham+D159687 n=8 panels B–F, Sham+Vehicle n=8, Sham+D159687 n=6 panel F, Sham+Vehicle n=4, Sham+D159687 n=3 panels G and H.

Fig. 2

D159687 treatment rescued contextual and cue fear conditioning deficits in TBI animals at 3 months post-surgery. (A) Contextual and cue recent recall at 24 hr after training. TBI animals treated with vehicle froze significantly less than sham animals or TBI animals treated with D159687. No significant differences were observed in baseline freezing on the training day. ***p<0.001 vs Training, ^bp<0.01, ^cp<0.001 vs TBI+Vehicle, repeated measures two-way ANOVA with Tukey’s HSD correction. (B) Contextual and cue remote recall at 1 month after training. The deficits in contextual and cue recall were persistent in TBI+Vehicle animals as compared to sham or TBI+D159687 animals. ^ap<0.05, ^bp<0.01 vs TBI+Vehicle, one-way ANOVA with Tukey’s HSD correction. Mean±SEM, Sham n=14, TBI+Vehicle n=10, TBI+D159687 n=10.

Fig. 3

D159687 improved spatial reference memory in TBI animals. (A) Path length to reach the hidden platform during water maze acquisition. TBI+Vehicle animals took a longer path length to find the platform as compared to sham or TBI+D159687 animals. ***p< 0.001, repeated measures two-way ANOVA with Tukey’s HSD correction. (B) Path length to find the platform on the fourth day of water maze acquisition. **p<0.01, ***p<0.001 vs TBI+Vehicle, one-way ANOVA with Tukey’s HSD correction. (C) Time spent in each quadrant during the probe trial. TBI+Vehicle animals spent significantly less time in the target quadrant as compared to sham or TBI animals treated with D159687. ***p<0.001 vs TBI+Vehicle in the target quadrant, ^cp<0.001 vs other quadrants, two-way ANOVA with Tukey’s HSD correction. (D) Spatial working memory was significantly improved in TBI+D159687 animals as compared to TBI+Vehicle animals. **p<0.01, repeated measures two-way ANOVA with Tukey’s HSD correction. Mean±SEM, Sham n=14, TBI+Vehicle n=10, TBI+D159687 n=10.

Fig. 4

The PDE4D inhibitor D159687 reversed the deficits in basal synaptic transmission and late phase of LTP in area CA1 of the hippocampus at 3 months after TBI. (A) I-O responses were significantly reduced in hippocampal slices from TBI animals treated with vehicle as compared to sham animals or TBI animals treated with D159687 (100 nM). *p<0.05, ***p<0.001 vs TBI+Vehicle, ^ap<0.05 vs Sham, repeated measures two-way ANOVA with Tukey’s HSD correction. Sham n=14 slices/13 animals, TBI+Vehicle n=7 slices/6 animals, TBI+D159687 n=6 slices/6 animals. (B) Expression of LTP induced by high frequency tetanization (arrows, 4 trains of 1×100 Hz for 1 sec, separated by 5 min). Deficits in the late phase of LTP after TBI were rescued by treatment with D159687 (bar). *p<0.05 vs Sham, repeated measures two-way ANOVA with Tukey’s HSD correction. Sham n=7 slices/7 animals, TBI+Vehicle n=6 slices/6 animals, TBI+D159687 n=5 slices/5 animals. (C) Average of fEPSP slopes from 0–5, 50–60 and 170–180 min post-tetanization. *p<0.05, **p<0.01 vs TBI+Vehicle, one-way ANOVA with Tukey’s HSD correction. (D) Paired-pulse facilitation ratio was not significantly altered after TBI or D159687 treatment. Mean±SEM. Sham n=10 slices/10 animals, TBI+Vehicle n=6 slices/6 animals, TBI+D159687 n=6 slices/6 animals.

Fig. 5

Cortical and hippocampal atrophy at 5 months post-surgery. (A) Representative sections stained with H&E plus Luxol fast blue at bregma level −3.0 mm. Scale bar 1 mm. (B) Cortical atrophy was significantly increased in TBI animals treated with vehicle or D159687. (C) Significant hippocampal atrophy was observed in vehicle-treated TBI animals. A trend for hippocampal atrophy was observed in D159687-treated TBI animals (p=0.0551). *p<0.05, ***p<0.001 vs Sham, one-way ANOVA with post-hoc Tukey’s HSD correction. Mean±SEM, Sham n=14, TBI+Vehicle n=10, TBI+D159687 n=10.

Fig. 6

PDE4D expression in Cd11b⁺/CD45⁺ microglia at 3 months after TBI. Representative flow cytometry scatter plots from the ipsilateral (A, B) parietal cortex and (E, F) hippocampus of sham and TBI animals. Total microglia cell numbers significantly increased in the (C) parietal cortex, but not in the (G) hippocampus. PDE4D expression was significantly decreased in the microglia cell population in the (D) parietal cortex, but not in microglia within the (H) hippocampus. *p<0.05, **p<0.01 vs Sham, Student’s unpaired t-test. Mean ± SEM, n=3/group.

Fig. 7

cAMP levels were increased with D159687. (A) Treatment of naïve animals with D159687 at 3 mg/kg (i.p.) significantly increased cAMP levels in the parietal cortex and hippocampus. At the dose used in the behavior studies, 0.3 mg/kg D159687 also significantly increased cAMP levels in the hippocampus, but not in the parietal cortex. *p<0.05, **p<0.01 vs Vehicle, one-way ANOVA with Tukey’s HSD correction. Mean ± SEM, n=8/group. (B) Treatment of TBI animals with D159687 at 0.3 mg/kg (i.p.) significantly increased cAMP levels in the parietal cortex as compared to vehicle-treated TBI animals. *p<0.05 vs Vehicle, one-way ANOVA with Tukey’s HSD correction. Mean ± SEM, Sham+Vehicle n=4, TBI+Vehicle n=3, TBI+D159687 n=4.