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Phosphodiesterases (PDEs) are a superfamily of intracellular second messenger cyclic nucleotide hydrolyzing enzymes composed of 12 families. The Pde4 family has been implicated in depression and cognition, and PDE4 inhibitors have been evaluated as antidepressants and possible cognitive enhancers. Pde4d−/− mice show an antidepressant phenotype and learning enhancement on some tests, but not others as do mice treated with PDE4 inhibitors. Here, we report for the first time the behavioral phenotype of a new Pde4d knock-down (KD) rat model of PDE4D deficiency. Consistent with other data on PDE4D deficiency, Pde4d KD rats showed depression resistance in the Porsolt forced swim test and hyperreactivity of the acoustic startle response with no differential response on prepulse inhibition, suggesting no sensorimotor gating defect. Pde4d KD rats also exhibited a small exploratory activity reduction but no difference following habituation, and no enhanced spatial learning or reference memory in the Morris water maze. A selective improvement in route-based learning in the Cincinnati water maze was seen as well as enhanced contextual and cued fear conditioning and a more rapid rate of cued extinction from their higher freezing level that declined to wild-type (WT) levels only after ∼20 extinction trials. The rat model confirms Pde4d's role in depression but not in spatial learning or memory enhancement and shows for the first time higher fear conditioning and altered extinction compared with controls. The new model provides a tool by which to better understand the role of PDE4D in neuropsychiatric disorders and for the development of alternate treatment approaches.
Phosphodiesterases (PDEs) are a superfamily of intracellular second messenger cyclic nucleotide hydrolyzing enzymes consisting of 12 families (Conti et al. 2003; Houslay 2001). Each family has isoforms and some have posttranscriptional splice variants (Houslay 2001). PDE4A and 4D are expressed in cortex, olfactory bulb, hippocampus and brainstem (area postrema and nucleus tractus solitarius) (Perez-Torres et al. 2000). PDE4A, B and D are expressed in neurons, whereas 4C is not (Zhang 2009). PDEs are regulators of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) signaling and are implicated in some neurological and neuropsychiatric disorders (Siuciak 2008). Accordingly, they have become potential therapeutic targets (Siuciak 2008; Zhang 2009). PDE4 inhibitors such as rolipram show preclinical and clinical antidepressant efficacy (Li et al. 2009; Zhang et al. 2002; Zhang 2009), and preclinical evidence as cognitive (Burgin et al. 2010; Zhang 2009) and noradrenergic enhancers (Nishi et al. 2008). The PDE4s are cAMP-specific and act via protein kinase A (PKA)-phophoso-cyclic AMP-response binding (pCREB) protein and related cascades (Zhang 2009). Chronic treatment with PDE4 inhibitors increases brain-derived neurotrophic factor (BDNF) and neurogenesis, as do established antidepressants. Although PDE4 inhibitors show antidepressant efficacy clinically (Bobon et al. 1988; Fleischhacker et al. 1992; Hebenstreit et al. 1989; Zeller et al. 1984), they have problematic side effects (nausea) (Robichaud et al. 2001, 2002a,b).
Pde4d −/− mice (Dlaboga et al. 2006; Hansen et al. 2000; Jin et al. 1999; Li et al. 2011; Zhang et al. 2002, 2008) and rolipram-treated mice and rats show some overlapping effects. Rolipram-treated and Pde4d−/− mice show increased time in the target quadrant on Morris water maze (MWM) probe trials, increased novel object recognition (NOR) and increased passive avoidance latency. MicroRNA (miRNA)-induced Pde4d inhibition induces effects similar to those seen in Pde4d−/− and rolipram-treated mice (Li et al. 2011). Pde4d−/− mice also show antidepressant effects on the tail suspension test (Zhang et al. 2002) and forced swim test (FST) (Zhang et al. 2002, 2008; Zhang 2009) and rolipram-treated rats show increased responding on a differential reinforcement of low rates of response (DRL) schedule (Zhang et al. 2006). In addition, PDE4 inhibitors (rolipram and HT0712) are able to reverse novel object memory deficits in CREB-binding protein-deficient (CBP+/−) mice (Bourtchouladze et al. 2003), and rolipram reverses MK-801-induced radial arm and passive avoidance memory deficits (Zhang et al. 2000), and reverses MEK-ERK (MAP-ERK kinase-extracellular-related kinase) inhibitor-induced radial arm maze (RAM) memory deficits (Zhang et al. 2004).
There are also inconsistencies. Pde4d−/− mice show no differences in fear conditioning but show impaired retention at 24 h after unconditioned stimulus-conditioned stimulus (US-CS) pairing (Rutten et al. 2008). In the MWM, rats treated with the PDE4 inhibitors rolipram or DC-TA 46 show impaired probe trial performance with no differences on hidden or visible platform learning (Giorgi et al. 2004) in contrast to knock-out (KO) mice (Li et al. 2011). Pde4d−/− mice show no change in working memory in the RAM and fewer late (but not early) reference memory errors (Li et al. 2011). PDE4 inhibitors induce anxiolytic effects in rats (Silvestre et al. 1999) but no change (Imaizumi et al. 1994) or anxiogenic effects in mice (Zhang et al. 2008) and dogs (Heaslip & Evans 1995).
To address such differences, an alternate model was developed: a new PDE4D genetically modified rat, F344-Pde4dTn(sb-T2/Bart3)2.285Mcwi.
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- Materials and methods
The PDE4 family has been implicated in depression and multiple lines of evidence support this role. These include studies in mice using gene deletion, enzyme inhibitors and miRNA to KD the protein. Pde4d−/− mice show an antidepressant phenotype in the tail suspension test (Zhang et al. 2002) and FST (Zhang et al. 2002, 2008; Zhang 2009). Treatment of WT mice with PDE4 inhibitors rolipram, piclamilast, CPD840, MEM1018 and MEM1091 induced an antidepressant phenotype in the FST, but only rolipram and piclamilast decreased responses and increased reinforcement rates in an operant DRL paradigm (Zhang et al. 2006). In the present experiment, using an entirely different model of PDE4D deficiency, the Pde4d KD F344 rat, we confirmed an antidepressant phenotype in the FST. Hence, across two species, two genetic models and multiple pharmacological models, PDE4D reduction reliably induces immobility resistance, which is predictive of antidepressant activity and related changes in DRL testing. Moreover, human trials of PDE4 inhibitors show clinical antidepressant efficacy; however, this is accompanied by the complication of nausea which makes these drugs unacceptable until further molecular differentiation of the structure of such inhibitors can be designed to eliminate this untoward effect. Hence, the rat Pde4d-deficient model provides convergent evidence that PDE4D is a potentially important pharmacotherapeutic target that represents a new class of antidepressants that would benefit some of the many patients refractory to the therapeutic effects of existing antidepressants.
The Pde4d−/− mouse also shows evidence of cognitive enhancement. For example, these mice have been shown to exhibit greater target quadrant preference on memory trials in the MWM, increased preference for a new object over a familiar one in the NOR test, increased latency to remain on an elevated platform in the step-down inhibitory avoidance test and fewer reference memory errors for constantly unbaited arm entries on the last 2 days out of 14 in the RAM with no differences on trial-dependent performance in the daily constantly baited arms in an 8-arm apparatus; several of these effects were also seen in rolipram-treated mice (Li et al. 2011). On the other hand, another study showed no differences in Pde4d−/− mice on another type of learning, fear conditioning (Rutten et al. 2008), and rats treated with rolipram show impaired MWM learning and reference memory on probe trials (Giorgi et al. 2004). For the present data in the genetic Pde4d KD rat, we find a mixed pattern of cognitive effects. We tested the MWM more extensively than in any of the previous studies, testing the animals in three progressively more challenging phases of the task, acquisition with a 10-cm platform, reversal with a 7-cm platform and shift with a 5-cm platform. In addition, we used a maze that was proportionately more challenging than that used in the mouse and rat studies (Giorgi et al. 2004; Li et al. 2011). The mouse pool was 95 cm in diameter with an 8.5 × 15.5-cm platform for a search area ratio of 53.8:1. Moreover, impaired probe performance in rats treated with PDE4D inhibitors was observed in the MWM in a 180-cm pool with a 13 × 15-cm platform for a search area ratio of 130.5:1. By contrast, we used a 210-cm diameter pool with a 10-cm diameter platform during acquisition for a search area ratio of 441:1, and this ratio became even larger on reversal (900:1), and still larger on shift (1764:1). Despite this, we found no differences on any phase of the hidden platform learning trials. Nor did we find any reference memory change on probe trials given 24 h after each of the three learning phases for average distance to the platform site, crossovers or percent time or percent distance in the target quadrant. Hence, we found no evidence of enhanced or impaired spatial learning or spatial reference memory.
As mentioned, one study in Pde4d−/− mice showed no changes in 1-h contextual or cued fear conditioning, but showed impaired conditioning at 24 h in both contextual and cued fear (Rutten et al. 2008). In the present experiment with Pde4d KD rats, we did not conduct a 1-h fear conditioning test and tested at 24 h for contextual fear conditioning and at 48 h for cued fear conditioning. In contrast to Pde4d−/− mice, Pde4d KD rats showed enhanced contextual fear conditioning 24-h post-training. During the 48-h cued conditioning, Pde4d KD rats showed only a trend on the first retention trial. The most striking finding in the Pde4d KD rats was on subsequent cued extinction trials, in which the KD group showed higher percent time spent freezing with a prolonged retention of the immobility response compared with WT. However, the rate of extinction from the first to the last trial was greater in the KD group than in the WT group, requiring 20 trials to decline to WT levels. Hence, the issue of whether PDE4D enhances or impairs conditioned fear is not easily resolved, but the present data support enhancement of conditioning with a prolonged extinction of the response. This may have implications for the way PDE4D inhibitors might affect patients with depression but the precise clinical implications cannot be predicted based on this finding.
No other data are available on route-based egocentric learning after PDE4D reduction. In the present experiment, we tested this form of learning using the CWM. We did not find an overall change in route-based learning in the absence of distal cues in Pde4d KD rats but we did see fewer errors in the midportion of the learning curve and this effect was significant. In this midrange, the Pde4d KD rats learned to eliminate errors at a faster rate than WT, although WT rats later caught up and the groups ended the test at the same performance level.
We also found Pde4d KD rats to be slightly less exploratory in an open field during the first 5 and first 30 min but not after they had become fully habituated to the apparatus during the last 30 min of the 60-min test period. Pde4d−/− mice also show reduced open-field activity during a 5-min exploration test (Zhang et al. 2008). However, a subsequent experiment did not replicate this finding and showed no differences in 5 min of open-field exploration in Pde4d−/− mice (Li et al. 2011). Because the mouse studies did not test for longer periods, further comparisons on locomotor exploration and habituation are not possible.
We also found that Pde4d KD rats had a marked enhancement of the acoustic startle reflex with no indication that PPI was differentially affected. Although the magnitude of the startle facilitation effect observed in the Pde4d KD group was reduced in the presence of the 70-dB prepulse compared with no prepulse, and further reduced in the presence of the 76-db prepulse, further analysis in terms of percent inhibition revealed no differential response, i.e. the WT group showed a 48.4% startle inhibition at 70 dB vs. the Pde4d KD group that showed a 49.7% inhibition at this prepulse level. At the 76 dB prepulse level, the WT group showed a 61.8% startle inhibition and the Pde4d KD group a 61.7% startle inhibition; essentially identical degrees of inhibition. This indicates that despite the Pde4d KD rats being hyperreactive to the startle signal, they do not show a deficit in sensorimotor gating.
The Pde4d KD rat shows a clear behavioral phenotype and this should prove useful in research to further understand the role of PDE4D in brain function, especially in depression. This new model may also be valuable for understanding the role of PDE4D in the retention of conditioned fear responses that may have therapeutic value when used appropriately. While the Pde4d KD rat is not a complete gene deletion, it should nonetheless be a valuable model in further analyses of this protein and can be used in conjunction with the Pde4d−/− mouse and pharmacological inhibitors to more fully elucidate how this important enzyme may be manipulated for treatment purposes.