Inhibition of phosphodiesterase 2 increases neuronal cGMP, synaptic plasticity and memory performance

Neuropharmacology 47 (2004) 1081–1092 www.elsevier.com/locate/neuropharm

Inhibition of phosphodiesterase 2 increases neuronal cGMP, synaptic plasticity and memory performance Frank G. Boess a,1, Martin Hendrix b,
, Franz-Josef van der Staay a,2, Christina Erb a,3, Rudy Schreiber a,4, Wilma van Staveren c, Jan de Vente c, Jos Prickaerts c,5, Arjan Blokland d, Gerhard Koenig a,6 b

a Bayer Healthcare AG, Pharma Research CNS, 42096 Wuppertal-Elberfeld, Germany Bayer Healthcare AG, Pharma Research-Chemical Research, Forschungszentrum Aprather Weg. D-42096 Wuppertal, Germany c Department of Psychiatry and Neuropsychology, University of Maastricht, 6200 MD Maastricht, The Netherlands d Department of Neurocognition, University of Maastricht, 6200 MD Maastricht, The Netherlands

Received 23 March 2004; received in revised form 2 June 2004; accepted 28 July 2004

Abstract An essential element of the signalling cascade leading to synaptic plasticity is the intracellular second messenger molecule guanosine 30 ,50 -cyclic monophosphate (cGMP). Using the novel, potent, and selective inhibitor Bay 60-7550, we show that the enzyme 30 ,50 -cyclic nucleotide phosphodiesterase type 2 (PDE2) is responsible for the degradation of newly synthesized cGMP in cultured neurons and hippocampal slices. Inhibition of PDE2 enhanced long-term potentiation of synaptic transmission without altering basal synaptic transmission. Inhibition of PDE2 also improved the performance of rats in social and object recognition memory tasks, and reversed MK801-induced deficits in spontaneous alternation in mice in a T-maze. Our data provide strong evidence that inhibition of PDE2 can improve memory functions by enhancing neuronal plasticity. # 2004 Elsevier Ltd. All rights reserved. Keywords: Phosphodiesterase 2; PDE2; cGMP; Long-term potentiation; LTP; Memory; Learning

1. Introduction The nitric oxide/guanosine 30 ,50 -cyclic monophosphate (NO/cGMP) signal transduction pathway has an important role during synaptic plasticity. Nitric oxide
Corresponding author. Tel.: +49-202-365288; fax: +49-202358149. E-mail address: [email protected] (M. Hendrix). 1 Present address: Neuroscience Discovery Research, Eli Lilly and Company Limited, Lilly Research Centre, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK. 2 Present address: ID Lelystad, Institute for Animal Science and Health BV, Lelystad 8200 AB, The Netherlands. 3 Present address: Merz GmbH, 60318 Frankfurt, Germany. 4 Present address: Roche Pharmaceuticals, Palo Alto, CA 94304, USA. 5 Present address: Johnson & Johnson PRD, B2340 Beerse, Belgium. 6 Present address: Envivo Pharmaceuticals, Cambridge, MA 02139, USA.

0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.07.040

synthase (NOS), the enzyme that generates NO, is activated by Ca2+-influx through glutamate receptors of the N-methyl-d-aspartate (NMDA)-type on postsynaptic neurons (Garthwaite, 1991). NO stimulates both post- and presynaptic guanylyl cyclase (GC), leading to the formation of the second messenger cGMP (Boulton et al., 1995; De Vente and Steinbusch, 1992). In turn, cGMP directly opens cyclic nucleotide-gated ion channels (Savchenko et al., 1997) and activates cGMP-dependent protein kinase (PKG), which phosphorylates synaptic proteins (Wang and Robinson, 1997). Inhibitors of NOS, GC, or PKG block longterm potentiation (LTP) of hippocampal synaptic transmission, a physiological correlate of learning and memory processes (Bo¨hme et al., 1991; O’Dell et al., 1991; Schuman and Madison, 1991; Zhuo et al., 1994; Boulton et al., 1995; Lu et al., 1999). Thus, NO may serve as a retrograde signal that closes an important feedback loop during use-dependent synaptic plasticity

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in the hippocampus (reviewed by Collingridge and Bliss, 1995; Hawkins et al., 1998). While marked reductions in LTP have been demonstrated in knock-out mice that lack both endothelial and neuronal NO synthase (Son et al., 1996), the role of NO as a retrograde messenger in LTP is controversial (discussed in Holscher, 1997; Hawkins et al., 1998). Changes in the strength of the postsynaptic response during LTP as a consequence of the activation of cAMP-dependent protein kinase (PKA) and calcium/calmodulin-dependent kinase include increased glutamate receptor density and function (reviewed by Lynch, 2004). Several substrate proteins of these kinases are also phosphorylated by PKG which could result in similar changes in postsynaptic function through the NO/cGMP/PKG pathway (Wang and Robinson, 1997; Ahem et al., 2002). NOS inhibitors impair memory performance in onetrial passive avoidance in chicks (Holscher and Rose, 1993) and spontaneous alternation in mice (Yamada et al., 1996). Local injection of cGMP analogues can ameliorate the deficit induced by NOS-inhibition (Yamada et al., 1996). Intra-hippocampal injections of 8-Br cGMP improved the ability of rats to remember objects (Prickaerts et al., 2002a), as well as retention performance in a passive avoidance task (Bernabeu et al., 1996). Furthermore, cGMP levels in the hippocampus of rats increased transiently during the first 30 min of a passive avoidance task (Bernabeu et al., 1996). These findings support an essential role of cGMP in early memory formation. An important part of the signal transduction process is the rapid degradation of cGMP by cyclic nucleotide phosphodiesterases (PDEs). At least 21 PDE genes have been identified and subgrouped into 11 families (Soderling and Beavo, 2000; O’Donnell, 2000). PDE4, PDE7 and PDE8 hydrolyse cAMP, while PDE5, PDE6 and PDE9 hydrolyse cGMP; PDE1, PDE2, PDE3, PDE10 and PDE11 can hydrolyse both cAMP and

cGMP. The strong expression of PDE2 in neurons of the hippocampus and cortex (Repaske et al., 1993) suggests that this enzyme may control intraneuronal cGMP and cAMP levels in areas that are important for memory formation and storage. A characteristic feature of PDE2 is the positive cooperativity of the substrate cGMP—low levels of cGMP enhance the rate of cAMP hydrolysis by a factor of 5–6 (Martins et al., 1982). Under basal conditions, PDE2 activity in neurons is low, but it is stimulated by the acute increase in cGMP levels following GC activation during signal transduction. Therefore, inhibition of brain PDE2 may selectively increase cGMP and cAMP levels in active synapses and could thus influence synaptic plasticity and memory formation. We investigated the role of PDE2 in neuronal signal transduction and memory, using the highly potent and selective PDE2 inhibitor 2-(3,4-dimethoxybenzyl)-7{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methyllimidazo[5,1-f][1,2,4]triazin-4(3H)-one (Bay 60-7550). To this end, we determined the effects of Bay 60-7550 on cGMP levels in neurons in primary culture and in slices, on hippocampal LTP, and on cognitive function in rats and mice, assessed using an object recognition task, social memory, and a T-maze spontaneous alternation task.

2. Methods 2.1. Materials The novel PDE2 inhibitor 2-(3,4-dimethoxybenzyl)7-{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (Bay 60-7550) (Table 1) and the GC stimulator Bay 41-8543 (Stasch et al., 2002) were synthesized by the Chemistry Department of Pharma Research, Bayer AG (Wuppertal, Germany).

Table 1 Selectivity of Bay 60-7550 Enzyme

Source

Bay 60-7550 IC50 (nM)

n

Selectivity for PDE2

PDE2A PDE2 PDE1C PDE1 PDE3B PDE4B PDE5 PDE5A PDE7B PDE8A PDE9A PDE10A PDE11A

Human, recombinant Bovine, purified Human, recombinant Bovine, purified Human, recombinant Human, recombinant Human, purified Human, recombinant Human, recombinant Human, recombinant Human, recombinant Human, recombinant Human, recombinant

4:7  1:0 2:0  0:7 240  35 108  15 >4000 1830  840 583  60 704  148 >4000 >4000 >4000 940  400 >4000

7 12 7 12 3 3 12 9 3 2 2 3 2

50-fold 50-fold 800-fold 350-fold 100-fold 150-fold 800-fold 800-fold 800-fold 200-fold 800-fold

IC50 values are given as mean  standard error of the mean of n experiments.

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2.2. PDE inhibition and selectivity assays PDE2 was purified from bovine heart, PDE1 from bovine aorta and PDE5 from human platelets (Saenz de Tejada et al., 2001). Human recombinant PDEs were expressed in SF9 cells using the pFASTBAC baculovirus expression system. The IC50 of Bay 607550 at PDE2 was determined with [3H]-cAMP as radioactive substrate in the presence of 1 lM cGMP. Selectivity assays with PDE1 were performed in the presence of Ca2+ (3 mM) and Calmodulin (0.1 lM). [3H]-cAMP was used as substrate for PDE1, PDE3B, PDE4B, PDE7B, PDE8A and PDE10A; [3H]-cGMP for PDE5 and PDE9A. Test compounds were dissolved in DMSO and diluted in assay buffer (20 mM Tris/ HCl, 5 mM MgCl2, 0.1 mg/ml bovine serum albumin, pH 7,5; final DMSO concentration 0.1%). Reactions were carried out in duplicate in 96-well plates for v 15 min at 30 C in a final volume of 100 ll. PDE activity was measured using the [3H]-cAMP and [3H]-cGMP Scintillation Proximity Assay1 system (Amersham, Little Chalfont, England). Binding to human recombinant adenosine A1 receptors expressed in CHO cells and A2A receptors expressed in HEK293 cells was determined using 1 nM [3H]DPCPX (Libert et al., 1992) and 50 nM [3H]CGS21680 (Varani et al., 1996), respectively. Binding to the agonist binding site of GABAA receptors in rat brain membranes was performed using 1 nM [3H]muscimol (Martini et al., 1983) and binding to the benzodiazepine binding site was assayed using 1 nM [3H]flunitrazepam (Speth et al., 1979). Binding to AMPA-, kainate- and NMDA-type glutamate receptors in rat brain membranes was determined using 5 nM [3H]AMPA (Olsen et al., 1987), 5 nM [3H]kainic acid (London and Coyle, 1979) and 2 nM [3H]CGP-39653 (Sills et al., 1991), respectively. For all glutamate receptors, 1 mM l-glutamic acid was used to define non-specific binding. Effects on human recombinant histamine H1 and H3 receptors expressed in CHO-K1 cells were assessed using 1 nM [3H]pyrilamine (De Backer et al., 1993) and 3 nM [3H]R()-a-methylhistamine (Yanai et al., 1994), respectively. Human recombinant M1, M2, M3, M4 and M5 muscarinic acetylcholine receptors expressed in Sf9 cells were all assayed using 0.3 nM [3H]methylscopolamine and 1 lM atropine to determine non-specific binding (Buckley et al., 1989). Binding to human recombinant 5-hydroxytryptamine (5-HT) 5-HT3 receptors expressed in HEK-293 cells was determined using 0.7 nM [3H]GR-65630 (Boess et al., 1997b). Human recombinant 5-HT2A, 5-HT2B and 5-HT2C receptors expressed in CHO cells were assayed using 0.5 nM [3H]ketanserin, 1 nM [3H]lysergic acid diethylamide (LSD) and 1 nM [3H]mesulergine, respectively (Bonhaus et al., 1995; Wolf and Schutz, 1997). Binding to human recombinant 5-HT6 receptors

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expressed in Hela cells was determined using [3H]LSD (Boess et al., 1997a). The affinity for 5-HT4 receptors in guinea pig striatal membranes was determined using 0.7 nM [3H]GR-113808 (Grossman et al., 1993). Binding to human recombinant 5-HT1A receptors and 5HT7 receptors expressed in CHO cells was determined using 1.5 nM [3H]8-OH-DPAT (Martin and Humphrey, 1994) and 5.5 nM [3H]LSD (Shen et al., 1993), respectively. The effect on human recombinant acetylcholinesterase activity was tested by spectrophotometric quantitation of thiocholine generated from the substrate acetylthiocholine (Nadarajah, 1992) and on monoaminooxidase A and B (MAO-A and -B) activity using 4-hydroxyquinoline generated from the substrate kynuramine (Urban et al., 1991). The adenosine deaminase assay was conducted at different adenosine concentrations ranging from 3 to 100 lM in v 10 mM phosphate buffer, pH 7.4, at 25 C, in the presence of 0.9 U adenosine deaminase and a LineweaverBurk plot was constructed. EHNA was tested at 1, 3 and 10 nM, Bay 60-7550 at 10 lM. The Ki value for EHNA was calculated by plotting Km =Vmax versus inhibitor concentration. 2.3. cGMP and cAMP accumulation in neurons Rat and mouse primary cortical cells were cultured in 96-well plates (150 000 cells/well) as described previously (Suchanek et al., 1998). On day 3, 50% of the medium was replaced with fresh medium. On days 6–7, cells were washed once in HBS (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1.0 mM MgCl2, 5.6 mM glucose, 8.6 mM HEPES, pH 7.4) and incubated for v 20 min at 37 C (5% CO2) with Bay 60-7550 (in 100 ll HBS) in the presence or absence of the GC stimulator Bay 41-8543 or the adenylyl cyclase stimulator forskolin. Intracellular cGMP content was measured with an enzyme-linked immunosorbent assay system (cGMP and cAMP EIA Systems, Amersham). 2.4. Cyclic nucleotide levels and immunohistochemistry in hippocampal slices Hippocampus slices (400 lm) were prepared from male Wistar rats (aged 6–7 weeks, Charles River, Sevenum, The Netherlands) using a Campden Vibroslicer, as described previously (Van Staveren et al., 2001). Slices were incubated for 40 min with different concentrations of Bay 60-7550 in aerated Krebs-bicarbonv ate buffer (35.5 C). In the last 10 min, slices were incubated with or without the NO donor sodium nitroprusside (SNP, 0.1 mM). The slices were processed for cGMP-immunohistochemistry or cAMP and cGMP radioimmunoassays as described before. Data were analysed by ANOVA, supplemented with post-hoc comparisons (Student Newman Keuls test).

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2.5. LTP measurements Seven to eight-week-old male Wistar rats (Tierzucht Scho¨nwalde GmbH) were killed by a blow to the neck. After decapitation, the brain was quickly removed and placed in to ice-cold artificial cerebrospinal fluid (ACSF) with the following composition (in mM): NaCl 124, KCl 4.9, MgSO4 1.3, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25.6, d-glucose 10, saturated with 95% O2, 5% CO2, pH 7.4. Both hippocampi were isolated and transverse hippocampal slices (400 lm thickness) were prepared using a tissue chopper with a cooled stage. The slices were transferred into a submerged type recording chamber, where they were allowed to recover for at least one hour before the experiment started. The chamber was constantly perfused with ACSF at a rate v of 2.5 ml/min at 33  1 C. Bay 60-7550 was dissolved in DMSO and diluted in ACSF (final concentration 0.003%) and was applied 20 min before until 20 min after the tetanus. Synaptic responses were elicited by stimulation of the Schaffer collateral-commissural fibres in stratum radiatum of the CA1 region using lacquer-coated stainless steel stimulating electrodes. Glass electrodes (1–4 MX) were placed in the apical dendritic layer to record field excitatory postsynaptic potentials (fEPSPs). The initial slope of the fEPSP was used as a measure of synaptic strength. The stimulus strength of the test pulses was adjusted to 30% of the EPSP maximum. During baseline recording, 3 single stimuli (10-s interval) were averaged every 5 min. Once a stable baseline was established, LTP was induced by a weak tetanization paradigm (Wilsch et al., 1998) consisting of four paired pulses with an interpulse interval of 10 ms applied with a pulse width of 0.2 ms at the theta frequency of 5 Hz (i.e. every 200 ms). Data were analysed by repeated measures ANOVA. 2.6. Object memory Four-month-old male Wistar rats and 6-month-old male C57BL/6J mice (Charles River, Sevenum, The Netherlands) were housed individually in type III (rats) or type II (mice) Makrolon cages during testing. The animals were tested in a circular arena, 83 cm in diameter for rats, and 43 cm for mice. The front half of the side wall was transparent; the rear half was grey. In a first trial (T1:3 min) two similar objects were placed in a symmetrical position 10 cm (rats) or 5 cm (mice) away from the grey wall. Four different sets of objects were used (Prickaerts et al., 2002b). In a second trial (T2:3 min), two dissimilar objects (one with a familiar shape and a new one) were presented. The time spent exploring each object during T1 and T2 was recorded. Independent groups of rats were tested (12 rats were used for each treatment group). All mice (n ¼ 24) were tested in all treatment conditions using a within subjects design with three or more days between treatments. Vehicle (EtOH:Solutol:H2O—5:10:85), or 0.3, 1,

or 3 mg kg1 Bay 60-7550 was administered p.o. immediately after T1. Memory was assessed at T2, after a 24-h retention interval. An index measure of discrimination (d2) between the new and the familiar objects was calculated as d2 ¼ ðexploration time new objectexploration time familiar object)/(total exploration time in T2). In rats, the effects of Bay 60-7550 on memory performance during T2 were analysed by analysis of variance (ANOVA) with the factor treatment, followed by Fischer’s least significant difference (LSD) post hoc comparison. In mice, a repeated measures ANOVA over treatment was used, followed by one-sample t-statistics. 2.7. Social recognition Adult (4–5 months) male Wistar rats (HsdCpb:WU) and juveniles (4–5 weeks) (Harlan-Winkelmann, Borchen, Germany) were housed in groups of three in type IV (adult) or type III (juvenile) Makrolon cages. The animals were randomly assigned to their treatment groups. Independent groups of rats were tested (10 rats were used for each treatment group). The task consisted of two trials, separated by a 24-h retention interval. During the first trial (T1), the adult rat was confronted with an unknown juvenile rat for 2 min. The amount of time spent by the adult animal inspecting the juvenile was registered. Bay 60-7550 (0.3–6 mg kg1) was given orally, immediately after T1, in a volume of 1 ml kg1 (EtOH:Solutol:H2O—5:10:85). After a retention interval of 24 h, inspection time was measured in a second trial (T2) for 2 min. The percent reduction in social investigation time was used as the measure of memory improvement. 2.8. T-maze continuous alternation Male C57BL/6JIco mice (IFFA CREDO, l’Arbresle, France) were housed in groups of 10 in type III Makrolon cages. Experiments were performed according to Gerlai (1998). Spontaneous alternation was defined as a visit to the other of the two goal arms of a T-maze than that visited in the previous trial. Testing of a mouse consisted of one single session that was terminated as soon as 14 free-choice trials were completed or 25 min had elapsed. The percent alternations out of the total number of free-choice trials was calculated. Independent groups of mice were tested (10 mice were used for each treatment group). MK-801 was administered i.p. in 0.9% NaCl solution. Bay 60-7550 or vehicle (EtOH:Solutol:H2O—5:10:85) was given orally 30 min before the session (application volume 5 ml kg1). Treatment effects were analysed using ANOVA, supplemented with LSD post hoc comparisons. Student’s t-test was used to evaluate whether the percent alternations per treatment condition deviated from chance level (50%).

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3. Results 3.1. Bay 60-7550—a selective PDE2 inhibitor Bay 60-7550 inhibited the activity of PDE2 purified from bovine heart with an IC50 value of 2:0  0:7 nM (n ¼ 12) and human recombinant PDE2 with an IC50 value of 4:7  1:0 nM (n ¼ 7). Bay 60-7550 showed 50fold selectivity for PDE2 compared to PDE1 and more than 100-fold selectivity compared to PDE5 and the other PDEs tested (PDE3B, PDE4B, PDE7B, PDE8A, PDE9A, PDE10A, PDE11A, see Table 1, Fig. 1A). Bay 60-7550 had an IC50 of >10 lM (less than 50% inhibition at 10 lM) for the following receptors and enzymes:adenosine receptor subtypes A1, A2A, GABAA receptor agonist and benzodiazepine binding site, glutamate receptors (AMPA, kainate, NMDA subtypes), histamine receptor subtypes H1, H3, muscarinic acetylcholine receptor subtypes M1–M5, serotonin receptor subtypes 5-HT1A, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5HT4, 5-HT6, 5-HT7, acetylcholinesterase, monoamine oxidase MAO-A, MAO-B. Bay 60-7550 did not show any effects on adenosine deaminase at concentrations of up to 10 lM, while EHNA inhibited adenosine deaminase with a Ki of 1.5 nM. The Ki value measured for the adenosine deaminase inhibition by EHNA corresponds very well to the values of 0.8–1.6 nM reported in the literature (Vargeese et al., 1994; Caiolfa et al., 1998). 3.2. Effects on cGMP in neuronal cultures and slices Bay 60-7550 alone (up to 1 lM) did not affect the cGMP levels in rat cortical neurons in culture, and the GC stimulator Bay 41-8543 (1 lM) alone increased cGMP levels only slightly. However, incubation of neurons with Bay 41-8543 (1 lM) plus Bay 60-7550 (1 nM–1 lM) resulted in a significant increase in cGMP levels at concentrations as low as 1 nM (compared to incubation with Bay 41-8543 alone), reaching levels 20times baseline at 100 nM (Fig. 1B). Similar results were obtained with rat hippocampal neurons (data not shown). Similar effects were observed in mouse cortical neurons, where Bay 41-8543 (100 nM) plus Bay 60-7550 (1 nM–1 lM) significantly increased cGMP levels (Fig. 1C). Bay 60-7550 increased the cAMP accumulation stimulated by 2 lM of the adenylyl cyclase stimulator forskolin at 100 nM–1 lM (Fig. 1D). Hippocampal slices did not show appreciable levels of cGMP-like immunoreactivity under control conditions (Fig. 2A). Incubation with Bay 60-7550 (10 nM–1 lM) increased cGMP-like immunoreactivity (Fig. 2B–E), especially in the presence of the NO-donor sodium nitroprusside (SNP; 100 lM) (Fig. 2F–J). cGMP-like immunoreactivity was highest in the perforant path, next to varicose fibres the hippocampus.

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Double-labelling studies with anti-synaptophysin antibodies revealed limited co-localization of cGMP and synaptophysin immunoreactivity (Fig. 2K–M). Measurement of cGMP and cAMP levels in hippocampal slices by radioimmunoassay confirmed that Bay 60-7550 increased cGMP and cAMP levels in a dosedependent fashion (Fig. 3A,B). In the presence of SNP (100 lM), Bay 60-7550 (10 nM–10 lM) significantly increased cGMP levels compared to control. To induce significant changes in cAMP levels, a 10-fold higher concentration of Bay 60-7550 was necessary (in the presence of SNP). 3.3. Hippocampal long-term potentiation To investigate the consequences of PDE2 inhibition on synaptic plasticity in rat hippocampal slices, we measured the effect of Bay 60-7550 on sub-maximal long-term potentiation in the CA1 region induced by a weak tetanic stimulation of the Schaffer-collateral pathway. Treatment with Bay 60-7550 (10 or 100 nM) starting 20 min before the weak tetanic stimulation caused a significantly larger potentiation of the field excitatory postsynaptic potential (fEPSP) slope than that observed in control slices stimulated in a similar fashion (Fig. 4). At a concentration of 100 nM Bay 60-7550, this increase persisted over the whole period measured (120 min), while at 10 nM, significant differences were observed up to 40 min after the LTP inducing stimulus. The lowest concentration of Bay 607550 tested (1 nM), did not change the potentiation of the fEPSP slope compared to control. Basal synaptic transmission was not affected by the tested concentrations of Bay 60-7550. 3.4. Effects on learning and memory The object recognition task is based on the spontaneous behaviour of rodents to explore a novel object more than a familiar one (Ennaceur and Delacour, 1988; Prickaerts et al., 2002b). After a 24-h retention interval between first (T1) and second (T2) exposure to an object, vehicle-treated animals did not discriminate between the familiar and a novel object. Bay 60-7550, given orally immediately after T1, improved the memory performance of rats (F 3;44 ¼ 11:40; p < 0:01; Fig. 5A). Post hoc analysis revealed that 1 and 3 mg kg1 were effective in improving memory. Bay 60-7550 also improved the performance of mice in this task (F 3;92 ¼ 4:17, p < 0:01; Fig. 5B). Post hoc analysis revealed that object memory was improved after treatment with 0.3 and 1 mg kg1. In the social memory task (Thor and Holloway, 1982), the recognition performance of an adult rat is measured as the difference in time spent investigating a juvenile during the first (T1) and a second encounter

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Fig. 1. Effect of Bay 60-7550 on enzyme inhibition and cellular cGMP levels. (A) Bay 60-7550 inhibits human recombinant PDE2A (u), PDE1C (m) and PDE5A (O). Values are the means  SEM of three experiments, (B) Bay 60-7550 (in the presence of the GC stimulator Bay 41-8543) increases cGMP levels in rat cortical neurons, and (C) in mouse cortical neurons, (D) Bay 60-7550 increases cAMP levels in rat cortical neurons (in the presence of the adenylyl cyclase stimulator forskolin). Neurons were grown in 96-well plates (150 000 neurons/well). Values shown are fmol cGMP per well (B, C) and fmol cAMP per well (D), (E) Structure of Bay 60-7550, (F) Structure of cGMP.

p < 0:01 different from GC- or forskolin-stimulated control value.

(T2). After an interval of 24 h (T2), normal untreated rats do not remember the juvenile and the investigation time is the same as that during T1. Bay 60-7550 improved the recognition performance of adult rats (F 3;35 ¼ 3:79, p < 0:05; Fig. 5C) at T2. Post hoc LSD comparisons confirmed that Bay 60-7550 (1 and 3 mg kg1) reduced the social investigation time at T2, i.e. improved the memory performance of adult rats. The lowest dose was ineffective. In a second experiment, Bay 60-7550 (0.6- 6 mg kg1) also improved recognition performance (F 3;36 ¼ 5:08, p< 0:01; Fig. 5D).

Post hoc LSD comparisons confirmed that the doses of 2 and 6 mg kg1, but not the lowest dose, reduced the social interaction time at T2. The T-maze continuous alternation task depends on intact hippocampal functioning and measures predominantly spatial working memory (Gerlai, 1998). Vehicle-treated mice had an alternation rate higher than the chance level (t19 ¼ 7:86; p < 0:001), whereas mice injected with the NMDA receptor blocker MK801 (0.04 mg kg1) 30 min before the test had a lower continuous alternation rate (ANOVA: F 4;95 ¼ 9:52,

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Fig. 2. Localization of cGMP-immunoreactivity in rat hippocampal slices incubated with Bay 60-7550. cGMP-immunostaining in the CA1 area is shown by fluorescence microscopy (A–J). Slices were incubated with Krebs-incubation buffer alone (A) or in the presence of 0.01–10 lM Bay 60-7550 (B–E) or with 0.001–10 lM Bay 60-7550 in the presence of 0.1 mM SNP (F–J). Panels (K–M) demonstrate double staining of cGMP and synaptophysin by confocal microscopy in the CA3 area after treatment with 1 lM Bay 60-7550 and SNP. Bar in (J) indicates 100 lm for (A–J); bar in (M) indicates 20 lm for (K–M).

p < 0:001). Different doses of Bay 60-7550 (0.3–3 mg kg1) were tested for their ability to reverse this effect of MK-801. The highest dose of Bay 60-7550

(3 mg kg1) fully antagonized the MK-801-induced deficit (Fig. 6), restoring the alternation rate to above the chance level (t19 ¼ 5:43, p < 0:001).

4. Discussion

Fig. 3. Effect of Bay 60-7550 on cGMP and cAMP levels in rat hippocampal slices. Slices were incubated with Krebs-incubation buffer alone or Bay 60-7550 (10 nm–10 lM) in the absence (u) or presence of 0.1 mM SNP (v). cGMP (A) and cAMP, (B) levels were measured by radioimmunoassay. Each value is the mean (SEM) of three animals (n ¼ 3).
p < 0:05;

p < 0:01 different from control (0 M or 0 M with SNP).

Owing to its high potency and excellent selectivity profile, Bay 60-7550 is a very useful tool for the study of PDE2-dependent processes. To date, the physiological actions of PDE2 have been characterized with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (Mery and Fischmeister, 1994; Podzuweit et al., 1995), which inhibits PDE2 with an IC50 of 1 lM. However, in contrast to Bay 60-7550, EHNA is also a very potent adenosine deaminase inhibitor (IC50  2 nM), which complicates the interpretation of results obtained with this compound. The dose-dependent increase in cGMP levels in cortical neurons incubated with Bay 60-7550 (1 nM–1 lM) plus Bay 41-8543 (1 lM; a stimulator of GC) suggests that PDE2 has a major role in the degradation of cGMP in neurons under conditions of GC stimulation. In the absence of GC stimulator, Bay 60-7550 (1 lM) typically did not affect intracellular cGMP accumulation, which is consistent with the high Km value of PDE2 for cGMP and cAMP (10 and 30 lM, Martins

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Fig. 5. Effects of Bay 60-7550 on object recognition and social memory. Bay 60-7550 improved object recognition performance in rats (A), and mice (B), measured as an increase in the discrimination index d2 [d2 ¼ ðexploration time new objectexploration time familiar object)/(total exploration time)]. Independent groups of rats were tested (12 rats were used for each treatment group). All mice (n ¼ 24) were tested in all treatment conditions using a within subjects design with three or more days between treatments. Bay 60-7550 improved social memory in rats (C and D) measured as a reduction of the time a juvenile is investigated by an adult rat during the second encounter. Independent groups of rats were tested (10 rats were used for each treatment group).
p < 0:05 different from vehicle control (Fischer’s LSD post hoc comparison). #p < 0:05 different from zero (one-sample t-statistics). Fig. 4. Effect of Bay 60-7550 on hippocampal long-term potentiation. Treatment with 100 nM Bay 60-7550 (A) increased the LTP in the CA1 region of the hippocampus over the whole period measured (120 min). Ten nanomolar Bay 60-7550, (B) showed significant effects up to 40 min after the LTP-inducing stimulus, while 1 nM, (C) was no longer effective (values are means  SEM of 12–14 slices; repeated measures ANOVA,

p < 0:01). Basal synaptic transmission was not affected. Application of Bay 60-7550 is indicated by horizontal bar: 20 min before and after a weak tetanic stimulation of the Schaffer collateral pathway.

et al., 1982). PDE2 activity is low under basal conditions, but increases in the presence of higher cGMP concentrations (after GC stimulation) due to an allosteric cGMP binding site. This property of PDE2 is

important for the regulation of neuronal cGMP levels. Although PDE2 is able to degrade cAMP and cGMP with similar Km and Vmax in vitro (Martins et al., 1982), we observed alterations in cAMP levels in cortical neurons (in the presence of forskolin) and in hippocampal slices (with or without SNP) only at higher concentrations of Bay 60-7550 (100 nM or 1 lM, Figs. 1D and 3). This is probably due to the presence of several other cAMP metabolizing PDEs in these cells. Our results are consistent with the observation that the PDE2/adenosine deaminase inhibitor EHNA enhanced the NMDA receptor-induced elevation of cGMP (but not cAMP) levels in rat cortical and

F.G. Boess et al. / Neuropharmacology 47 (2004) 1081–1092

Fig. 6. Effect of Bay 60-7550 on the MK-801 induced deficit in the continuous alternation task. Mice treated with vehicle only and mice treated with MK-801 plus 3 mg kg1 Bay 60-7550 alternated above chance level. Administration of 3 mg kg1 Bay 60-7550 antagonized the MK-801 induced deficit.
p < 0:05 different from the vehicletreated control group, #p < 0:05 better than chance level. Independent groups of mice were tested (10 mice were used for each treatment group).

hippocampal neurons in primary culture (Suvarna and O’Donnell, 2002). In contrast, NMDA-receptorinduced increases in cAMP levels were enhanced by the PDE4 inhibitor rolipram. Thus selective inhibition of PDE2 will lead primarily to an increase in neuronal cGMP levels after GC stimulation. The PDE2/adenosine deaminase inhibitor EHNA also potentiated the cGMP increase in rat striatal neurons treated with diethylamine (Wykes et al., 2002). If a localized increase in cGMP levels, subsequent to NMDA receptor activation, is indeed important for synaptic plasticity during learning and memory, inhibition of PDE2 may selectively increase cGMP concentration in the synapses involved in this process. Inhibition of PDE2 activity may, in fact, be part of a signal transduction pathway that is active under physiological conditions: incubation of PC12 cells with nerve growth factor (NGF) resulted in their differentiation to a neuronal phenotype and in a decrease in endogenous PDE2 activity by 50% within 24 h and by 100% after 72 h (Bentley et al., 2001). Interestingly, NGF did not decrease PDE2 mRNA or protein expression. However, several phosphoproteins co-immunoprecipitated with an epitopetagged PDE2A2-construct only after NGF-stimulation, which suggests that an NGF-regulated complex controls PDE2 activity. Bay 60-7550 increased cAMP and cGMP levels in hippocampus slices, and increased cGMP immunoreactivity in the hippocampus (Fig. 2), cortical areas, the caudate putamen, and several subnuclei of the

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thalamus (data not shown). In the hippocampus, cGMP levels were increased in the perforant pathway, next to varicose fibres throughout the hippocampus. The limited colocalization of cGMP and synaptophysin immunoreactivity suggests that cGMP levels may be increased in a subset of presynaptic terminals, while the main cGMP increase takes place in other (postsynaptic) parts of the neurons. Bay 60-7550 also significantly increased LTP in the CA1 region but did not affect basal synaptic transmission. These results suggest that inhibition of PDE2 increases neuronal cGMP levels after GC activation and increases the use-dependent enhancement of synaptic function. The effect on synaptic efficacy may be due to a PKG-mediated increase in neurotransmitter release (Arancio et al., 1995, 2001). An alternative interpretation could be changes on the postsynaptic side due to PKG-mediated phosphorylation of ion channels or other elements of signal transduction pathways (Wang and Robinson, 1997; Ahem et al., 2002), consistent with the results of many studies that show changes in the strength of the postsynaptic response during LTP (reviewed by Lynch, 2004). Our results support the postulated role of NO/ cGMP signal transduction in synaptic plasticity (Zhuo et al., 1994; Arancio et al., 1995; Boulton et al., 1995; Lu et al., 1999; Monfort et al., 2002) and show that PDE2 is an important element of this pathway. The involvement of cAMP and protein kinase A in different aspects of LTP and memory formation has been well documented (e.g. Otmakhov et al., 2004; Lynch, 2004). We cannot exclude the possibility that the effects of PDE2 inhibition on LTP and the different learning tasks are at least partially mediated by an elevation of cAMP levels. The fact that PDE2-mediated hydrolysis of cAMP is stimulated by increased cGMP-levels provides a possibility for cross-talk between the two signal transduction pathways and inhibition of neuronal PDE2 may increase both cGMP and cAMP levels. As expected for a drug that specifically affects the synaptic connections involved in memory formation, Bay 60-7550 improved the object memory performance of normal unimpaired rats and mice in a dose-dependent manner. It also improved social recognition memory in rats. The observation that Bay 60-7550 is active on administration after the first trial in both the object memory and social recognition task suggests that PDE2 inhibition has a positive effect on the consolidation of novel memories. Earlier studies have shown that high cGMP levels caused by treatment with PDE5 inhibitors or direct injection of 8-Br-cGMP improve the memory performance of mice in a passive avoidance task (Baratti and Boccia, 1999) and of rats in an object recognition task, most likely due to an effect on the consolidation process (Prickaerts et al., 1997, 2002a, 2002b). The behavioural effects of Bay 60-7550

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in the object recognition task in rats were essentially similar. However, while the pattern of expression of PDE2 (Repaske et al., 1993), GC (Matusoka et al., 1992; Burgunder and Cheung, 1994) and NOS (Bredt et al., 1991) in the brain is consistent with their role in a common signal transduction pathway, PDE5 mRNA expression is low in the hippocampus and cortex (Loughney et al., 1998). In addition, the PDE5 inhibitor zaprinast did not increase NMDA-induced cGMP accumulation in hippocampal neurons (Suvarna and O’Donnell, 2002), suggesting that PDE5 inhibitors may influence behaviour via vascular or other non-neuronal mechanisms. Stimulation of the NMDA receptor leads to an increase of cGMP in the hippocampus (Vallebuona and Raiteri, 1994). NMDA receptor blockade with e.g. MK-801 impaired learning and memory performance in several cognition tests (Castellano et al., 2001). Using the mouse continuous alternation task, we showed that 3 mg kg1 Bay 60-7550 could fully antagonize the MK-801-induced decrease in spontaneous alternation of mice in the T-maze. These results provide further evidence for the connection between NMDA receptor activation and cGMP-mediated signal transduction and show that deficits in this pathway can be overcome by inhibition of PDE2. There is a good agreement in the active doses (1 and 3 mg/kg) between the rat object recognition and the rat social memory tests. In the mouse T-maze spontaneous alternation task, similar doses of Bay 60-7550 (3 mg/kg) reversed the MK-801-induced impairment. In the mouse object recognition task, effects were already observed at lower concentrations (0.3 and 1 mg/kg), while 3 mg/kg was no longer active. The fact that Bay 60-7550 was not active in the mouse object recognition test at 3 mg/kg is interesting, since it was active at this concentration in the rat object recognition test and in the other behavioural models. This observation could be attributed to the involvement of different neuronal systems in the two mouse models, the presence of the NMDA-receptor channel blocker MK-801 in the mouse T-maze spontaneous alternation task and to species differences in the pharmacological or pharmacokinetic properties of Bay 60-7550. An altered NO/cGMP signalling pathway may reduce synaptic plasticity during ageing and in memory disorders. GC activity is reduced in the superior temporal cortex of patients with Alzheimer’s disease (Bonkale et al., 1995), and a decrease in [3H]-cGMP binding in the hippocampus may reflect a loss of PKG protein or function (Bonkale et al., 2000). Furthermore, treatment of rat astrocytes with b-amyloid peptides or injection of b-amyloid peptides into the hippocampus of adult rats decreases the expression of soluble GC (Baltrons et al., 2002). The resulting diminished synaptic plasticity may contribute to the memory deficits

observed in patients with Alzheimer’s disease. We have shown here that the potent and selective PDE2 inhibitor Bay 60-7550 increases cGMP levels in neurons and enhances LTP in the CA1 region of the hippocampus. Bay 60-7550 improved early consolidation of social recognition memory in rats and object memory in rats and mice. In addition, Bay 60-7550 reversed MK801induced deficits in T-maze continuous alternation in mice. Increasing or restoring normal neuronal cGMP levels with the help of PDE2 inhibitors may, therefore, represent a novel therapeutic approach to improve memory performance in conditions in which the NO/ cGMP signal transduction pathway is impaired, such as in Alzheimer’s disease and other memory disorders.

Acknowledgements The LTP studies were conducted in the laboratories of Dr. U.H. Schro¨der and Professor K. Reymann, Forschungsinstitut Angewandte Neurowissenschaften (FAN), Magdeburg, Germany. A. Sik, Maastricht, is acknowledged for contributions to the object recognition studies. K. Selbach is acknowledged for contributions to the social recognition studies. This work is dedicated to our former colleague, the late Ulrich Niewoehner.

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