Behavioural Pharmacology
Phosphodiesterase 2 and 5 inhibition attenuates the object memory deficit induced by acute tryptophan depletion

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Abstract

The underlying mechanism of short-term memory improvement after inhibition of specific phosphodiesterases (PDEs) is still poorly understood. The present study aimed to reveal the ability of PDE5 and PDE2 inhibitors, that increase cyclic guanosine monophosphate (cGMP) and both cyclic adenosine monophosphate (cAMP) and cGMP, respectively, to reverse an object recognition deficit induced by acute tryptophan depletion. Acute tryptophan depletion is a pharmacological challenge tool to lower central serotonin (5-hydroxytryptamine; 5-HT) levels by depleting the availability of its dietary precursor tryptophan. Short-term object memory was tested in male Wistar rats by exposing them to the object recognition task. First, the effects of acute tryptophan depletion upon object recognition 2 h after administration of the nutritional mixture were established. Subsequently, acute tryptophan depletion was combined with the PDE5 inhibitor vardenafil (1, 3 and 10 mg/kg) or with the PDE2 inhibitor BAY 60-7550 (0.3, 1 and 3 mg/kg), 30 min prior to testing. Acute tryptophan depletion significantly lowered plasma tryptophan levels and impaired object recognition performance. Vardenafil (3 and 10 mg/kg) and BAY 60-7550 (3 mg/kg) were able to attenuate the acute tryptophan depletion induced object recognition impairment. Thus, both PDE5 and PDE2 inhibition improved short-term object recognition performance after an acute tryptophan depletion induced deficit. The underlying mechanisms, however, remain poorly understood and further studies are needed to determine whether the present findings can be explained by a direct effect of enhanced cAMP and cGMP levels upon 5-HT activity, or even other neurotransmitter systems, and possibly an interaction with synthesis of nitric oxide or effects upon cerebral blood flow function.

Introduction

The second messenger molecules cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) play an important role in intracellular signalling and appear to be differentially involved in learning and memory processes (Bernabeu et al., 1996, Bernabeu et al., 1997, Prickaerts et al., 2002a, Rutten et al., 2007b). Both cAMP and cGMP are selectively hydrolyzed by phosphodiesterase (PDE) enzymes and inhibition of the PDEs appears to be a reliable method to improve memory processes by increasing the levels of either cAMP, cGMP or both (Blokland et al., 2006).

From the eleven subclasses of PDEs, enhanced memory performance has been demonstrated after inhibition of PDE2 (Boess et al., 2004, Rutten et al., 2007b), PDE4 (Zhang et al., 2005, Rutten et al., 2006) and PDE5 (Prickaerts et al., 2004, Devan et al., 2006). Only recently, pro-cognitive effects have also been observed after PDE9 inhibition (Van der Staay et al., 2008) and inhibition of PDE10 is gaining increasing interest for the improvement of cognitive deficits, specifically those related to schizophrenia (Schmidt et al., 2008). The PDE inhibitors (PDE-Is) type 4 and 5 selectively increase central cAMP and cGMP, respectively. PDE2 breaks down both cAMP and cGMP, thus, inhibition leads to enhanced concentrations of both second messengers (Boess et al., 2004). Whereas several mechanisms may explain the cognitive enhancing properties of PDE inhibition for long-term memory, the underlying mechanism for short-term memory improvement is still poorly understood (Boess et al., 2004, Devan et al., 2004, Rutten et al., 2007a).

Elevated levels of cAMP and cGMP enhance long term potentiation, which is assumed to be the underlying substrate of memory formation (Bliss and Collingridge, 1993), through activation of the cAMP/PKA/CREB (Impey et al., 1996) and cGMP/PKG/CREB pathway (Lu et al., 1999), respectively. This requires gene transcription or protein synthesis and provides an explanation for improvement of long-term memory processes after PDE inhibition (Blokland et al., 2006), not however for short-term memory enhancement. To date, short-term memory improvement after PDE2 inhibition has only been demonstrated in mice exposed to a spontaneous alternation task, in which the PDE2-I 2-(3,4-dimethoxybenzyl)-7-{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methyl imidazo[5,1-f][1,2,4]triazin-4(3H)-one (BAY 60-7550) was able to fully reverse the working memory deficit induced by the N-methyl-d-aspartate (NMDA) receptor blocker MK-801 (Boess et al., 2004). Improved object recognition performance was also found in rats after treatment with the less selective PDE5-I zaprinast, which reversed a memory deficit induced by the nitric oxide synthase (NOS) inhibitior 7-nitroindazole (Prickaerts et al., 1997). In a learning task for rats, NOS inhibition (NOS-I) with N(omega)-nitro-l-arginine methyl ester (L-NAME) also induced impaired performance, which was attenuated after treatment with the highly selective PDE5-I sildenafil (Devan et al., 2006). Activation of presynaptic cGMP by elevated levels of nitric oxide (NO), which is a retrograde messenger (Murad et al., 1978), is short-lasting without the requirement of protein synthesis or gene transcription and might thus underlie short-term memory processes.

In general, a direct link has been proposed between the levels of cAMP and changes in neurotransmitter (acetylcholine, noradrenaline, serotonin and histamine) release and activity (Silvestre et al., 1999, Lourenco et al., 2006), which could underlie the improvement of short-term memory after inhibition of PDE4 (Rutten et al., 2006, Rutten et al., 2007a). Along similar lines, involvement of the serotonin (5-hydroxytryptophan; 5-HT) system was demonstrated in a recent study in which the highly selective PDE4-I rolipram was tested for object recognition performance in a serotonergic deficit model induced by acute tryptophan depletion (Rutten et al., 2007a). Rolipram reversed the acute tryptophan depletion induced short-term memory deficits, suggested to be due to an interaction with specific neurotransmitter systems.

An acute decrease of central 5-HT concentrations can be accomplished by applying the acute tryptophan depletion method. Acute tryptophan depletion is a 5-HT challenge tool that manipulates the availability of the essential amino acid tryptophan, the dietary precursor of 5-HT (Young, 1993, Bell et al., 2005, Neumeister et al., 2005). By administering a tryptophan-free nutritional mixture, plasma tryptophan levels are depleted and less of the precursor is available for synthesis into 5-HT (Fernstrom, 1983, Fadda et al., 2000). The acute tryptophan depletion method is widely used both clinically and pre-clinically as a tool to investigate the implication of the 5-HT system in affective disorders (Booij et al., 2003).

Acute tryptophan depletion induced memory deficits have been observed in both healthy and serotonergic vulnerable human subjects (Klaassen et al., 1999, Riedel et al., 1999, Riedel, 2004, Booij et al., 2005, Evers et al., 2005, Jans et al., 2007b, Sambeth et al., 2007). Moreover, as mentioned above, Rutten et al. (2007a) demonstrated that acute tryptophan depletion significantly reduces object recognition performance in Wistar rats both 1 h and 3 h after administration of the nutritional mixture. Similar short-term memory deficits induced by acute tryptophan depletion in the rat object recognition task have also been demonstrated by others (Lieben et al., 2004b, Lieben et al., 2005, Jans et al., 2007a). Thus, the acute tryptophan depletion method appears to be a reliable deficit model in which memory enhancing compounds, including PDE-Is, can be tested (Rutten et al., 2007a).

The aim of the present study was to further explore the link between PDE2 and PDE5 inhibition, the 5-HT system and changes in short-term memory performance. In addition to the short-term memory enhancing effects of the PDE4-I rolipram (Rutten et al., 2007a), we investigated the ability of the PDE5-I vardenafil and the PDE2-I BAY 60-7550 to reverse an acute tryptophan depletion induced impairment in object recognition performance.

Section snippets

Animals

A total of 23 six-month-old male Wistar rats (Charles River, The Netherlands) were used, weighing between 450 and 500 g at the start of the experiments. On arrival, animals were housed individually in standard Makrolon™ (Type III) cages on sawdust bedding and maintained on a 12:12-hr light-dark schedule (lights on from 18:00 h-06:00 h) in a temperature controlled (21 ± 2 °C) and air-conditioned testing room. Animals were housed in the room where testing took place with background noise being

Plasma

The percentage change of the ratio tryptophan/∑LNAA in plasma is shown in Fig. 1. Acute tryptophan depletion decreased the ratio over time [F(1, 14) = 399.72; P < 0.001] and this decrease over time appeared to be different for the treatment conditions [F(4, 14) = 12.96; P < 0.001]. An overall treatment effect was found [F(4, 14) = 8.026; P < 0.01]. Post hoc analysis showed that tryptophan+ treatment significantly differed from all tryptophan- treatments. No difference was found between the tryptophan-

Discussion

In the present study, acute tryptophan depletion was applied to impair short-term object recognition in rats and to be able to investigate the ability of PDE2 and PDE5 inhibitors to restore short-term memory deficits. Acute tryptophan depletion was established by administering a single dose of 10 ml/kg of a tryptophan-free protein-carbohydrate mixture, which resulted in a substantial decrease of the ratio tryptophan/∑LNAA in plasma after 2 h and significantly impaired object recognition

Acknowledgement

E.L.v.D. and J.P. are supported by the EU Framework 6 Integrated Project NEWMOOD (LSHM-CT-2004-503474).

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