Strain differences in paroxetine-induced reduction of immobility time in the forced swimming test in mice: Role of serotonin

Abstract

We studied the antidepressant-like effect of paroxetine in strains of mice carrying different isoforms of tryptophan hydroxylase-2 (TPH-2), the enzyme responsible for the synthesis of brain serotonin (5-HT). The effect of paroxetine alone and in combination with pharmacological treatments enhancing or lowering 5-HT synthesis or melatonin was assessed in the forced swimming test in mice carrying allelic variants of TPH-2 (1473C in C57BL/6 and 1473G in DBA/2 and BALB/c). Changes in brain 5-hydroxytryptophan (5-HTP) accumulation and melatonin levels were measured by high-performance liquid chromatography. Paroxetine (2.5 and 5 mg/kg) reduced immobility time in C57BL/6J and C57BL/6N mice but had no such effect in DBA/2J, DBA/2N and BALB/c mice, even at 10 mg/kg. Enhancing 5-HT synthesis with tryptophan reinstated the antidepressant-like effect of paroxetine in DBA/2J, DBA/2N and BALB/c mice whereas inhibition of 5-HT synthesis prevented the effect of paroxetine in C57BL/6N mice. The response to paroxetine was not associated with changes in locomotor activity, brain melatonin or brain levels of the drug measured at the end of the behavioral test. These results support the importance of 5-HT synthesis in the response to SSRIs and suggest that melatonin does not contribute to the ability of tryptophan to rescue the antidepressant-like effect of paroxetine.

Introduction

The selective serotonin (5-HT) reuptake inhibitors (SSRIs) are widely used in the treatment of depressed patients. However, they are not or only partially effective in a fraction of patients (Fava, 2003, Stimpson et al., 2002, Thase and Rush, 1995). The reasons are substantially unknown, though pharmacogenetic studies have linked the response to SSRIs to polymorphisms in various genes coding for 5-HT mechanisms, particularly the promoter of the 5-HT transporter molecule (Serretti and Artioli, 2004).

Inbred mouse strains, such as C57BL/6J, C57BL/6N, DBA/2J, DBA/2N and BALB/c used in the present study, have greatly contributed to our knowledge of the genetic determinants of behaviour, particularly in pre-clinical psychiatric research (Crawley et al., 1997, Jacobson and Cryan, 2007). Previous studies showed wide differences across mice strains in the response to SSRIs in the forced swimming and tail suspension tests (Crowley et al., 2005, David et al., 2003, Lucki et al., 2001), two procedures commonly used to assess the antidepressant potential of compounds in mice and rats (Porsolt et al., 1977, Steru et al., 1985). This quite likely implies genetic differences as one of the factors contributing to the response to antidepressant drugs. We set out to characterize the neurochemical substrate/s accounting for these differences and suggested that the recently discovered isoform of tryptophan hydroxylase (TPH), TPH-2 (Walther et al., 2003), the enzyme responsible for the synthesis of brain 5-HT, is a potential candidate. Allelic variations of human tph-2 have been linked to an increase in the risk of mood disorders, poor response to selective 5-HT reuptake inhibitors, enhanced reactivity of the amygdala to emotional stimuli, and suicide (Brown et al., 2005, Zhang et al., 2005, Zill et al., 2004a, Zill et al., 2004b). The 1473G allele of the mouse TPH-2, linked to a lower 5-HT synthesis rate, is segregated to DBA/2 and BALB/c mice while C57BL/6 and 129/Sv mice are homozygous for the 1473C allele (Cervo et al., 2005, Zhang et al., 2004). Citalopram, a selective inhibitor of the 5-HT transporter molecule (Hyttel, 1977, Pollock, 2001, Pozzi et al., 1999), reduced immobility time in the forced swimming test in C57BL/6J and 129/Sv mice but had no effect on DBA/2J and BALB/c mice (Cervo et al., 2005). In addition, citalopram inhibited 5-HT synthesis in the brain of DBA/2J and BALB/c mice less than in C57BL/6J and 129/Sv mice (Cervo et al., 2005). Because there is evidence that non-serotonergic mechanisms might contribute to the antidepressant-like effects of several SSRIs (but not citalopram; Cryan et al., 2004), it is important to assess whether the findings with citalopram (Cervo et al., 2005) can be extended to other SSRIs. Thus, the antidepressant-like effect of paroxetine, a potent inhibitor of 5-HT reuptake and a widely used antidepressant drug (Rickels et al., 1992, Thomas et al., 1987), was assessed in strains of mice differing for the allelic form of TPH-2 associated to “high” and “low” 5-HT synthesis rate. The role of 5-HT was further assessed by evaluating the response to paroxetine and paroxetine plus tryptophan in the forced swimming test in mice given the 5-HT synthesis inhibitor p-chlorophenylalanine (pCPA) (Koe and Weissman, 1966) or the 5-HT precursor l-tryptophan (Grahame-Smith, 1964, Weber and Horita, 1965). 5-HTP accumulation after aromatic l-amino acid decarboxylase inhibition with m-hydroxybenzylhydrazine (Carlsson and Lindqvist, 1978) was used as an indicator of 5-HT synthesis after various pharmacological treatments. The potential contribution of differences in the pharmacokinetics of paroxetine across strains was also taken into account by measuring brain levels of the drug at the end of the behavioral test.

Melatonin is synthesized from tryptophan via 5-HT through two enzymatic steps involving the N-acetylation through serotonin N-acetyltransferase to obtain N-acetylserotonin, which is methylated by hydroxyindole-O-methyltransferase to form melatonin (Axelrod and Weissbach, 1961, Weissbach et al., 1960). Exogenous melatonin, with few exceptions (Bourin et al., 2004, Dubocovich et al., 1990), reduces immobility time in the rat and mouse forced swimming test (Micale et al., 2006, Raghavendra et al., 2000, Wong and Ong, 2001) and mouse tail suspension test (Prakhie and Oxenkrug, 1998). In addition, tryptophan markedly increased the levels of blood (Esteban et al., 2004, Jaworek et al., 2004, Leja-Szpak et al., 2004) and brain (Crespi et al., 1994) melatonin in the rat. Thus, we also addressed the role of this hormone in tryptophan-induced rescue of the behavioral effect of paroxetine by combining inactive doses of paroxetine and melatonin and measuring melatonin levels in the brain of DBA/2N mice given tryptophan alone or in association with other treatments as an indicator of the conversion of tryptophan into melatonin.