Serotonin transporter density and anxiolytic-like effects of antidepressants in mice

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Abstract

Background

Chronic treatment with the dual serotonin/noradrenaline reuptake inhibitor (SNRI) duloxetine reduces the density of serotonin transporter sites in cortex and engenders an anxiolytic-like response. To determine the reproducibility of these effects and their generality to other antidepressants we compared the effects of chronic duloxetine treatment with another SNRI, venlafaxine, and two selective serotonin reuptake inhibitors, paroxetine and fluoxetine.

Methods

Separate groups of mice were administered vehicle, fluoxetine (15 mg/kg), paroxetine, duloxetine or venlafaxine (10 mg/kg) perorally twice daily for 28 days and tested in the mouse zero-maze and in motility cages on days 21 and 22, respectively, to determine effects on anxiety and motor activity. On day 28 brains were analysed for serotonin transporter (SERT) density in cortex and noradrenaline transporter (NET) density in cortex and hippocampus.

Results

Duloxetine and fluoxetine both reduced SERT density in cortex and induced anxiolytic-like effects. Paroxetine had an identical profile, but it is unclear if this drug down-regulated the SERT since extensive washing of cortical tissue did not remove all drug. Venlafaxine had no effect on behavioural or biochemical parameters. Only duloxetine reduced NET density in cortex, although not hippocampus.

Conclusions

The reduction in SERT density and anxiolytic-like effects with duloxetine, fluoxetine and, potentially, paroxetine suggest that down-regulation of the SERT may be a relevant mechanism in therapeutic response to these antidepressants.

Introduction

Although selective serotonin reuptake inhibitors (SSRIs) and serotonin and noradrenaline reuptake inhibitors (SNRIs) are effective in the treatment of mood disorders such as depression and anxiety, clinical improvement is only seen after 3–6 weeks of treatment (Nutt, 2005). This delayed onset of clinical efficacy in mood disorders occurs despite the fact that SSRIs and SNRIs acutely inhibit the serotonin transporter (SERT, Hrdina et al., 1985, Benmansour et al., 2002), and in the case of SNRIs also the noradrenaline transporter (NET, Rehavi et al., 1982, Benmansour et al., 2004). Therefore, events or adaptations beyond the acute inhibition of monoamine transporters must be necessary for clinical efficacy. The necessity for down-regulation/desensitisation of somatodendritic 5-HT1A autoreceptors in the raphe nucleus and 5-HT1B autoreceptors located on the terminals of serotonergic neurons, after chronic but not acute antidepressant treatment have been proposed as potential mechanisms to explain the delay in therapeutic efficacy (Rausch et al., 2006, Clark and Neumaier, 2001).

A direct role for the primary target of many antidepressants, i.e. the SERT, has been implicated in affective disorders. For example, Lesch et al. (1996) showed genetic variation in the human SERT, viz a common polymorphism in the promoter region of the gene coding for SERT, leading to short and long variants. The consequence is a greater expression of SERT in subjects homozygous for the long allele compared to those with the short variant as assessed in-vitro using human lymphoblast cell lines, post mortem tissue and also from brain imaging studies (Lesch et al., 1996, Greenberg et al., 1999, Heinz et al., 2000); although it is important to emphasize that not all studies report such a relationship between polymorphic variant and SERT expression (Zalsman et al., 2005, Mann et al., 2000). Nonetheless, some studies have shown an association between the short allele form of SERT and heightened anxiety in man (Lesch et al., 1996, Lesch and Mossner, 1998, Katsuragi et al., 1999). Similarly, SERT knockout mice demonstrate heightened anxiety in some animal models (Holmes et al., 2003).

Recently we (Troelsen et al., 2005) demonstrated that the SNRI duloxetine engendered an anxiolytic-like response in the mouse zero-maze after chronic treatment for 21 days (10 mg/kg, PO twice a day). In these same animals there was a significant reduction in the density of the SERT in frontal cortex whereas there was no effect on NET density in the hippocampus. These findings have been verified by Gould et al. (2006a) who administered duloxetine to rats via osmotic mini-pumps and demonstrated a reduction in SERT density but not NET density in the dentate gyrus region of the hippocampus. Thus despite the difference between the studies in terms of species, route of administration and sacrifice time after treatment the selective effect of duloxetine on SERT density are qualitatively the same.

In addition we previously showed a reduction in the functional status of somatodendritic 5-HT1A autoreceptors and terminal 5-HT1B autoreceptors in animals treated chronically with duloxetine (Troelsen et al., 2005) by demonstrating a truncated hypothermic response to systemically administered 8-OHDPAT and mCPP, respectively (Maj and Moryl, 1992, Maj and Moryl, 1993, Hagan et al., 1997). In contrast to duloxetine, chronic treatment with either the SSRI citalopram or the tricyclic antidepressant amitriptyline had no effect in the zero-maze despite both treatment regimens similarly resulting in a truncated hypothermic response to 8-OHDPAT and mCPP in mice, which was equal to or greater than that attained with chronic duloxetine treatment. Moreover, whilst both citalopram and amitriptyline reduced cortical SERT density the magnitude of this effect was small (∼ 20%) compared to that seen with duloxetine (∼ 70%). We suggested that the lack of an anxiolytic response to citalopram and amitriptyline was due to a modest reduction in SERT density engendered by these drugs compared to duloxetine.

Since citalopram has a short half-life in mice (Fredricson, 1982) compared to duloxetine (Troelsen et al., 2005), lower in-vivo exposure to the former drug may have precluded seeing behavioural and biochemical effects demonstrated with duloxetine. However, rather than re-test citalopram at higher doses, for greater length or increased frequency, in the current study we opted to test the SSRIs paroxetine and fluoxetine which have longer half-lives in mouse than citalopram (Hirano et al., 2004, Hirano et al., 2005, Holladay et al., 1998, Bourin et al., 2001), to verify the assertion that a reduction in SERT density is necessary for an anxiolytic-like response in the mouse zero-maze. Previous studies in rats have shown that chronic administration of SSRIs to rats via osmotic mini-pumps reduces SERT but not NET density in various brain regions (Benmansour et al., 1999, Benmansour et al., 2004). Furthermore, it was surprising that in our previous study we saw no effect of the SNRI duloxetine on NET density in the hippocampus. Therefore we also assessed the effects of these compounds on NET density in the hippocampus and cortex.

Since the SNRI duloxetine was active in our animal model of anxiety after chronic treatment, we were interested to ascertain if another SNRI venlafaxine, which is marketed as an anxiolytic (Nutt, 2005), would also induce an anxiolytic-like response in mice and, if so, whether this drug also reduced SERT density. Although both venlafaxine and duloxetine are SNRIs, venlafaxine has 100–300 fold lower affinity for the SERT and NET compared to duloxetine (Bymaster et al., 2001). Whether these potency differences between the two SNRIs are relevant to the parameters of the present study are unclear, although Gould et al., 2006a, Gould et al., 2006b have demonstrated that unlike duloxetine, venlafaxine administered to rats for 21 days via osmotic mini-pumps does not engender any change in the density of either the SERT or NET in various brain regions.

It is worth noting that relative to the effects of acute antidepressant administration, few studies have assessed the effects of chronic antidepressant treatment in animal models of anxiety, and little data exist on the effects of venlafaxine and duloxetine per se in anxiety models. Moreover, an anxiolytic-like response to antidepressants of various classes after chronic treatment in animal models has rarely been reported (see review by Borsini et al., 2002, Li et al., 2006, Ripoll et al., 2006). Also, although others have shown that chronic treatment with antidepressants downregulates the SERT in adult rodents (Benmansour et al., 1999, Hirano et al., 2005) and suggested this may be relevant to their therapeutic effect this has not generally been vindicated with the use of available animal models of anxiety or depression (but see Thakker et al., 2005). Therefore, the current study combines behavioural measures of anxiety and motor activity in mice with determination of SERT density in cortex, as well as NET density in hippocampus and cortex, after chronic treatment with fluoxetine, paroxetine, duloxetine and venlafaxine.

Section snippets

Animals and housing

Female NMRI mice (Harlan Scandinavia) weighing 20–25 g were housed and habituated for 7 days before experiments in Macrolon III cages (20 × 40 × 18 cm) holding 6 mice per cage, with all cages enclosed within a Scantainer (Scanbur A/S, DK). Such group housing and isolation from male mice is known to suppress estrus in female mice, i.e. the normal 4–5 day cycling in female mice is prolonged (Whitten 1959 reviewed in Ma et al., 1999, University of California, Breeding information). Moreover, under

Effect of chronic treatment with antidepressants on zero-maze performance in mice (Fig. 1)

From Fig. 1 it is clear that chronic treatment with some of the antidepressants for 21 days engendered an anxiolytic-like effect in the mouse zero-maze. Specifically, there was a significant main effect of treatment on the time spent by animals in the two open areas (F[4,71] = 7.5, p < 0.001). Post hoc Dunnett's test clearly showed that mice treated with fluoxetine (p < 0.01), paroxetine (p < 0.001) and duloxetine (p < 0.01) spent more time in the open areas compared to vehicle treated animals (Fig. 1A).

Discussion

The current study replicates our findings with duloxetine in a recent publication (Troelsen et al., 2005), where we demonstrated that 3–4 weeks administration of this SNRI induced an anxiolytic-like response in mice and a reduction in SERT density in mouse cortex. Importantly, the current study adds some weight to the idea that a reduction in SERT density after chronic treatment with some monoamine reuptake inhibitors might lead to an anxiolytic-like response. Thus, like duloxetine the two

Acknowledgements

We are grateful to Ulla Borberg and Lisbeth Barnbæk for excellent technical assistance.

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