Molecular and Cellular Pharmacology
Time-dependent effects of escitalopram on brain derived neurotrophic factor (BDNF) and neuroplasticity related targets in the central nervous system of rats

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Abstract

Chronic treatment with antidepressants affects several proteins linked to neuroplasticity, particularly brain derived neurotrophic factor (BDNF): this leads eventually to their therapeutic effects. It is possible that also for putative early therapeutic onset, antidepressants may act by promoting cellular adaptations linked to neuroplasticity. Escitalopram, known to be already effective in preclinical models of depression after 7 days, allowed us to investigate whether two effective treatment regimens (7 and 21 days) may contribute to synaptic plasticity by acting on BDNF signalling. We focused our attention on two regulators of BDNF transcription, CREB and CaRF (calcium responsive factor), and on kinases, CaMKII, ERK1/2 and p38 MAPK, linked to BDNF that play a distinctive role in synaptic plasticity. We evaluated whether the effects of escitalopram on these targets may be different in brain areas involved in the depressive symptomatology (hippocampus, frontal and prefrontal cortex). Here we demonstrate that escitalopram regulates intracellular pathways linked to neuroplasticity at both the time points evaluated in an area-specific manner. While the two escitalopram-treatment regimens failed to affect gene expression in the rat frontal cortex, 7 days of treatment with escitalopram activated intracellular pathways linked to BDNF and increased the levels of Pro-BDNF in the rat prefrontal cortex. Moreover, 21 days of treatment with escitalopram decreased CREB/BDNF signalling while increasing p38 levels in the rat hippocampus. Even if further experiments with different antidepressant strategies will be needed, our data suggest that escitalopram efficacy may be mediated by early and late effects on synaptic plasticity in selective brain areas.

Introduction

In recent years more and more interest has focused on the molecular mechanisms involved in neuroplasticity for its possible implication in the treatment of major depression (Krishnan and Nestler, 2008). In particular, selective serotonin reuptake inhibitors (SSRIs), currently among the most used antidepressants (ADs) in clinical practice, trigger a complex biochemical cascade mediating the actions of the neurotrophin brain derived neurotrophic factor (BDNF) and leading ultimately to promote neuronal plasticity (Duman & Monteggia, 2006, Martinowich & Lu, 2008).

We used as a pharmacological tool the S-enantiomer of citalopram, escitalopram, the SSRI with the highest selectivity for the serotonin transporter (Owens et al., 1997, Owens et al., 2001) to gain further insights into antidepressant actions in the CNS and to explore molecular mechanisms that can be responsible for an earlier appearance of the therapeutic effect. In fact this drug has been shown to be effective as early as after 7 days of treatment in several behavioural models of depression (Capone et al., 2006, Montgomery et al., 2001, Sanchez et al., 2003, Reed et al., 2009).

Taking advantage of this peculiar feature we tested the hypothesis that different temporal molecular mechanisms may be involved in mediating escitalopram effects by using two treatment regimens. We treated healthy rats with escitalopram for 7 or 21 days at a dose that has been shown to be active (10 mg/kg) and examined the temporal effects on neuroplasticity related targets of this SSRI in key areas involved in the etiopatogenesis of depression and in mediating the outcome of the ADs: hippocampus, frontal and prefrontal cortex (Holmes & Wellman, 2009, Kennedy et al., 1997, Manji & Duman, 2001, Mayberg, 2003, Nestler et al., 2002). In doing so, we followed a four-step approach: first, we evaluated the effects of the two treatment regimens on gene expression of BDNF and of cAMP response element binding (CREB) protein that acts as a regulator of BDNF expression (Blendy, 2006, Martinowich & Lu, 2008).

Our attention was then focused in those areas where at least one of the two treatment regimens was able to affect gene expression. As second step, we evaluated the protein levels of BDNF (mature and Pro-BDNF) and CREB to assess whether changes in gene expression could be coupled to changes also in protein levels.

Third, protein kinases acting as downstream targets of BDNF signalling, participating at least in regulating CREB activation and that likely play a role in mediating antidepressant behavioural effects were investigated. In particular, calcium/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated kinases (ERKs) and p38 MAPK total and phosphorylated (activated) levels were measured to assess whether these kinases could be regulated by these two treatment regimens (Barbiero et al., 2007, Gourley et al., 2008, Huang & Reichardt, 2001, Miller & Raison, 2006). Fourth, we investigated the effects of escitalopram on another transcription factor, the calcium responsive transcription factor (CaRF), which regulates BDNF neuronal expression in a calcium selective manner (Tao et al., 2002),after 7 and 21 days of treatment with escitalopram. The present study was undertaken to test whether this complex interconnected pathway, may present a temporal and area-specific profile in response to escitalopram treatment.

Section snippets

Animals

Experiments were performed on adult male Sprague–Dawley albino rats of 8 weeks of age at the beginning of the experimental procedure (Charles River, Calco, Italy). Animals were housed 3 per cage in polycarbonate cages (28 × 17 × 12 cm) in a temperature- and humidity-controlled environment on a 12-h light–dark cycle (lights on at 6.00 a.m.) with ad libitum access to food and tap water. The procedures used in this study were in strict accordance with the European legislation on the use and care of

Results

The effects of a subchronic (7 days) or a chronic (21 days) treatment with escitalopram (10 mg/kg) on BNDF and neuroplasticity related targets were investigated in areas playing a role in the response of ADs. For a comprehensive summary of the results see Supplementary Tables 1 and 2.

Discussion

Even if an improvement of clinical symptoms appears in the earlier weeks after the beginning of an antidepressant (AD) treatment, the significant improvement in mood appears over a longer time. This delay in the therapeutic outcome of antidepressants leads to the hypothesis that long-term adaptative responses of the CNS need to be evoked to treat major depression (Castrén, 2004). Solid research efforts have been spent to evaluate the effects of chronic (3–4 weeks) AD treatments on synaptic

Conclusions

Next, we will evaluate the functional and behavioural impacts of the molecular effects evoked by escitalopram using specific tests in animal models, in an attempt to link the observed changes on neuroplasticity related targets to behavioural outcomes induced by escitalopram. Another compelling aim will be to compare the effect of escitalopram on these neuroplasticity related targets to those of other AD treatments acting on the serotonergic system, and also on other neurotransmitter systems, in

Acknowledgment

This work has been supported in part by an unrestricted grant Lundbeck. We are greatful to Mr. Ian David Haworth for English language support.

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    1

    Alboni S., Benatti C. and Capone G. equally contributed to this work.

    2

    Present address: Department of Experimental Medicine, University of Insubria, Via Rossi 9, 21100 Varese, Italy.

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