Galanin (1-15) enhancement of the behavioral effects of Fluoxetine in the forced swimming test gives a new therapeutic strategy against depression

Highlights

• GAL(1-15) enhances the antidepressant-like effects induced by Fluoxetine.

• 5-HT1A receptors participate in the GAL(1-15)/FLX interactions.

• GAL(1-15)/FLX interactions affected binding and 5-HT1AR mRNA levels in hippocampus.

Abstract

The pharmacological treatment of major depression is mainly based on drugs elevating serotonergic (5-HT) activity. Specifically, selective 5-HT reuptake inhibitors, including Fluoxetine (FLX), are the most commonly used for treatment of major depression. However, the understanding of the mechanism of action of FLX beyond its effect of elevating 5-HT is limited. The interaction between serotoninergic system and neuropeptides signaling could be a key aspect. We examined the ability of the neuropeptide Galanin(1-15) [GAL(1–15)] to modulate the behavioral effects of FLX in the forced swimming test (FST) and studied feasible molecular mechanisms. The data show that GAL(1-15) enhances the antidepressant-like effects induced by FLX in the FST, and we demonstrate the involvement of GALR1/GALR2 heteroreceptor complex in the GAL(1-15)-mediated effect using in vivo rat models for siRNA GALR1 or GALR2 knockdown.

Importantly, 5-HT1A receptors (5HT1A-R) also participate in the GAL(1-15)/FLX interactions since the 5HT1AR antagonist WAY100635 blocked the behavioral effects in the FST induced by the coadministration of GAL(1-15) and FLX. The mechanism underlying GAL(1-15)/FLX interactions affected the binding characteristics as well as the mRNA levels of 5-HT1A-R specifically in the dorsal hippocampus while leaving unaffected mRNA levels and affinity and binding sites of this receptor in the dorsal raphe. The results open up the possibility to use GAL(1-15) as for a combination therapy with FLX as a novel strategy for treatment of depression.

Introduction

Major Depression is the most frequent mood disorder, with a lifetime prevalence that has been reported to range from 7% to 21% (Albert et al., 2011). It is associated with a substantial functional impairment, diminished quality of life, increased burden both for patients and caregivers, as well as with a higher risk of mortality.

Although several neurotransmitter systems and brain areas have been implicated in this pathology, the pharmacological treatment of major depression is mainly based on drugs elevating serotonergic (5-HT) activity (Blier and de Montigny, 1994, Jans et al., 2007). Specifically, selective 5-HT reuptake inhibitors (SRRIs) are the most commonly used for treatment of major depression. In particular, Fluoxetine (FLX) is usually chosen for the treatment of symptoms of depression (Artigas, 2013). However the understanding of the mechanism of action of FLX beyond its effect of elevating 5-HT is limited. Recent studies have also implicated several neuropeptides in the pathophysiology of depression in rodents and in the possibility to use them as a combined treatment with antidepressant drugs.

In this context, the neuropeptide Galanin (GAL) (Tatemoto et al., 1983) is of particular interest. GAL is widely distributed in neurons within the Central Nervous System (CNS) and three GAL receptor (GALR) subtypes, GALR1-3 with a high affinity for GAL have been cloned (Branchek et al., 2000, Mitsukawa et al., 2008). GALR1 and GALR3 mainly activate inhibitory G proteins Gi/Go, while GALR2 mainly couples to Gq/G11 to mediate excitatory signaling (Branchek et al., 2000, Wang et al., 1997).

GAL participates in mood regulation and depression in rodents (Bellido et al., 2002, Juhasz et al., 2014, Wang et al., 2016, Weiss et al., 1998). Thus, the activation of GALR1 and GALR3 results in a depression like behavior while stimulation of GALR2 leads to anti-depressant-like effects (Bartfai et al., 2004, Kuteeva et al., 2008, Lu et al., 2005). Recently, it has been described that GAL and its receptor GALR3 are differentially methylated and expressed in brains of major depression subjects in a region- and sex-specific manner (Barde et al., 2016).

In this context, GAL modulates 5-HT1A receptor (5-HT1AR) function at autoreceptor and postsynaptic level in the brain. In the dorsal raphe (DR), intracerebroventricular (icv) GAL induced a time-dependent reduction in affinity and an increase in the 5-HT1AR autoreceptor density(Razani et al., 2001). At the post-synaptic level, GAL reduced the affinity of the 5-HT1AR in the ventral limbic cortex (Fuxe et al., 1988, Hedlund and Fuxe, 1996). Moreover, in hypothermia, locomotor activity and passive avoidance, icv GAL blocked post-synaptic 5-HT1AR function (Kehr et al., 2002, Misane et al., 1998, Razani et al., 2001). The antagonistic GALR-5-HT1AR interactions described in the brain, especially in the limbic forebrain regions and in the dorsal raphe, has been suggested to be of high relevance for depression (Fuxe et al., 1988, Hedlund and Fuxe, 1996, Kehr et al., 2002, Misane et al., 1998, Razani et al., 2001).

Not only GAL but also the N-terminal fragments like GAL(1-15) are active in the CNS (Diaz-Cabiale et al., 2007, Diaz-Cabiale et al., 2010, Diaz-Cabiale et al., 2005, Hedlund and Fuxe, 1996). Recently we described that GAL(1-15) induces strong depression-related and anxiogenic-like effects in rats and these effects were significantly stronger than the ones induced by GAL (Millon et al., 2015). The GALR1-GALR2 heteroreceptor complexes in the dorsal hippocampus and DR, areas rich in GAL(1-15) binding sites (Hedlund et al., 1992) were involved in these effects (Millon et al., 2015) and demonstrated also in cellular models (Borroto-Escuela et al., 2014).

GAL(1–15) is also able to enhance the antidepressant effects induced by the 5-HT1AR agonist 8-OH-DPAT in the forced swimming test (FST) (Millon et al., 2016), effect that was again significantly stronger than the ones induced by GAL. Importantly, the mechanism underlying this action involved interactions at the receptor level in the plasma membrane with changes also at the transcriptional level. Thus, GAL(1–15) affected the binding characteristics as well as the mRNA levels of 5-HT1AR in the dorsal hippocampus and DR (Millon et al., 2016).

In view of these results the purpose of the current study was to assess the ability of GAL(1–15) to modulate the behavioral effects of FLX. We have analyzed the effect of GAL (1–15) on FLX-mediated responses in a behavioral test of depression. We tested the involvement of GALR2 in the GAL (1–15) effect with the selective GALR2 antagonist M871 and using an in vivo rat model for siRNA GALR2 knockdown. The role of the GALR1 in the behavioral actions of GAL(1–15) were analyzed using an in vivo rat model for siRNA GALR1 knockdown. Moreover, to study wether the effects of GAL(1–15) on FLX action were mediated via 5-HT1AR, we have analyzed the effect of the 5-HT1AR antagonist WAY100635 in the GAL(1-15)-mediated effect in the FST. We have also determined the binding characteristics and mRNA levels of 5-HT1AR in the DR and dorsal hippocampus after GAL(1-15)-FLX administration.