Behavioural pharmacologyAnxiolytic-like effect of mirtazapine mediates its effect in the median raphe nucleus
Introduction
Mirtazapine, an antagonist of central α2-adrenergic autoreceptors and heteroreceptors, and postsynaptic 5-hydroxytryptamine2 (5-HT2) and 5-HT3 receptors, achieves its unique therapeutic effect by enhancing noradrenaline and serotonin release (Millan, 2006, Yamauchi et al., 2012). Several clinical trials demonstrated the effectiveness of mirtazapine in the treatment of not only depression but also anxiety disorders, including post-traumatic stress disorder (Davidson et al., 2003), generalized anxiety disorder (Gambi et al., 2005), panic disorder (Ribeiro et al., 2001) and social anxiety disorder (Van Veen et al., 2002). Although its beneficial effects in relieving anxiety symptoms and curing anxiety disorders have been reported consistently, the mechanism by which mirtazapine exerts its anxiolytic effect has not been fully clarified.
Evidence from animal experiments also supports the anxiolytic-like effect of mirtazapine. The systemic administration of mirtazapine decreases conditioned freezing behavior in the contextual fear conditioning model (Kakui et al., 2009). A number of studies have demonstrated the reliability of the fear conditioning test as a behavioral paradigm with which to clarify the mechanisms involved in fear and anxiety and to evaluate the efficacy of therapeutic agents such as SSRIs and benzodiazepines in the treatment of anxiety disorders (Inoue et al., 2011). Studies using an in vivo microdialysis technique have found that the acute systemic administration of mirtazapine increases extracellular serotonin concentrations in the hippocampus but not the prefrontal cortex of rats (Yamauchi et al., 2012), suggesting that the anxiolytic effect of mirtazapine might be mediated by the facilitation of central 5-HT neurotransmission in the hippocampus.
In contextual fear conditioning, the amygdala and hippocampus are two main brain structures implicated in the acquisition, expression and retrieval of fear conditioning (LeDoux, 2000, Maren, 2008). In addition, the median raphe nucleus (MRN) also has an important role in the acquisition and expression of conditioned fear (Almada et al., 2009, Avanzi et al., 2003, Borelli et al., 2005). Lesions or inactivation of the MRN were shown to reduce the expression of contextual fear conditioning (Borelli et al., 2005, Silva et al., 2004). The MRN includes a great density of α1-adrenoceptors and α2-adrenoceptors in addition to the high density of 5-HT receptors (Day et al., 1997, Kia et al., 1996, Rosin et al., 1993, Talley et al., 1996). Administration of the α2-adrenergic agonist, clonidine, into the MRN decreases the level of 5-HT in this nucleus, whereas the administration of an α2-adrenergic antagonist into the MRN enhances 5-HT release (Adell and Artigas, 1999). These results suggest that α1- and α2-adrenoceptors in the MRN may affect contextual conditioned fear by increasing 5-HT release. However, little is known about the sites of anxiolytic action of mirtazapine in the brain.
The aim of this study is to clarify the brain regions where mirtazapine acts as an anxiolytic in contextual conditioned fear. In this study, we examined the effects of intracranial injections of mirtazapine administered directly into the amygdala, hippocampus or MRN on the expression of contextual conditioned fear by using freezing as an index of fear.
Section snippets
Animals
Male Sprague-Dawley rats (260–320 g) were obtained from the Shizuoka Laboratory Animal Center (Shizuoka, Japan). The rats were housed in polypropylene cages with wood shavings on the floor, four animals per cage, with free access to food and water. The room temperature was kept at 22±2 °C. The subjects were maintained on a 12-h light/dark cycle (light phase: 06:30-18:30). Experiments began after a one-week period of acclimatization. All experiments were performed between 08:00 and 13:00, except
Histology
Tissue damage was not apparent in either the drug group or the vehicle group. Fig. 1 depicts the sites of drug injection into the MRN (A), dorsal hippocampus (B) and amygdala (C). The histological results were plotted on representative sections taken from the rat brain atlas of Paxinos and Watson (1997).
Effect of mirtazapine microinjection into the MRN on the expression of contextual conditioned freezing (Fig. 2A)
A unilateral mirtazapine microinjection into the MRN given 10 min before testing significantly reduced conditioned freezing compared with the vehicle group [t(15)=3.996, P<0.01].
Effect of mirtazapine microinjection into the dorsal hippocampus on the expression of contextual conditioned freezing (Fig. 2B)
Bilateral
Discussion
In the present study, we investigated the target brain sites where mirtazapine, a NaSSA, exerts its anxiolytic-like effect in the contextual fear conditioning test. The local microinjection of mirtazapine (3 μg/site) into the MRN, but not into the hippocampus or amygdala, reduced the expression of contextual conditioned freezing significantly. Moreover, intra-MRN treatment with mirtazapine did not change motor activity compared with the vehicle controls, thereby excluding non-specific motor
Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific Research No. 24591673 (T. Inoue) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
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