Research ReportRGS2 mediates the anxiolytic effect of oxytocin
Highlights
► Oxytocin (OT) increased RGS2 levels in the amygdala of virgin mice. ► Acute restraint stress induced RGS2 expression in the amygdala. ► OT antagonist attenuated stress-induced RGS2 expression. ► Restraint stress did not increase RGS2 levels in the amygdala of OTR KO mice.
Introduction
OT is the classical reproductive hormone in female mammals, promoting uterine contractions during labor and milk ejection during lactation (Gainer and Wray, 1994, Neumann, 2001). OT also acts as a neurotransmitter/neuromodulator to regulate a range of central nervous system functions in both males and females, including emotional (Neumann, 2008), parental (Numan and Insel, 2003), affiliative (Insel and Shapiro, 1992), and sexual (Argiolas and Gessa, 1991) behaviors, as well as spatial and social cognition (Bielsky and Young, 2004, Tomizawa et al., 2003). Moreover, OT is an important regulator of anxiety (Bale et al., 2001, Blume et al., 2008, Neumann et al., 2000a) and of stress-coping circuitries (Ebner et al., 2005, Huber et al., 2005). For instance, OT released in the hypothalamus mediates mating-induced anxiolysis in rats (Waldherr and Neumann, 2007). Psycho-social or physical stressors like forced swimming and restraint stress evoke the release of OT in various areas known to be involved in the modulation of stress mechanisms, including the amygdala and the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei (Ebner et al., 2000, Ebner et al., 2005, Wigger and Neumann, 2002). In humans, moreover, intranasal OT promotes trust, and reduces the level of anxiety, possibly at the level of the amygdala (Heinrichs et al., 2003, Kirsch et al., 2005, Kosfeld et al., 2005, Labuschagne et al., 2010, Slattery and Neumann, 2010).
The amygdala is a region of the brain particularly relevant to the processing of behavioral and neuroendocrinal stress responses (Gray, 1996), especially with respect to the oxytocinergic system (Bale et al., 2001, Neumann et al., 2000a). Within the amygdala, specifically in the medial and central (CeA) subnuclei, a substantial number of oxytocinergic fibers (Sofroniew, 1983) and OT receptors have been detected (Barberis and Tribollet, 1996, Gimpl and Fahrenholz, 2001), suggesting that locally released OT is a potential mediator of the complex stress response. Indeed, local blockade of OT receptors within the amygdala resulted not only in altered emotionality (Bale et al., 2001, Neumann, 2002) but also in a dis-inhibition of the hypothalamo-pituitary-adrenal (HPA) axis of rats (Neumann et al, 2000b).
There is no doubt that OT in its activated state is an endogenous neuromodulator with an anxiolytic effect in response to stress. However, the precise mechanism of OT receptor-mediated effects and the involvement of subsequent intracellular signaling cascades, for example in the anxiolytic effects of OT, are only beginning to be elucidated (Blume et al., 2008). There is an OT receptor, which is a G-protein-coupled receptor (GPCR) that couples to a complex intracellular signaling pathway (van den Burg and Neumann, 2011). The binding of OT with the receptor activates the MAP kinase cascade both in vitro (Tomizawa et al., 2003) and in vivo (Blume et al, 2008) and induces the phosphorylation of cAMP response element-binding protein (CREB), a transcription factor critical to neuronal development, synaptic plasticity and memory formation (Han et al., 2007, Lonze et al., 2002, Tomizawa et al., 2003). These results suggest the anxiolytic effect of OT to occur through the regulation of gene expression.
Regulator of G-protein signaling (RGS) proteins are key modulators of G protein-coupled receptor signaling by virtue of their ability to accelerate the intrinsic GTP hydrolysis activity of G subunits (Hollinger and Hepler, 2002, Hollinger and Hepler, 2004). RGS2 was one of the first mammalian RGS proteins to be identified, (Siderovski et al., 1996) and stimulates GTPase activity through interaction with Gqα in vitro (Heximer et al., 1997, Hollinger and Hepler, 2002). Previous studies have shown that RGS2 modulates anxiety in both mice and humans (Cui et al., 2008, Flint, 2003, Oliveira-Dos-Santos et al., 2000, Smoller et al., 2008, Yalcin et al., 2004). For instance, the use of transgenic and knockout mice as well as quantitative trait locus (QTL) techniques in the laboratory has led to the identification of candidate genes related to fear- and anxiety-related behaviors (Norrholm and Ressler, 2009). In mice, a QTL on chromosome 1 is associated with anxiety-related phenotypes (Flint, 2003); the principal quantitative trait gene for this linkage signal has been identified as RGS2 (Yalcin et al., 2004). In addition, RGS2 gene polymorphisms have been associated with panic disorder (Leygraf et al., 2006) and completed suicides (Cui et al., 2008).
The aim of the present study was to clarify the molecular mechanism behind the local anxiolytic effect of OT in the amygdala. We first identified RGS2 as a gene whose expression is up-regulated by OT and investigated whether RGS2 expression was increased in OT-applied amygdala slices and lactating mice. Moreover, we examined whether acute restraint stress induced OT secretion and RGS2 expression in CeA of female mice, resulting in an anxiolytic effect. Finally, a deficiency and blockade of the OT receptor was found to abrogate these effects of acute restraint stress.
Section snippets
Up-regulated genes in primary cultured neurons treated with oxytocin
We first examined up-regulated genes in primary cultured neurons treated with OT by a microarray analysis. Five genes were up-regulated more than two-fold compared with control neurons (Table 1). The greatest increase was shown by the RGS2 gene (Table 1). Moreover, we examined up-regulated genes in the central amygdala (CeA) of lactating mice at postpartum 7 days by a microarray analysis. Only RGS2 was up-regulated more than two-fold among the five genes in the CeA of lactating mice compared
Discussion
The present study provided the following four important findings (Table 2). First, OT induced RGS2 expression in the CeA of female mice. Second, repeated restraint stress also induced RGS2 expression in the CeA. Third, RGS2 levels in the CeA correlated with anxiolysis. Fourth, restraint stress neither increased RGS2 levels in the CeA nor had an anxiolytic effect in OT receptor-deficient or OT receptor antagonist-injected mice.
Psycho-social or physical stressors like restraint stress and forced
Subjects
Female C57BL6 mice aged 10 to 12 weeks were used for all experiments except those with OT receptor (OTR)-deficient mice.
OTR-deficient mice (OTR-KO) were purchased from Deltagen (San Mateo, CA, USA) (Matsushita et al., 2010). The wild-type (WT) female littermates were used as controls. Originally in an equal mix of the C57BL/6 and 129X1/SvJ strains (RW4 embryonic stem cell line), they have been repetitively backcrossed for 4 generations with C57BL/6 mice (from Deltagen). The females aged 10 to 12
Acknowledgments
This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant-in-aid for Scientific Research from the Ministry of Health, Labor and Welfare of Japan.
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