br Acknowledgements br Introduction Anxiety and depression
Introduction Anxiety and depression are major components of maintaining the cycle of addiction, and are responsible for the negative reinforcement of drug seeking behaviors (Sarnyai et al., 1995, Koob and Le Moal, 2008). Both rats and humans exhibit increased anxiety states during withdrawal from amphetamine (Cantwell and McBride, 1998, Schuckit et al., 1999, Srisurapanont et al., 1999a, Srisurapanont et al., 1999b; Srisurapanont, 2001; Romanelli and Smith, 2006, Shoptaw et al., 2009, Barr et al., 2010, Vuong et al., 2010). There is currently no FDA-approved treatment for the negative affect and increased anxiety states exhibited during psychostimulant withdrawal, thus there is great need to identify potential pharmacological targets. Corticotropin-releasing factor (CRF) is strongly implicated in negative affect and anxiety states (Radulovic et al., 1999, Takahashi, 2001, Bale and Vale, 2004, Bale, 2005, Lukkes et al., 2009b). Activation of the CRF2 receptors within the serotonergic cell body region, the dorsal raphe nucleus (dRN), increases serotonin neuronal activity and release of serotonin in the limbic system (Pernar et al., 2004, Forster et al., 2006, Forster et al., 2008, Scholl et al., 2010). Furthermore, CRF2 receptors are up-regulated in the dRN of rats for up to 6 weeks of amphetamine withdrawal, with no change in expression of dRN CRF1 receptors (Pringle et al., 2008). Infusions of CRF into the dRN augments serotonin release in the central nucleus of the amygdala (CeA) of amphetamine pre-treated rats, as compared to CRF-infused controls, via CRF2 receptor-dependent mechanisms (Scholl et al., 2010). Importantly, anxiety-like behaviors of rats undergoing amphetamine withdrawal can be reduced by CRF2 receptor antagonism directly within the dRN (Vuong et al., 2010). Therefore, CRF2 receptor antagonists may be important in the treatment of withdrawal-induced anxiety to prevent the ongoing cycle of addiction. However, it is not known whether widespread antagonism of central CRF2 receptors throughout the entire po1 mg will be effective in reducing anxiety states during amphetamine withdrawal. This is important to understand, given that any potential therapeutics targeting CRF2 receptors will not be directly infused into the dRN, but instead will access CRF2 receptors throughout the entire brain as a result of systemic administration. The global brain distribution of CRF2 receptors is limited (as compared to CRF1 receptors), but expression is highest in regions relevant to fear and anxiety-like behaviors, including the hypothalamus, amygdala, lateral septum (LS) and bed nucleus of the stria terminalis (BNST; Chalmers et al., 1995, Radulovic et al., 1999, Liu et al., 2004, Henry et al., 2006, Skórzewska et al., 2011, Takahashi et al., 2011, Lebow et al., 2012, Ventura-Silva et al., 2012, Elharrar et al., 2013). Therefore, in addition to the dRN, centrally administered CRF2 receptor antagonism will affect multiple brain regions that have the potential to affect anxiety states during amphetamine withdrawal. Whether activation of central CRF2 receptors enhances or reduces anxiety-like behaviors has been tested using genetic models and pharmacological manipulation, with conflicting results. For example, CRF2 receptor knockout mice show hypersensitivity to swim stress and displayed increased anxiety behaviors compared to wild-type mice (Bale et al., 2000, Kishimoto et al., 2000), suggesting that central CRF2 antagonism would increase anxiety behaviors. Conversely, ventricular administration of the CRF2 receptor antagonist, antisauvagine-30 (ASV) decreases anxiety-like behaviors of rats when compared to vehicle infusion (Takahashi, 2001, Takahashi et al., 2001). Given the conflicting findings, and the fact that previous research has not tested the central effects of CRF2 receptor antagonism in a pre-stressed model, the current studies determined whether central CRF2 receptor antagonism is effective at reducing anxiety-like behaviors of rats during amphetamine withdrawal. We also examined whether expression of CRF1 and CRF2 receptors in the CeA, LS and BNST is altered by either amphetamine or saline pre-treatment, in order to elucidate which regions aside from the dRN may facilitate behavioral changes following central CRF2 receptor antagonism.