Alternatively procedural factors may have contributed to

Alternatively, procedural factors may have contributed to the conflicting results. For instance, in order to have a more circumscribed area of drug diffusion, a volume of 0.05μl was injected in experiment 1b, which is four times lower than the one used in the mentioned plus-maze investigation. Besides, for a more precise identification of the injection sites, Veratridine slices in our experiment were immunostained for the NADPH-diaphorase, which is massively found in the dlPAG, but not in the dmPAG or lPAG, allowing, therefore, a clearer demarcation among these subfields (Carrive and Paxinos, 1994). Finally, in the study of Borelli and Brandao (2008) the effects of CRF in each of the PAG columns were assessed separately and not in the same experimental session as here. This may have compromised data comparison in their study, given well-known fluctuation in baseline activity of control subjects between experiments (Hogg, 1996). One important finding of the current study is that activation of dPAG CRF2 receptors by urocortin 2 did not interfere with escape expression in the two tests employed, but significantly impaired inhibitory avoidance learning in the elevated T-maze. It should be noted that although the escape threshold of control animals in the in experiment 2b was lower than in the other experiments with the electrical stimulation of dPAG, it is unlike that a floor effect may have masked a pro-escape effect of urocortin 2. It has been shown that the variation in escape threshold (intensity of electrical current after drug injection minus intensity before drug) may assume values below zero, as observed, for instance, after bicuculline administration in the dPAG (Casarotto et al., 2010). Evidence from both pharmacological and knockdown studies corroborates with the anxiolytic effect found here after CRF2 receptor activation. For instance, it has been shown that CRF2 receptor deficient mice are hypersensitive to stress and exhibit enhanced anxious behavior (Bale et al., 2000, Kishimoto et al., 2000). Intracerobroventricular injection of urocortin 3, a highly selective CRF2 receptor agonist, increases open-arms exploration in rats exposed to the elevated plus-maze (Valdez et al., 2003). Results such as these have led to the proposal that CRF1 and CRF2 receptors play opposing roles in anxiety regulation (Bale, 2005) and that the former ligand site may facilitate recovery of stress response, acting to inhibit initial CRF1-induced aversive consequences (Reul and Holsboer, 2002, Risbrough and Stein, 2006). However, other evidence in the literature indicates that this may be a simplistic model, and many factors such as drug dose, brain location, time, stress basal level and type of behavior measured may influence the direction of CRF2-evoked effects (Henry et al., 2006, Risbrough and Stein, 2006). This seems to be the case here where an opposing influence of CRF1 and CRF2 receptors was evidenced on avoidance, but not escape regulation. Such a sort of mismatch has been also seen for CRF1 and CRF2 receptor influence on the magnitude and plasticity of defensive startle response in mice. Thus, Risbrough et al. (2004) showed that whereas the two CRF receptors act in concert to increase startle reactivity, they work in opposition to regulate the flexibility of startle, measured by inhibition of startle by sensory stimuli (i.e. prepulse inhibition). Therefore, while the role played by CRF1 receptors in enhancing defensiveness has been widely acknowledged, the influence of CRF2 in this process seems to be more complex (for a recent review, see Janssen and Kozicz, 2013).
Role of the funding source This work was supported by National Counsel of Technological and Scientific Development (CNPq); Foundation for Research Support of the State of Sao Paulo (FAPESP), Brazil (Grant number: 2010/07286-9).
Conflict of interest
Acknowledgments
Introduction Corticotropin-releasing factor (CRF) is a 41-amino acid polypeptide that plays a central role in coordinating the endocrine, immune, autonomic, and behavioral responses to stress [1], [2], especially in physiological regulation of the endocrine system related to the hypothalamic–pituitary–adrenal (HPA) axis. When physiological or psychological stress occurs, CRF is synthesized and secreted from the hypothalamic paraventricular nucleus (PVN) and binds to CRF receptors in the pituitary gland. This triggers secretion of adrenocorticotropin (ACTH) from the anterior lobe of the pituitary. ACTH stimulates glucocorticoid secretion from the adrenal glands into blood, and glucocorticoid exerts a negative feedback on the activity of HPA axis [1], [3], [4]. In the disease state, hyperactivation of HPA axis and hypersecretion of CRF have been reported to be related to the pathogenesis of anxiety and depression [5], [6], [7], and modulation of CRF signaling has been investigated as a new treatment strategy for those mood disorders. Two CRF receptors have been identified, named as CRF1 and CRF2. CRF1 receptors are expressed in the mammalian gastrointestinal (GI) tracts, anterior pituitary located outside of the blood-brain barrier (BBB), and the brain regions related to emotion and cognitive processes, such as amygdala, hippocampus, and cerebral cortex [8], [9], [10], whereas the distribution of CRF2 receptors is more restricted [11], [12]. In addition, CRF1 receptor knockout mice show less behaviors related to anxiety and depression compared with those in wild-type mice [13], [14], [15]. Thus, CRF1 receptor antagonists have been recognized as promising candidates for new anxiolytics and antidepressants.