Alternatively procedural factors may have contributed to the

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, 2188 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 Nicotine dependence drives the habit of smoking, which causes a heavy load of disease and death, as smoking is one of the largest contributors to preventable morbidity and mortality (GBD 2015 Risk Factors Collaborators, 2016; World Health Organization(WHO), 2017). Inhaled nicotine reaches the brain, where it binds and activates nicotinic acetylcholine receptors (Hurst et al., 2013; Zoli et al., 2015) in several brain regions, including the mesocorticolimbic regions involved in the reward system (Picciotto and Kenny, 2013). Nicotine stimulates dopamine release from neurons originating in the ventral tegmental area (VTA) and terminating in the nucleus accumbens and prefrontal cortex (PFCx). Repeated nicotine exposure causes long-lasting adaptations of dopaminergic transmission that mediate the motivation to maintain nicotine self-administration despite the known harmful effects. The long-term effects of nicotine that support compulsive use require the activation, desensitization, and up-regulation of the nicotinic receptor to mediate alterations in the activity of the neuronal circuitry of the mesocorticolimbic system (De Biasi and Dani, 2011; Pistillo et al., 2015). The changes in synaptic function are based on the regulation of transcriptional and epigenetic modulations that sustain the onset of addiction (Pistillo et al., 2015). Among the systems which mediate the neurobiological adaptations supporting addiction, a role has been proposed for the corticotropin releasing factor (CRF) system (Koob, 2010; Zorrilla et al., 2014). CRF (also known as CRH) is a 41-amino acid neuropeptide that has a role in coordinating the endocrine, autonomic, and behavioural response to stress through the regulation of the hypothalamic-pituitary-adrenal stress system (Bale and Vale, 2004). In addition to the stress-response regulation related to its hypothalamic expression, a wider set of functions were discovered in association with CRF expression in numerous brain areas which demonstrate its relevance in anxiety, depression, and addiction (Bale and Vale, 2004; Hauger et al., 2006; Zorrilla et al., 2014). The CRF family includes three additional members called urocortin 1, urocortin 2 and urocortin 3, which show a more confined expression ocurring mainly in hypothalamic and brainstem structures (Hauger et al., 2006). CRF released from synaptic vesicles plays a neuromodulatory role that varies depending on whether the neuropeptide and its receptor are expressed on glutamatergic or GABAergic neurons, which leads to different responses in distinct brain regions (Henckens et al., 2016). CRF binds to two G-protein-coupled receptors, CRF1R and CRF2R, with a 4- to 20-fold higher affinity towards CRF1R, thus activating signal transduction pathways including cyclic AMP–protein kinase A, mitogen-activated protein kinases, and other pathways. These signals result in modulating the transcription of downstream target genes, synaptic transmission, and plasticity, thus mediating short- and long-term effects (Hauger et al., 2006; Henckens et al., 2016). Within the conceptual three-stage framework for addiction, comprising of binge/intoxication, reward, and withdrawal (Koob and Volkow, 2016, 2010), a role for the CRF system has been mainly proposed in the withdrawal phase in association with negative affect, dysphoria, and anxiety feelings (Baiamonte et al., 2014; Bruijnzeel et al., 2012, 2009; Cohen et al., 2015; George et al., 2007; Koob, 2010; Marcinkiewcz et al., 2009; Zhao-Shea et al., 2015). However, a significant role in the reward component cannot be ruled out (Brielmaier et al., 2012; Lemos et al., 2012; Peciña et al., 2006).