CRF binds to CRF and
CRF binds to CRF1 and CRF2 receptor subtypes, triggering downstream cascades and leading to stimulatory G protein (GsP) activation. Once activated, GsP undergoes a structural change provoking the separation of the subunits Gα and Gβγ. The Gα subunit induces the production of the second messenger cAMP (cyclic adenosine monophosphate) by adenylyl cyclase enzyme. cAMP activates cAMP-dependent protein kinase A (PKA), leading to a series of downstream events which involve transcription factor CREB (cyclic AMP-responsive element binding protein) phosphorylation and, consequently, neuron activation (Chang et al., 1993, Chen et al., 1993, Lovenberg et al., 1995, Perrin et al., 1995, Vita et al., 1993). Moreover, Kageyama et al. (2007) showed that CRF acts through the cAMP/PKA pathway. Actually, CREB phosphorylation seems to be a pivotal process related to neuronal activation. For instance, previous studies have shown that this event is augmented in threatening situations, such as predatory hunting and predator odor tests (Adamec et al., 2003, Adamec et al., 2009, Kabitzke et al., 2011). Nevertheless, Stern et al. (2011) proposed that CRF-induced CREB phosphorylation can also be accomplished through an accessory process that involves Gβγ subunit activation and posterior mitogen-activated protein kinase kinase/mitogen-activated protein kinase (MEK/MAPK) pathway activation, which phosphorylates CREB. Besides these two pathways, CREB may be phosphorylated by other kinase proteins such as CaMK (calmodulin kinase) and protein kinase C (PKC) (Hauger et al., 2006, Silva et al., 1998). Thus it remains unclear through which intracellular mechanisms CRF changes defensive behavior when this peptide activates CRF receptors in the mPFC. Although CRF1 and CRF2 receptors are distributed in several streptozotocin areas, it is important to note that CRF1, but not CRF2, is strongly expressed in the mPFC (Chalmers et al., 1995, Steckler and Holsboer, 1999, Van Pett et al., 2000).
Therefore, in view of (i) the contrasting effects on anxiety produced by low and high doses of CRF in the mPFC (Ohata and Shibasaki, 2011), (ii) the large density of CRF1 receptor in the mPFC (Steckler and Holsboer, 1999) and (iii) the distinct pathways through which CRF may phosphorylate CREB (Hauger et al., 2006, Kageyama et al., 2007, Stern et al., 2011), this study was conducted to investigate the effects of CRF peptide (as a CRF receptor agonist) and CRF1 receptor antagonist (CP376395) on anxiety of mice exposed to the EPM (Experiments 1 and 2). To investigate whether the effects of CRF on anxiety-like behavior depend on the intracellular cAMP/PKA pathway, mice received intra-mPFC injections of a PKA inhibitor (H-89) alone (Experiment 3) or prior to local injection of CRF (Experiment 4) and were exposed to the EPM.
Materials and methods
Discussion Intra-mPFC CRF increased anxiety-like behaviors in mice subjected to the EPM, by reducing open-arm exploration (i.e. percent open-arm entries and percent open-arm time) without affecting the general level of activity (i.e. closed-arm entries) in the maze. These spatiotemporal results were confirmed by the analysis of complementary behavior, which revealed some important changes provoked by higher doses of CRF (75 and/or 150pmol) in the ethological parameters (namely, a decrease in the frequency of unprotected SAP, unprotected HD and OAEE and an increase in the frequency of protected SAP). These effects of CRF on complementary measures confirm the enhancement of open-arm aversion, since mice spent less time on the potentially threatening areas of the maze. The present results corroborate a previous finding that intra-mPFC injections of CRF increase defensive behavior in rats exposed to the light-enhanced startle test (Bijlsma et al., 2011). In line with those and the present results, intra-mPFC injections of CRF intensified the anxiogenic-like effect induced by restraint stress in rats exposed to the EPM (Jaferi and Bhatnagar, 2007). Nonetheless, the present results partially corroborate a recent study conducted by Ohata and Shibasaki (2011), who reported that the effects of intra-mPFC injections of ovine CRF (oCRF), a highly selective CRF1 receptor agonist (Eckart et al., 2001, Hillhouse and Grammatopoulos, 2006, Reul and Holsboer, 2002), on anxiety are dose-dependent. Briefly, while low doses (0.05μg which corresponds to 10pmol) of oCRF produced anxiogenesis, high doses (1.0μg or 210 pmol) of this CRF1 agonist led to the opposite effect, i.e. anxiolysis in rats exposed to the EPM (Ohata and Shibasaki, 2011). By contrast to these findings, the present results showed that while the lowest dose of CRF (37.5pmol or 0.18μg) did not change any behavioral parameters of mice in the EPM, the highest dose (150pmol or≈0.74μg) produced a robust anxiogenic-like effect, suggesting that at least within of this telencephalic area, CRF increases (rather than decreases) anxiety-like behavior in mice exposed to the EPM. Given that oCRF displays higher affinity to CRF1 receptors than human,rat CRF (h,rCRF, the drug used in the present study) (Eckart et al., 2001, Hillhouse and Grammatopoulos, 2006, Reul and Holsboer, 2002), one could assume that lower doses of oCRF might produce anxiogenic-like effects similarly to that observed with higher doses of h,rCRF. Evidence corroborating this assumption is found in previous studies in which oCRF and h,rCRF were injected into the midbrain periaqueductal gray (PAG) of mice exposed to two different tests of anxiety. Briefly, while low doses of oCRF (0.03 and 0.1μg) produced anxiogenic-like effects in a prey (mouse)–predator (rat) interaction (Carvalho-Netto et al., 2007), intra-PAG injections of h,rCRF enhanced anxiety-related behavior only at higher doses (≈0.74μg) in mice exposed to the EPM (Miguel and Nunes-de-Souza, 2011). However, very recently, Pentkowski et al. (2013) showed that intra-mPFC injections of cortagine (0.05μg and 0.1μg), a highly selective CRF1 receptor agonist (Tezval et al., 2004), reduced (rather than increased) the defensive behavior of mice exposed to a predator. Thus, at least in mice, the role of the endogenous CRF at CRF1 receptors located within the mPFC in the modulation of defensive behavior probably depends on the type of threatening situation (EPM or predator) confronting them.