CRF binds to CRF and CRF receptor subtypes triggering
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 Busulfan 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 The consistent anti-anxiety effects obtained with intra-mPFC injections of CP 376395 strongly suggest a tonic role of CRF at CRF1 receptors located within this limbic brain area in the modulation of anxiety in the mouse on the EPM. Intra-mPFC injection of CP 376395 led mice to explore the potentially aversive areas of the EPM to a greater degree. When given alone, all three doses (0.75, 1.5 and 3.0nmol) of this highly selective CRF1 antagonist attenuated spatiotemporal (%OE and %OT) and complementary (uHD, uSAP, OAEE) measures of anxiety without changing general locomotion (i.e., closed-arm entries). The present results are in line with previous findings showing an anxiolytic-like effect following the deletion of the CRF1 receptor gene in mice (Contarino et al., 1999, Smith et al., 1998, Timpl et al., 1998). Briefly, those authors showed that the lack of CRF1 receptor activity attenuates anxiety-like behavior of mice exposed to the EPM and light/dark box models. The hypothesis raised above, in that the role of endogenous CRF at CRF1 receptors located within the mPFC in the modulation of defensive behavior depends on the type of threatening situation confronting the animals, is strengthened by the lack of effect of local infusions of the acidic-astressin [Glu11,16]Ast, a CRF1 antagonist (Eckart et al., 2001), on the behavior of mice exposed to a predator (Pentkowski et al., 2013). Differently from what we showed in this study with the mouse EPM, those authors reported that intra-mPFC injections of 0.1 or 0.2μg of [Glu11,16]Ast did not change any behavioral measure in mice subjected to the rat exposure test.