br Materials and methods br Results
Materials and methods
Discussion Intra-BNST injections of NOC-9 (at all doses) were able to produce freezing and anxiogenic-like effects. Freezing was observed immediately after the NO donor injection and lasted about 2–3min (data not shown). When exposed to the EPM 5min after intra-BNST NOC-9 injection, mice displayed anxiogenic-like behavior. Given that the BNST has a dense population of NMDA glutamate receptors (Guimaraes et al., 2005, McElligott and Winder, 2008) as well as CRF neurons (Cummings et al., 1983, Ju et al., 1989, Morin et al., 1999, Sakanaka et al., 1987, Swanson et al., 1983), we investigated whether the anxiogenic-like effect induced by the increase in NO release (induced by local injection of NOC-9) would depend on glutamate and/or CRF actions at their respective receptors. NOC-9 produces an intracellular reaction at physiological pH, triggering a NO release without peroxynitrite formation, that could damage neuronal membranes (Seccia et al., 1996). The aversive effect of NOC-9 has already been highlighted in previous studies involving its microinjection in other alltrans sites, such as the inferior colliculus, amygdala and PAG (Braga et al., 2009, Miguel et al., 2012). For instance, intra-PAG injections of NOC-9 elicited explosive motor behaviors (e.g., jumping and running) followed by freezing and pain inhibition (Miguel et al., 2012). However, as observed in the present study, when injected into the BNST, NOC-9 seems to elicit more subtle defensive behaviors. Although this NO donor produces freezing, it seems to be unable to provoke jumping and running when injected into the BNST. One could argue that the enhancement of NO production might induce freezing and anxiety wherever a NO donor is injected into any brain limbic structures. Recent results with NOC-9 injections into the medial prefrontal cortex (mPFC) have suggested that this assumption is not entirely true. Intra-mPFC injections of NOC-9 induce anxiety in mice exposed to the EPM, however this NO donor did not provoke any other anxiogenic-like behavior (results not published). Together, these findings corroborate previous evidence showing that defensive behavior would be coordinated by a hierarchical brain defensive system (McNaughton and Corr, 2004). Those authors have argued that while the anxiety state is mediated mainly by forebrain structures (e.g. prefrontal dorsal stream, posterior cingulate, septo-hipocampal system, and amygdala), the fear state involves more caudal structures (e.g. medial hypothalamus and periaqueductal gray). Although it would be expected that both rostral and caudal limbic structures play a role in the modulation of anxiety and fear, the BNST seems to be a forebrain area involved in the modulation of more subtle defensive behaviors. Curiously, as far as we know, the effects of NOC-9 injection into the BNST on defensive behavior had never been tested prior to our study. We have recently demonstrated that fear-like behaviors (e.g., jumping, running, freezing) induced by intra-PAG injection of NOC-9 were completely blocked by prior local injection of NBI 27914, a CRF1 antagonist, suggesting that NO production may lead to CRF release within this midbrain area. The hypothesis that the aversive effects induced by NO production could be mediated by CRF1 receptors within the BNST was tested in the present study through combined local injections of CP 376395 and NOC-9 in mice. We decided to focus on CRF receptor type 1 given the results obtained by our group in the PAG (Miguel and Nunes-de-Souza, 2011) and amygdala (not published) showing a role of CRF1 (but not CRF2) on the modulation of anxiety-like behavior. Interestingly, while intra-BNST CP 376395 was incapable of changing the freezing induced by local injection of NOC-9 (Fig. 5A), this CRF1 antagonist attenuated the anxiogenic-like behavior produced by the NO donor in mice exposed to the EPM. Although statistics had revealed only a tendency for pretreatment×treatment interaction (p=0.06), post hoc tests confirmed that CP 376395 impaired the effects of NOC-9 on anxiety indices (Fig. 5B), without changing closed-arm entries, a widely used measure of general activity (Cruz et al., 1994, Rodgers and Johnson, 1995). These results indicate that the anxiogenic-like effects produced by NOC-9 in the BNST are attenuated by the blockade of CRF1 receptors, suggesting that the neuropeptide CRF plays an important role in the modulation of anxiety-related behaviors induced by NO release. Evidence showing that NO may release CRF was shown by Raber et al. (1995) who described an increase of CRF release into the amygdala and hypothalamus after treatment with nitroprusside, a NO donor. However, as shown in the present study, CRF1 receptors located within the BNST do not seem to be involved in the modulation of the NO-induced freezing. This lack of effects of CP 376395 is in contrast to those observed when the CRF1 receptors are blocked in the midbrain PAG. Briefly, intra-PAG NOC-9-induced freezing was absent in mice that received prior local injection of NBI 27914, a CRF1 receptor antagonist (Miguel et al., 2012).