ryanodine In the present study we investigated

In the present study, we investigated the role played by CRF1 and CRF2 receptors located within the mouse PAG on the anxiogenic and antinociceptive effects produced by local infusion of CRF. To block CRF receptors, we used the selective CRF1 and CRF2 ryanodine antagonists, respectively, NBI 27914 ((5-chloro-4-(N-cyclopropyl)methyl-N-propyl)-2-methyl-6-(2,4,6-trichlorophenyl) aminopyridine) (Hammack et al., 2003, Jochman et al., 2005), and antisauvagine-30 (Sahuque et al., 2006, Takahashi et al., 2001).
Materials and methods
Results
Discussion Intra-dPAG microinjections of CRF reduced open arm exploration without affecting general activity (closed-arm entries) in the EPM. This result corroborates previous studies demonstrating that intra-PAG CRF enhances anxiety-like indices in the EPM in rats (Borelli and Brandão, 2008, Martins et al., 1997) as well as in mouse anxiety tests such as the mouse defense test battery (MDTB) and rat exposure test (RET, Carvalho-Netto et al., 2007). Here, the anxiogenic effects of CRF (i.e., reduction in% open-arm entries and% open-arm time) were completely blocked by a prior intra-dPAG injection of NBI 27914, a CRF1 receptor antagonist. Importantly, closed-arm entries, a widely used measure of general activity level (e.g., Cruz et al., 1994, Rodgers and Johnson, 1995), remained unchanged in animals treated with NBI 27914, combined either with CRF or with vehicle, suggesting that the NBI 27914 effects were selective in reducing the anxiogenic effects produced by CRF. The reduction in the closed-arm entries observed with the lower dose of CRF (Fig. 2A) seems to be an isolated effect, since the higher dose of the neuropeptide did not confirm this effect, either when injected alone (Fig. 2A) or combined with vehicle (Figs. 3A and 5A). Indeed, in addition to preventing the anxiogenic-like effects of intra-dPAG CRF on direct measures of open arm exploration (see above), intra-dPAG NBI 27914 also prevented the anxiogenic-like increase in protected SAP and reduction in unprotected SAP as well as tended to reduce unprotected HD and open arm end exploration seen in response to CRF treatment. Importantly, intra-dPAG NBI 27914 was completely devoid of intrinsic behavioral activity under present test conditions. Taken together, the present results suggest that the antianxiety effect following deletion of CRF1 receptor gene in mice observed elsewhere (Contarino et al., 1999, Smith et al., 1998, Timpl et al., 1998) might be at least in part due to a dysfunction of PAG CRF1 receptor activity. Intra-dPAG injections of CRF also reduced the nociceptive response induced by formalin injection into the hind paw. CRF-treated animals showed a reduction in the time spent on licking the formalin-injected paw, characterizing an antinociceptive role played by this neuropeptide in the mouse PAG. Presently, we do not know how CRF elicits antinociception in the PAG. Possibly, CRF activates neurons that belong to the descending inhibitory system of pain (Fields and Basbaum, 1999), which, in turn, inhibit nociceptive afferents in the dorsal horn of the spinal cord (Harris, 1996, Kharkevich and Churukanov, 1999, Millan, 2002). Similar to what was observed in anxiety-like indices, intra-dPAG injection of NBI 27914 completely blocked the antinociceptive effect of CRF, suggesting, for the first time, that CRF1 receptors located within this midbrain structure play a role in the modulation of nociception. Both anxiogenic and antinociceptive effects produced by CRF seem to be selective for the CRF1 receptor, since intra-dPAG microinjection of ASV 30, a CRF2 receptor antagonist, altered neither the anxiety nor the antinociception response. Intra-dPAG ASV 30 was also unable to prevent the anxiogenic (observed in both conventional and ethological anxiety-related measures) and antinociceptive effects produced by local infusion of CRF. Thus, despite their presence in all columns of the PAG (Steckler and Holsboer, 1999), CRF2 receptors do not play a role in anxiety, assessed in the EPM, or in nociception assessed in the formalin test in mice.