br Results br Discussion In this study we

Results
Discussion In this study, we investigated the role of galanin and GAL2 receptors on anxiety-like behaviours mediated by the dorsal hippocampus of rats. We showed that locally administered galanin (1 nmol) decreased exploration of the open arms in the EPM, suggesting an anxiogenic-like effect. The dorsal hippocampus of rats expresses more GAL2 than GAL1 receptors (O’Donnell et al., 1999) and, apparently, does not exhibit GAL3 receptors (Mennicken et al., 2002). Based on this particular prolyl hydroxylase inhibitor of galanin receptors in the dorsal hippocampus, we hypothesized that the anxiogenic-like effect of galanin would be mediated by GAL2 receptors. Indeed, intra-dorsal hippocampal administration of M871, a selective GAL2 antagonist, produced an anxiolytic-like effect in rats exposed to the EPM, at least at the higher dose tested (3 nmol). This anxiolytic-like effect of the GAL2 antagonist points to a tonic effect of galanin on anxiety, mediated by the dorsal hippocampus. Neither galanin nor M871 affected locomotor activity (number of closed arm entries), thereby excluding nonspecific motor interference in the EPM behaviours. The next set of experiments we designed to confirm that GAL2 receptors are responsible for the anxiogenic-like effect of galanin in the dorsal hippocampus. For this approach, we initially chose a dose of M871 (1 nmol) that had no effect alone. Unexpectedly, pretreatment with this dose of M871 in the dorsal hippocampus did not counteract the anxiogenic-like effect of galanin in the EPM. We did not test the higher dose of M871 (3 nmol) due to its ability to induce an anxiolytic effect per se. Our next step was to evaluate the association of galanin and an intermediate dose of M871 (2 nmol), both drugs infused in the dorsal hippocampus of rats. We showed that previous administration of M871 2 nmol blocked the anxiogenic-like effect of galanin in the EPM, supporting the involvement of GAL2 receptors in this effect. Other studies have shown an anxiogenic role of galanin 1) by infusion of the neuropeptide in the amygdala (Moller et al., 1999), 2) by intracerebroventricular infusion of the fragment galanin 1–15 (Millón et al., 2015), or 3) by activation of DRN GAL1 receptors (Morais et al., 2016). On the other hand, several studies have demonstrated that galanin mediates anxiolytic-like effects in different conditions and brain regions (Barrera et al., 2006, Morais et al., 2016, Narváez et al., 2015a, Narváez et al., 2015b, Rajarao et al., 2007, Reyes-Alcaraz et al., 2016, Sciolino et al., 2012, Soares et al., 2016a, Soares et al., 2016b, Morais et al., 2016, Silote et al., 2013). Altogether, these results support the statement that the effects of galanin on anxiety depend on the brain region, animal model, dose and galanin receptors involved (Barrera et al., 2005, Holmes and Picciotto, 2006, Soares et al., 2016a, Soares et al., 2016b). The exact pre- and post-synaptic localization of galanin receptors in the dorsal hippocampus is not described (Kinney et al., 2009, Lang et al., 2015). However, most of the galanin-positive fibres in the dorsal hippocampus overlap with noradrenergic terminals originating in the locus coeruleus (Xu et al., 1998). Therefore, it is possible that galanin is released together with noradrenaline under stress conditions such as exposure to a new environment, e.g., the EPM. The release of endogenous galanin could be sufficiently strong to promote physiological consequences. Corroborating this idea, Millón et al. (2015) also described an anxiolytic-like effect of the GAL2 antagonist M871 administered by i.c.v. infusion in rats tested in an open field. If GAL2 receptors are heteroreceptors localized post-synaptically in the hippocampus neurons and are coupled to Gq proteins, their activation would stimulate neurotransmitter release. In fact, previous studies showed an increase of acetylcholine release in the dorsal hippocampus with galanin infusion into the CA1 region (Yoshitake et al., 2011) or with exposure to a novel environment (Giovannini et al., 2001). Though this idea is speculative, it raises the possibility that the anxiogenic-like effect of galanin could occur due to the greater release of acetylcholine. Even though the role of acetylcholine on anxiety in the hippocampus is not completely elucidated, some studies suggest that acetylcholine may increase anxiety through the hippocampus (Mineur et al., 2013, Zarrindast et al., 2011, Zarrindast et al., 2013).