Subsequently we decided to test whether the orexin pathway w
Subsequently, we decided to test whether the orexin pathway was involved (Delhanty et al, 2012, Delhanty et al, 2013, Pei et al, 2014). Orexin neuronal systems are known to control limbic and neocortical structures (Bonnavion, de Lecea, 2010, Sakurai, 2007). Orexin receptors and possibly orexin synthesis are present in the hippocampus, a region of great importance for temporal lobe epilepsy (Marcus et al, 2001, Morales et al, 2006). Previously, a link between DAG and the orexin pathway was shown in relation to feeding (Toshinai et al., 2006) and the orexin family seems to be involved in epileptic mechanisms (Akbari et al, 2014, Akdogan et al, 2008, Doreulee et al, 2010, Erken et al, 2012, Kortunay et al, 2012, Rejdak et al, 2009). It is thought that orexin release in the hippocampus may be excitatory (Akbari et al, 2011, Ida et al, 1999, Selbach et al, 2004, Wayner et al, 2004). The effect of simultaneous orexin receptor antagonism in epileptic seizure models was never investigated, thus we first determined whether blocking the effect of orexins via the dual orexin receptor antagonist almorexant affected pilocarpine-induced limbic seizures. We found that dual orexin receptor blockade in the hippocampus did not significantly affect seizure activity when compared to the control group. This may imply that the excitatory nature of the orexins does not necessarily result in the promotion of epileptic seizures. For instance, the neuropeptide ghrelin has been documented to be excitatory in the hippocampus yet it has repeatedly been shown that it possesses anticonvulsant properties when tested in different seizure models (Portelli et al., 2012a). Another Protease Inhibitor Cocktail (100X in DMSO, EDTA plus) could be that orexin-A and orexin-B seem to have opposing actions in epilepsy (Akbari et al, 2011, Doreulee et al, 2010, Ida et al, 1999, Selbach et al, 2004, Wayner et al, 2004), and by blocking both receptors simultaneously the excitatory effect of one ligand is being hindered by the inhibitory effect of the other. The concentration of almorexant that was chosen to sufficiently block the hippocampal orexin receptors failed to prevent DAG from being significantly anticonvulsant when compared to the control group. Seeing that the orexin pathway is unlikely to be involved in DAG's anticonvulsant effects, we decided to perform additional experiments with ghrelin receptor deficient mice in order to investigate the possible involvement of the ghrelin receptor in DAG's anticonvulsant effects. As expected, both the C57Bl/6 mice and the ghrelin receptor wild type mice groups showed a significantly decreased vulnerability to pilocarpine-induced seizures when administered with DAG. Unexpectedly however we noted that DAG's beneficial effect on seizure thresholds in these mice was absent in the ghrelin receptor deficient mice. These results point to the notion that, at least in pilocarpine-induced seizures, the ghrelin receptor seems to be involved in DAG's mechanism of action. Thus, it appears that DAG is a functional endogenous agonist of the central ghrelin receptor with known physiological effects in the fields of diabetes as well as in epileptic seizures. Further investigations should be performed to investigate how DAG affects the ghrelin receptor to attenuate pilocarpine-induced acute seizures. We have previously shown that ghrelin attenuates seizures by desensitizing (and internalizing) the ghrelin receptor (Portelli et al., 2012b). Here ghrelin was administered locally in the hippocampus in rats and systemically in mice. In the hippocampus, ghrelin was continuously administered for 2 hr prior to pilocarpine initiation, whereas in mice ghrelin was systemically administered 30 min before pilocarpine intravenous infusion. Despite a number of studies that have also described anticonvulsant effects following ghrelin administration (Portelli et al., 2012a), there is still debate on whether ghrelin has an important role in epileptic mechanisms (Biagini et al, 2011, Biagini et al, 2011, Lucchi et al, 2013). The only plausible reason for the differences in our results and that of the group of Biagini is perhaps the timing of the ghrelin injection. It could be that administration of ghrelin 30 min prior to pilocarpine administration, as opposed to 10 min as described by Biagini et al. (2011) and Lucchi et al. (2013), allowed ghrelin more time to reach the central ghrelin receptors to desensitize/internalize the receptors, resulting in an anticonvulsant effect in our case. Although it is known that acylated peptides cross the blood–brain barrier more quickly than their unacylated counterparts (Pardridge, 2012), it still remains a matter of debate whether the anticonvulsant effect following systemic ghrelin administration is in fact due to ghrelin acting on the central ghrelin receptors or due to the fact that ghrelin can be quickly converted to DAG (Delhanty et al, 2015, Satou et al, 2011). Whether DAG acts similarly to ghrelin or else activates different pathways when bound to the ghrelin receptor remains to be scrutinized. Apart from that, DAG should be tested in other models of epileptic seizures to determine whether it is a significant anticonvulsant peptide.