Discussion
The present study demonstrates, for the first time, the existence of an interaction between Galanin (GAL) and neuropeptide y (NPY) in the dentate gyrus (DG) of the ventral hippocampus upon the intranasal administration of GALR2 and Y1R agonists. Intranasal (i.n.) administration offers exciting potential to bypass the blood-brain barrier for the direct delivery of peptides and protein therapeutics rapidly into the CNS, supported by robust evidence in preclinical and clinical trials (Lochhead & Thorne, 2012; Rawal, Patel, & Patel, 2022). This method will be crucial as novel therapies continue to be studied in clinical trials and has several benefits, such as fewer side effects than peripheral administration and the comfort of non-invasiveness application (Chapman et al., 2013; Crowe & Hsu, 2022). Accordingly, intranasal esketamine has recently come into the market as an antidepressant, but its use is limited due to potential neurotoxicity, psychocomimetic side effects, potential abuse and interindividual variability in treatment response (Langmia, Just, Yamoune, Muller, & Stingl, 2022).
Following intranasal GALR2 and Y1R agonists co-administration we observed an increased cell proliferation in the ventral dentate gyrus (DG) of the hippocampus by using the proliferating cell nuclear antigen (PCNA). In agreement, we have recently observed the ability of the co-agonist treatment to enhance the cell proliferation in the DG of the dorsal hippocampus at 24 hours (Borroto-Escuela et al., 2022; Mirchandani-Duque et al., 2022). Interestingly, enhanced resilience in a model of depression was recently demonstrated by genetically boosting neurogenesis in the ventral hippocampal DG (Planchez et al., 2021). Furthermore, the molecule P7C3 increased cell proliferation in the hippocampal DG associated to antidepressant effects in rodents and primates (Bauman et al., 2018; Walker et al., 2015). Moreover, intranasal administration of the Y1R agonist alone increased cell proliferation in the ventral DG in this work. However, we observed that the Y1R agonist lacks effects on cell proliferation in the dorsal DG (Borroto-Escuela et al., 2022; Mirchandani-Duque et al., 2022). These results confirm the functional differences described between ventral and dorsal parts and a differential role for NPY in these subregions of the hippocampal formation (Baptista & Andrade, 2018; Lee, Kim, Cho, Kim, & Park, 2017). Regarding the intranasal administration of the GALR2 agonist alone, we observed no effects on ventral hippocampal cell proliferation. Previously, it was reported that GalR2/3 mediated the proliferative and trophic effects of GAL (Abbosh et al., 2011), indicating in subsequent studies a role for GALR3 (Khan, Khan, Runesson, Zaben, & Gray, 2017). However, these studies were performed in vitroconditions, exhibiting significant differences in systems in vivo .
At cellular level, these effects on hippocampal cell proliferation after GALR2 and Y1R agonists intranasally co-administered seem to be mediated by increased BDNF expression on the ventral hippocampal DG. BDNF, a member of neurotrophins, has a pivotal role in increasing neurogenesis through changes in proliferation and cell survival (Miranda et al., 2019). Recent evidences showed that physical exercise protects the brain from depressive symptoms through increasing hippocampal neurogenesis combined with BDNF (Murawska-Cialowicz et al., 2021). Thus, therapeutics that promote the close correlation between dentate neurogenesis and BDNF, as seen under the GALR2 and Y1R agonist combination, may be the key to preventing or curing depression. In this regard, our data are consistent with previous evidences on the BDNF-related neuroprotective effect of NPY in models of neurodegeneration (Corvino et al., 2012; Croce et al., 2011).
These cellular effects induced by GALR2 and the Y1R agonist were achieved in hippocampal neuronal cells by studying GALR2/Y1R heteroreceptor complexes upon agonist coactivation of both receptor protomers. We observed an increase of GALR2/Y1R heteroreceptor complexes by using in situ PLA upon combined co-incubation with GALR2 and Y1R agonists. We have previously reported the presence of GALR2/Y1R heteroreceptor complexes in HEK cells and several limbic brain regions, including the amygdala and the dorsal hippocampus (Borroto-Escuela et al., 2021; Narvaez et al., 2016; Narvaez et al., 2018; Narvaez et al., 2015). Additionally, we confirmed how 5HT1A-FGFR1 heteroreceptor complexes significantly stimulated hippocampal plasticity linked to antidepressant-like actions (Borroto-Escuela et al., 2012; Narvaez et al., 2020). Besides, GALR2 and Y1R agonist co-incubation promoted an increasement of the neurites length in hippocampal neuronal cells at 24 hours, where BDNF might be a common mechanism in our in vivo and in vitro experiments. In agreement, it was shown that BDNF exerted a promoting effect on dendritic outgrowth in primary hippocampal cultures and the hippocampus (H. I. Kim, Lim, Choi, Kim, & Choi, 2022; Park et al., 2016).
The functional outcome was certified by demonstrating the enhancement of the antidepressant-like response after GALR2 and Y1R agonists intranasal co-administration on the forced swimming (FST) test at 24 h. In previous reports, the intranasal infusion of the Y1 agonist (Serova et al., 2017) and NPY (Nahvi et al., 2021) in rats or NPY in humans (Mathe et al., 2020) induced antidepressants effects at least for 24 hours. In agreement, the N-methyl-d-aspartate receptor antagonist, Ketamine or the group II metabotropic glutamate (mGlu2/3) receptor antagonist, LY341495 were shown to exhibit antidepressant-like effects after a single injection in the FST in rats at 24hours (Gigliucci et al., 2013; Koike & Chaki, 2014). We found that the intranasal administration of the GALR2 agonist alone lacks antidepressant-like effects at 24 hours. Species-specific differences between rats and mice in antidepressant responses have been reported (Polis, Fitzgerald, Hale, & Watson, 2019). Thus, previous studies indicating that the intranasal infusion of an spexin-based GALR2 agonist induced antidepressant-like effects was performed on mice at 2-3 hours (Yun et al., 2019). Moreover, we found that the GALR2 antagonist M871 counteracted the enhanced response observed, as previously reported after intranasal GALR2 and Y1R agonists (Borroto-Escuela et al., 2022). In this previous work we confirmed that the behavioral effects were independent of the motor activity since GALR2, Y1R agonist or their intranasal coadministration not showed locomotor alterations. In agreement with our findings, ventral hippocampus was involved in the antidepressant effects of NPY in posttraumatic stress disorder (Sabban & Serova, 2018). Accordingly, we may speculate that the molecular mechanisms underlying the antidepressant-enhancing actions of the Y1R and GALR2 agonists at 24 hours might be mediated by enhancing the signaling of these two protomers in the Y1R–GALR2 heterocomplexes in the neurogenic zone of the ventral hippocampus. Moreover, our data support the contribution of BDNF in this mechanism, as observed in vivo and in vitroexperiments. Besides, the TrkB antagonist ANA-12 was shown to counteract the antidepressant effects of ketamine at 24 hours (Ribeiro et al., 2020). Interestingly, running was shown to enhance BDNF signaling and neuronal proliferation on the ventral hippocampus related with antidepressant effects (Murawska-Cialowicz et al., 2021).
Taken together, the intranasal infusion of Y1R and GALR2 agonists may promote cell proliferation in the ventral hippocampal dentate gyrus and the induction of the BDNF neurotrophic factor. These effects may be mediated by Y1R–GALR2 heteroreceptor complexes to mediate increased neurites outgrowth observed on neuronal hippocampal cells. Accordingly, these cellular effects may be linked to the enhanced antidepressant effects observed. In this way, through a reorganization of the signaling in this Y1R–GALR2 heteroreceptor complex, including the homo-receptor complex associated with altered hetero/homo-signaling might mediate the increased antidepressant actions. Our data may suggest the development of new heterobivalent o multitargeting agonist pharmacophores acting on Y1R–GALR2 heterocomplexes in the ventral hippocampus for the novel therapy of mayor depression disorder or depressive-affecting diseases.