Generally speaking the regulation of GnIH on

Generally speaking, the regulation of GnIH on gonadotropins synthesis and release, although controversial, could be direct and/or indirect via GnRH, among others peptides. In this sense, different scenarios have been reported in vertebrates. In mammalian and avian species, GnIH fibers reach the median eminence and contact with GnRH neurons, suggesting both direct and indirect effects on gonadotropin regulation (Bentley et al., 2006). This is in accordance with some reports where GnIH receptors were detected in the gonadotropes and in GnRH1 neurons in the POA (Tsutsui, 2016, Tsutsui and Ubuka, 2016). However, in Ryuvidine and fish, this does not seem to be so clear. In fish, GnIH fibers were found in the neurohypophysis of goldfish (Sawada et al., 2002), sockeye salmon (Amano et al., 2006), sea bass (Paullada-Salmerón et al., 2016a), Nile tilapia (Ogawa et al., 2016), zebrafish (Spicer et al., 2017) and sole (Aliaga-Guerrero et al., 2018). However, in Indian major carp (Biswas et al., 2015), as in Cichlasoma dimerus (Di Yorio et al., 2016) adult fish, no fibers were detected at this level. This indicates that in these species, the effect of GnIH on pituitary hormones could be indirect. Alternatively, plasticity in the GnIH innervation could operate in the pituitary of these species depending on the sexual stage. In a previous work, it was demonstrated by immunohistochemistry that GnRH1, originated from preoptic area, represents the main hypophysiotropic variant in C. dimerus. GnRH1 expressing neurons are localized along the ventral surface of the forebrain and present an overlapping distribution with GnRH3 neurons (Pandolfi et al., 2005), as reported in other fish species (González-Martínez et al., 2002, Mohamed et al., 2005). In order to clarify the interactions between GnIH and GnRH1 neurons in this species, double labelling immunohistochemistry was performed. Surprisingly, the results obtained did not show axo-somatic, or fiber-fiber contacts in any of the analyzed areas, although GnIH-ir axons were observed close to GnRH1-ir fibers. Based on these results, it is suggested that GnIH does not directly interact with GnRH1 to inhibit gonadotropins release in this species. This is in agreement with the observed in the POA of tilapia (Ogawa et al., 2016), but contrast with the obtained with the hypophysiotropic variant (GnRH3) in zebrafish (Spicer et al., 2017). Taking into account that in our model species GnIH was not found innervating the pituitary neither GnRH1 neurons, we could speculate that GnIH potentially uses other pathways (kisspeptin, dopamine, neuropeptide Y, etc.) of the reproductive network to control gonadotropin secretion, as suggested in zebrafish (Spicer et al., 2017). Other possibility is that the interaction of GnIH-GnRH1 fibers show plasticity depending on the sexual stage. Finally, considering that a neurovascular regulation plus to the classical innervation of gonadotropes was demonstrated in zebrafish (Golan et al., 2015), it is possible that GnIH reaches the gonadotropes in C. dimerus via the vascular system, which is an interesting issue to be evaluated in future studies. GnRH2, also known as the “midbrain variant,” was described in almost all gnathostomes groups (Kavanaugh et al., 2008). Although it is not present or functional in some mammalian species, it is the most conserved GnRH variant in vertebrates (Pawson et al., 2003, Morgan and Millar, 2004, Kah et al., 2007). Studies in different vertebrate groups proposed that this variant is involved in the regulation of sexual and feeding behavior (Matsuda et al., 2008, Tostivint, 2011). On the other hand, several lines of evidence indicate that GnIH can also modulate behavior and feeding (Tsutsui and Ubuka, 2016). For example, in avians and mammals, intracerebroventicular injections of GnIH stimulates food intake (Tachibana et al., 2005, Johnson et al., 2007, Clarke et al., 2012, Tsutsui and Ubuka, 2016) and in hamsters, food restriction activates GnIH neurons (Klingerman et al., 2011). Also, it was demonstrated that GnIH administration can inhibit sexual motivation in hamsters (Klingerman et al., 2011, Piekarski et al., 2013) and rats (Johnson et al., 2007). However, very little is known about the role of GnIH in fish reproductive behavior. There is only one study regarding the administration of GnIH in sea bass that affects the diurnal to nocturnal ratio to locomotor activity along the reproductive cycle (Paullada-Salmerón et al., 2016c). In this context, the neuroanatomical and functional relationships between both neuropeptidergic systems were reported in vertebrate species from different groups. For example, in song birds GnIH neurons terminated in close proximity to GnRH2 neurons, which express GnIH receptors (Ubuka et al., 2008). Recently, a study performed in a teleost, the European sea bass, has revealed an inhibitory role of sea bass GnIH2 form on brain GnRH2 expression (Paullada-Salmerón et al., 2016b). In Indian major carp (Biswas et al., 2015) and sea bass (Paullada-Salmerón et al., 2016a) GnIH somas were localized in the midbrain, but in our model species, no GnIH somas were observed in this region where GnRH2 perikarya are localized. Despite this fact, we could demonstrate that several GnIH fibers in the NLT and in the MBT are in contact with GnRH2 fibers, suggesting a possible regulation. In fish, it has been evidenced that midbrain GnRH2 cells are bidirectionally connected to the pineal organ and modulate melatonin secretion (Servili et al., 2010, Servili et al., 2011), suggesting its involvement in the transduction of environmental effects in reproduction. Interestingly, the GnIH system of sea bass (Paullada-Salmerón et al., 2016a) and sole (Aliaga-Guerrero et al., 2018) also projects to the pineal organ. In addition, in birds, it was demonstrated that GnIH mediates the effects of photoperiod, as GnIH neurons express melatonin receptors and melatonin stimulates hypothalamic GnIH expression and release (Ubuka et al., 2005, Chowdhury et al., 2010). Thus, it remains to be elucidated in future studies whether the interactions between midbrain GnRH2 and GnIH reported in the present study represent a piece of a network involving the pineal organ and mediating the transduction of environmental information to the reproductive axis.