Meccanismi di Cross-talking tra i sistemi Orexinergico e GABAergico nei teleostei

Abstract

The neuropeptides hypocretins/orexins (ORX) are known to control state-dependent activities such as sleep-wakefulness, energy homeostasis and reward/addiction processes. To date, interests regarding ORXergic neuronal functions have mostly dealt with mammals, while only recently attention is beginning to be directed towards aquatic vertebrates. On this basis, the intention of the present study was to evaluate ORX-A effects on behavioral and neuronal activities in marine (the ornate wrasse, Thalassoma pavo) and freshwater (the goldfish, Carassius auratus) teleost fishes. To achieve such aims behavioral, molecular and neurodegenerative analysis were performed following intraperitoneal injections of not only the most effective doses of ORX-A, but also of GABAAR β subunit agonist muscimol (MUS) and antagonist bicuculline (BIC) as well as a classical benzodiazepine, diazepam (DZP), and a non benzodiazepine, zolpidem (ZOL), that are instead agonists of the GABAAR α subunits. Behavioral studies on explorative behaviors (swimming towards food sources; STF), free swimming (FS) and on resting states (R) displayed that ORX-A was responsible for very great (p<0.001) enhancements of STF in both teleosts while similarly very great and great (p<0.01) reduction of R was detected in wrasse and goldfish, respectively. Interestingly, ORX-A effects showed to be strongly influenced by environmental photoperiodic conditions as pointed out by behavioral and ORXR expression relationships in wrasses during constant light/dark conditions. In this case, ORX-A/photoperiodic-dependent motor behaviors might be related to a structural-functional dichotomy of some motor-controlling brain regions. Indeed during the day, ORX-A might be very likely promoting the awakening state via low-affinity ORXR sites at hypothalamic (HTH) and mesencephalic (MES) levels so inducing the promotion of STF and FS. On the other hand, during the night ORX-A might result in a promotion of R via activation of mostly telencephalic (TEL) high-affinity sites. As far as GABAAR influence is concerned, MUS was capable of inducing a moderate (p<0.05) increase in STF and this tends to not only underlie a highly conserved GABA stimulus on feeding but also that such an activity relies on the involvement of ORXR in most brain regions of both fish models. Moreover, it was also possible to observe that such a GABAAR agonist elicits a neuroprotection via ORXR as suggested by both the reduction of BIC-dependent mRNA down-regulations and the very low argyrophilic reactions in brain regions of MUS+BIC-treated fish. When the increase of STF was next checked to see whether it was linked to an enhancement of food consumption, I observed that ORX-A and GABAAR  drugs (MUS and BIC) did not modify this activity. On the other hand, GABAAR  subunits appeared to not only influence STF maneuvers but also food consumption as demonstrated by ZOL and DZP evoking very great increases in STF coupled with great and very great amounts of food consumption, respectively. Interestingly, also in 8 this case ZOL- and DZP-dependent hyperphagia seemed to be correlated to ORXR expression variations in mostly HTH and MES, brain areas that are involved in energy homeostasis and motor activities. At this point it was interesting to investigate if ORX- and GABAAR-induced STF effects were mediated through rewarding pathways like that reported for mammals. With such an aim, T-maze and Conditioned Place Preference (CPP) paradigms were performed. For this part, ORX-A accounted for a moderate improvement of T-maze performances that became great when associated to color visual stimuli, suggesting that ORX-A might be operating on memory processes also in fish. Moreover from CPP analysis in which ORX-A acted as a main agent responsible for great increase of color-dependent change in CPP, it seems that this neuropeptide and not GABAAR drugs, might be potentiating reward pathways in goldfish. Also in this case, behavioral performances are consistent with ORXR up-regulations in some TEL regions and in the nucleus of the posterior tuberculum, homologous of the amygdala plus hippocampus and ventral tegmental area that in mammals control learning and reward processes. It is worthy to note that DZP only accounted for a moderate increase in CPP change while BIC completely blocked ORX-A-induced change in CPP and this tends to point to a dual neurosignaling mechanism between ORX circuit and GABAAR / subunits on motivational-reward behaviors. Overall these results highlight, for the first time, not only an ORX neuronal driving force operating on feeding behaviors in goldfish, but above all its participation in the control of goal-oriented behaviors like in mammals.