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Materials and methods
Results
Discussion This study provide for the first time a report on GALP in fish, providing new information towards the distribution and functional characterization of the galaninergic system in these vertebrates. Our studies show the detailed neuroanatomical distribution of GALP in the faah inhibitors and gut of goldfish, and demonstrate that it has a stimulatory role on goldfish feeding behavior. To date, GALP distribution within the brain has only been described in mammals [2], [3], [4], [7], [8], [9]. In rats, GALP mRNA-expressing cells have been found within the Arc, distributed medially along the third ventricle, with the majority of cells being localized to the caudal portion of the Arc [3]. Fibres expressing GALP immunoreactivity have been found in various areas of the basal forebrain, including the paraventricular nucleus, medial preoptic area, bed nucleus of the stria terminalis, ventral part of the lateral septum, and periventricular nucleus [9]. Our immunohistochemical studies showed that GALP is present in many areas of the goldfish brain, including the hypothalamus. Within the hypothalamus, GALP and its receptor immunopositive cells were found in the NAT, NLTi, NPPv and NRL. The finding of GALP-like immunoreactivity in the NLT of the goldfish hypothalamus is in line with data in rats showing GALP mRNA in the mammalian Arc [3]. Moreover, the similar localization of GALP in the goldfish brain provides support for its central regulatory role. Approximately 40% of GALP neurons in the macaque hypothalamus have been found to coexpress with mRNA for the neuropeptide Y (NPY) Y1 receptor, believed to be responsible for the potent orexigenic actions of NPY [20]. The presence of both GALR1 and GALR2, primary receptors of GALP [2] in the brain and gut, suggests possible direct action of GALP in tissues involved in the regulation of metabolism. Collectively, these data suggests that GALP has a role in the regulation of energy homeostasis [19]. Whether GALP interacts with other appetite regulatory peptides to modulate energy balance in fish remain poorly understood. Apart from the hypothalamus, presence of GALP was detected in the goldfish telencephalon, optic tectum, torus longitudinalis and valvula of the cerebellum. The torus longitudinalis is a paired structure of the dorsal mesencephalon unique to actinopterygians that protrudes into the ventricle [25]. On the other hand, the valvula of the cerebellum is the most anterior subdivision of the cerebellum which lies within the mesencephalic ventricle, ventral to the optic tectum [26]. These two structures have been implicated in visual activity and eye movements [27], and in the processing of electrosensory information [28], respectively. A connection between the torus longitudinalis and the valvula of the cerebellum has been described in some species [29], and it has been postulated that the torus longitudinalis represents a main relay centre in premotor circuitry descending from the telencephalon to the brainstem [30], [31]. There are no studies available on a possible function of GALP in visual activity and/or in the processing of electrosensory information. The fact that this neuropeptide is present in brain areas previously reported to be involved in these processes provide evidences for possible roles of GALP in neural functions in fish. In the periphery, previous reports have shown the presence of GALP mRNAs in porcine small intestine [2] and in the rat seminiferous tubules [12]. Present immunostaining results revealed the presence of GALP and its receptor immunoreactive cells within the mucosa of the goldfish gut, in accordance with previous observations in pigs. Galanin, the main member of the family of peptides including GALP, has also been reported in the brain and intestine of goldfish [32], [33], [34]. The intestinal presence of the galaninergic system suggests a possible involvement of galanin and/or GALP in gastrointestinal processes, such as digestion and motility, among others. Further studies regarding these aspects would be of great interest.