br Materials and methods br Results br

Materials and methods
Results
Discussion In this study, we observed that PACAP2 immunoreactivity is mainly distributed in the telencephalon, hippocampus, and cerebellum of the zebrafish brain. This distribution pattern is similar to that reported in a previous study in which PACAP immunoreactivity was observed in the stargazer PF-03814735 by using stargazer PACAP-specific antibody [25]. We also observed that adcyap1b mRNA is highly expressed in the zebrafish telencephalon and diencephalon but poorly expressed in the cerebellum (Fig. 3B). PACAP2 is presumed to be expressed in the telencephalon and diencephalon because of the similarity in the patterns of immunoreactivity and mRNA expression. Conversely, the inconsistent patterns of immunoreactivity and mRNA expression in the cerebellum imply that cerebellar PACAP2 is delivered from another region of the brain. Furthermore, colchicine injection did not affect PACAP2 immunoreactivity in the cerebellum. These results suggest that cells of the cerebellum express no or low amounts of PACAP2 or that colchicine was not delivered to the cerebellum in an effective dose. To determine the source of PACAP2 in the cerebellum requires optimization of the colchicine concentration and injection area. The in situ hybridization method used to determine the distribution of adcyap1b mRNA must also be optimized. The pallium region of the telencephalon, the hippocampus, and the amygdala participate in memory systems in mammals [26]. In a comparative study of mouse and zebrafish brain regions, the mouse hippocampus and amygdala were found to correspond to the lateral and medial parts of the dorsal telencephalic area in zebrafish, respectively [27]. In goldfish, a telencephalon ablation study revealed that the lateral part of the dorsal telencephalic area is essential to forming temporal associative memories [28]. In vivo calcium imaging of whole zebrafish revealed that the telencephalon is activated during the retrieval of a behavioral program stored in long-term memory [29]. These reports strongly suggest that the telencephalon is an important neural center of memory systems, with conserved function through vertebrate evolution from teleosts to mammals. Here, we report that strong PACAP immunoreactivity was observed in the zebrafish telencephalon. Dense PACAP immunoreactivity was found in the hippocampus and amygdala of rat brain [18]. PACAP38 intracerebroventricular injection is reported to increase mnemonic processes, even at very low dosages, in rats [30]. These reports suggest that PACAP2 is involved in the memory system in zebrafish. Dense PACAP-immunoreactive fibers and perikarya have been observed in the hypothalamic nucleus in rodent, frog, and fish brains [31,32]. Our results showing PACAP2-immunopositive cells and fibers in the zebrafish hypothalamus are similar to those of these previous reports. The hypothalamus is closely associated with instinct behaviors. Ablation of both sides of the lateral region of the hypothalamus induces anorexia, and ablation of the ventromedial nucleus induces bulimia in rats and cats [33]. In the teleost, electro-stimulation of the hypothalamus induces feeding behavior in goldfish [34]. Therefore, the hypothalamus is considered to be the feeding center of the vertebrate brain. Further supporting these reports, numerous hypothalamic neuropeptides are implicated in the regulation of food intake and energy homeostasis in rodents and teleosts [35,36]. PACAP has been considered as an anorectic neuropeptide in vertebrates [37]. Previous reports indicate that PACAP plays a role in the regulation of feeding behavior in vertebrates including rodents [38], chicks [39], and goldfish [40]. These reports suggest that the anorexigenic action of PACAP has been evolutionarily conserved among vertebrates. Thus, hypothalamic PACAP2 may contribute to feeding regulation in zebrafish. We observed that PACAP2 immunoreactive fibers form a line from the hypothalamic region to the habenular nucleus (Fig. 5E–G). The habenular nucleus is involved in modulating motor activity, cognition, and emotional behavior, and the habenular structure is well conserved in all vertebrate animals [41,42]. A high level of PAC1R mRNA was detected in the habenular nucleus in the rat brain [18]. These reports suggest that PACAP2 is associated with cognition and emotional behavior in zebrafish.