In this study we describe the expression of

In this study, we describe the expression of galr2a and galr2b in zebrafish embryos using whole-mount in situ RNA hybridization. To further analyze the distribution of GALR2b and its interactions with galanin, we generated Tg(galr2b:egfp) zebrafish, which express EGFP under the control of a galr2b promoter. Investigation of CNS of transgenic reporter zebrafish revealed that galr2b:EGFP neurons are distributed, and interact with galanin-immunoreactive (galanin-IR) cells in various regions of the Castanospermine and spinal cord. We found that in some regions of the brain and spinal cord, galanin-IR nerve cells were not observed near galr2b:EGFP neurons, suggesting that GALR2b may interact with other ligands instead of galanin in these regions.
Materials and methods
Results
Discussion In the present study, we describe the expression of galr2a and galr2b genes in the zebrafish CNS, and identify the distribution of galr2b:EGFP neurons and their interactions with galanin-IR nerve cells by analyzing CNS of Tg(galr2b:egfp) zebrafish. This is the first report examining galr2a and galr2b expression and neuronal connections between GALR2b and galanin-IR nerve cells in zebrafish. In zebrafish embryos, expression of galr2a and galr2b was restricted to CNS. We found that galr2a was expressed at very low levels in a restricted area of the brain; however, galr2b was widely expressed in the brain and spinal cord in a pattern similar to mammalian galr2, indicating that zebrafish galr2b is functional ortholog of mammalian galr2 (Fig. 1). Immunohistochemical analysis of CNS of Tg(galr2b:egfp) zebrafish with anti-GAL antibody revealed that galr2b:EGFP neurons are distributed and interact with galanin-IR in the various regions of the brain and ventral spinal cord including the anterior commissure, postoptic commissure of the preoptic region, hypothalamus, and commissure of posterior tuberculum (Fig. 3). However, galanin-IR nerve cells were not observed near galr2b:EGFP neurons in the olfactory bulb (Fig. 3J), posterior tuberculum of the midbrain (Fig. 3H), dorsal area of the hindbrain (Fig. 3I), midbrain tegmentum (Fig. 3K), and spinal cord (Fig. 4L). Although GALR2b is a receptor for galanin, these data suggest that GALR2b may interact with other ligands instead of galanin in these regions. A possible candidate that interacts with GALR2b is spexin (SPX). SPX is a recently identified novel secreted neuropeptide, which is composed of 14 amino acids and highly conserved in different vertebrates [13], [19], [26], [29]. Several previous studies have revealed that SPX is widely expressed in the brain, skin, respiratory system, digestive system, reproductive system, and endocrine system, indicating that SPX may have multiple functions [17], [28]. Recently, it was reported that SPX is a functional agonist for GALR2 and GALR3 in humans as well as GALR2a and GALR2b in zebrafish. In this report, SPX has been shown to activate GALR2a and GALR2b in zebrafish in vitro by a ligand-receptor interaction assay [7]. Interestingly, zebrafish SPX has been shown to have higher potencies toward GALR2b than galanin, while GALR2a responds to SPX and GAL with similar potency, suggesting that SPX is a functional agonist for GALR2b in zebrafish [7]. Therefore, we hypothesize that galr2b:EGFP neurons may interact with SPX in the regions where galanin-IR nerve cells do not exist. To probe this hypothesis, we need to investigate the distribution and interactions between GALR2b and SPX neurons in the zebrafish CNS. Since neuropeptides mediate signals through specific GPCRs, it is important to analyze neuronal circuits including the neuropeptide and its receptor to understand the regulation and function of neuropeptides. In this study, we generated Tg(galr2b:egfp) zebrafish which expresses EGFP under the control of a galr2b promoter. We also have shown that galr2b:EGFP is expressed in the endogenous galr2b-expressing neurons and successfully analyzed the distribution and interactions of galr2b:EGFP and galanin neurons. Altogether, our data indicate that Tg(galr2b:egfp) zebrafish can be a valuable tool to study the functions of and neuronal circuit formation between GALR2b and its interacting neuropeptides.