Bullfrog (Rana catesbeiana) larvae weighing 5–7 g were collected from ponds in the suburbs of Toyama City, Japan. The developmental stages of the larvae were determined according to Taylor and Kollros (1946). Since the body size of obtained larvae at premetamorphic stages was very small, experiments were done using 100 larvae at prometamorphic stages (XI–XIX). As previously described (Shimizu et al., 2013), the animals were kept for 1–2 weeks under controlled light/dark conditions (12L/12D) with the water temperature maintained at 20–24°C. The larvae were fed every day at noon with a powder diet (Itosui Co., Tokyo, Japan) until used in experiments. Animal experiments were conducted in accordance with the Invasive Alien Species Act of Japan and the University of Toyama's guidelines for the care and use of alien and laboratory animals, and were done by approval of an ethics committee of the University of Toyama and permission of government authorities (No. 05000361).

In order to examine the effects of ICV administration of NPY and orexin A on food intake, and the effects of NPY receptor and orexin receptor antagonists on the actions of ICV-injected NPY and orexin A in prometamorphic larvae, the following chemicals were used. A NPY Y1-receptor antagonist, BIBP3226 (diphenylacetyl-D-Arg-4-hydroxybenzylamide, Bachem AG, Bubendorf, Switzerland) and a selective orexin receptor antagonist, SB334867 [N-(2-methyl-6-benzoxazolyl)-N′-1,5-naphthyridin-4-yl urea, Tocris Cookson Ltd., Bristol, UK], were purchased commercially, dissolved in dimethyl sulfoxide at 20–50 mM for storage, and diluted with 0.6% NaCl and 0.02% Na₂CO₃ solution (saline) before use. NPY (rat NPY, Peptide Institute Co., Osaka, Japan) and orexin-A (rat orexin-A, Peptide Institute) were also purchased commercially, dissolved in saline at 0.1 mM for storage at −80°C, and diluted with saline before use. 3-Aminobenzoic acid ethyl ester (MS-222) for anesthesia was obtained commercially from Sigma-Aldrich (St. Louis, MO, USA), and was prepared at concentration of 0.15%. Each animal was exposed to this solution for 2 min.

Details of the methods used for measuring food consumption in the larvae have been reported elsewhere (Matsuda et al., 2010; Shimizu et al., 2013, 2014a,b). As previously described (Shimizu et al., 2014b), two types of powder diet colored green and red, respectively (containing the same components as those described above), were obtained from Itosui Co., Tokyo, Japan. First, the test prometamorphic larvae were fed the green-colored diet and kept under laboratory conditions. Then, after a 24-h fast, an adequate amount of the red-colored food was made available at 3% of BW. Test substances were ICV-administered as described below, and after 15 min, each animal was decapitated and the gastrointestinal tract was removed. In our previous study, we observed food intake in intact and ICV-injected larvae during 60 min after recovery from anesthesia. Then, we determined that food intake is measured during first 15 min. The wet weight of the red-colored gastrointestinal contents was measured after removal of intestinal juice with tissue paper, and expressed as micrograms of food taken per gram BW. The experiments were conducted around noon.

In order to double-check the orexigenic actions of NPY and orexin A, and the antagonistic actions of BIBP3226 and SB334867 upon them (Shimizu et al., 2013, 2014b), we examined the effects of ICV administration of NPY, orexin A, BIBP3226, and SB334867 on food intake in prometamorphic larvae. Our previous studies have indicated that simultaneous injection of antagonists with peptides induces their antagonistic effects (Shimizu et al., 2013, 2014b).

We did not have an atlas for bullfrog larval brain. However, according to our previous experiments, same procedures have been done. Therefore, following sentences were mainly quoted from our previous paper (Matsuda et al., 2010; Shimizu et al., 2013, 2014b). “For ICV administration of test substances, each animal was placed in a stereotaxic apparatus under anesthesia with MS-222. A small area (~1 mm² square) of the parietal skull was carefully removed using a surgical blade (No. 19, Futaba, Tokyo, Japan), and then 0.1 μl/g BW (0.5–0.7 μl) of each test substance including Evans blue dye was injected into the third ventricle of the brain using a 10-μl Hamilton syringe with a 0.1-μl scale. The gap in the parietal skull was then filled with a surgical bonding agent (Aron Alpha, Sankyo, Japan). The accuracy of the injection site and volume was confirmed after the experiment by examining whether Evans blue dye was present in the ventricle without leakage. Control larvae in each experiment were injected with the same volume of saline in the same way as for the experimental group. Each larva that had received the ICV injection was placed individually in a small experimental tank (diameter 11 cm) containing 700 ml of tap water. After recovery from anesthesia, each larva was supplied with the red-colored food equivalent to 3% of its BW. After 15 min, the weight of the intestinal contents was measured as described above.”

“In order to check the effect of ICV injection of BIBP3226 on the orexigenic action of NPY at 10 pmol/g BW, half a microliter of BIBP3226 at 100 pmol/g BW was injected into the third ventricle of the brain of larvae as described above. The ICV-injected dose of BIBP3226 had been determined in previous experiments (Shimizu et al., 2013). Larvae in the control group were given injections of the same volume of saline-diluted dimethyl sulfoxide (vehicle). Following the administration of BIBP3226 or vehicle, either NPY at 10 pmol/g BW or saline was delivered by ICV injection. The feeding experiment was performed according to the procedures described above. In order to check the effect of ICV injection of SB334867 on the central actions of orexin A, 0.5 μl of SB334867 at 60 pmol/g BW in addition to orexin A at 6 pmol/g BW was injected into the third ventricle of the brain of the larvae. The ICV-injected dose of SB334867 had been determined by reference to a previous study (Shimizu et al., 2014b). Larvae in the control group were given an injection of the same volume of dimethyl sulfoxide diluted with saline.”

According to our previous experiments, same procedures have been done. Therefore, following sentences were mainly quoted from our previous paper (Shimizu et al., 2014b). “In order to examine the effect of ICV injection of SB334867 on the central action of NPY, 0.5 μl of SB334867 at 60 pmol/g BW in addition to NPY at 10 pmol/g BW was injected into the third ventricle of the larval brain. Larvae in the control group were given an injection of the same volume of dimethyl sulfoxide diluted with saline. The feeding experiment was performed according to the procedures described above.”

According to our previous experiments, same statistical analysis have been done. Therefore, following sentences were quoted from our previous paper (Kojima et al., 2009). “All results pertaining to the effect of receptor antagonists on food intake are expressed as the mean ± SEM. Statistical analysis was performed using two-way ANOVA with Bonferroni's method. Statistical significance was determined at the 5% level.”

ICV administration of NPY at 10 pmol/g BW stimulated cumulative food intake during the 15-min observation period in comparison with ICV injection of vehicle or BIBP3226 at 100 pmol/g BW. ICV injection of NPY at 10 pmol/g BW plus BIBP3226 at 100 pmol/g BW did not affect cumulative food intake during the 15-min observation period in comparison with ICV injection of vehicle (Figure 1). Interaction between treatments with NPY and BIBP3226 was significant by two-way ANOVA with Bonferroni's method (F = 5.83 and p = 0.023).

ICV administration of orexin A at 6 pmol/g BW enhanced cumulative food intake during the 15-min observation period in comparison with ICV injection of vehicle or SB334867 at 60 pmol/g BW. ICV injection of orexin A at 6 pmol/g BW plus SB334867 at 60 pmol/g BW did not affect cumulative food intake for 15 min after feeding in comparison with ICV injection of vehicle (Figure 2). Interaction between treatments with SB334867 and orexin A was significant by two-way ANOVA with Bonferroni's method (F = 8.05 and p = 0.009).

ICV administration of NPY at 10 pmol/g BW increased cumulative food intake during the 15-min observation period in comparison with ICV injection of vehicle or SB334867 at 60 pmol/g BW. ICV injection of NPY at 10 pmol/g BW plus SB334867 at 60 pmol/g BW did not affect cumulative food intake during the 15-min observation period in comparison with ICV injection of vehicle (Figure 3). Interaction between treatments with SB334867 and NPY was significant by two-way ANOVA with Bonferroni's method (F = 31.9 and p = 0.0001).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LG and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.