Intracranial Administration of Kisspeptin Peptides

Central administration of Kiss1 peptides was performed as described previously in Kizil and Brand (2011) and Ogawa et al. (2012)^{10 (link),64 (link)}. For whole brain gene expression assay, AB fish were anesthetized by immersion in 0.01% benzocaine (Sigma, USA) solution and intracranially administered with 1 μL of either kisspeptin1-15 (Kiss1; pyroglut-NVAYYNLNSFGLRY-NH₂; Open Biosystems) or kisspeptin2-10 (Kiss2; FNYNPFGLRF-NH₂; BioGenes) with two different doses (10^–12 and 10^–9 mol/fish in distilled water). For injection, the cranial bone close to the habenula above the left side of the anterior part of the optic tectum was incised using a sterilized barbed-end needle (BD Precision Glide 30G × 0.5″, BD Medical, New Jersey) (Fig. 1A), and through this incision,
Kiss1 solution was administered using a heat-pulled glass capillary micropipette attached with a microinjector (IM-9B, Narishige, Tokyo, Japan) (Fig. 1B). For control, the same volume of distilled water was administered. Fish in sham group received the puncture procedure without any administration.