Sb269970

Melperone hydrochloride (FUJIFILM Wako Pure Chemical Co., Osaka, Japan), ketanserin tartrate (FUJIFILM), methysergide maleate (Sigma-Aldrich, St. Louis, MO, USA), asenapine maleate (Sigma-Aldrich), and SB269970 (Sigma-Aldrich) were added to 10 μM FSW to inhibit the Serotonin/monoamine pathway. The inhibitors were applied 10 s before photoirradiation. S-Nitroso-N-acetyl-D,L-penicillamine (SNAP; FUJIFILM) was used as a nitric oxide (NO) donor (final concentration of 100 μM) [23 ]. SNAP was applied to the larvae 5 min before observation. 3,5-Difluorophenyl-acetyl-L-alanyl-L-S-phenylglycine T-butyl ester (DAPT; Sigma-Aldrich) was used as a γ-secretase inhibitor (final 20 mM). Serotonin (Tokyo Chemical Industry, Tokyo Japan) was dissolved to distilled water just before use and applied to culture (final 10 μM). The same volume of DMSO or seawater was applied as controls for chemical treatments.

Intrathecal injection was performed as described previously (Lapirot et al., 2009 (link)). One week after dural cannulation, rats received direct transcutaneous intrathecal injection before each IS application at 2 day intervals during the 7 day injection protocol. Under isoflurane-induced anesthesia (3% induction, 1.5% maintenance), a 25-G needle connected to a 25-μl Hamilton syringe was inserted percutaneously into the vertebral canal between L5 and L6. A tail-flick reaction denoted a successful puncture. A rat was injected intrathecally with 20 μl of 1% Chicago sky blue (C8678, Sigma-Aldrich, United States) to determine whether the drug injected intrathecally was able to reach the L6-S1 spinal cord level. After that, SB269970 (5-HT₇R antagonist, 7 μl, 5 mM dissolved in distilled water; Sigma-Aldrich, United States) or AS-19 (5-HT₇R agonist, 15 μl, 10 μM dissolved in saline; Sigma-Aldrich, United States) was administered intrathecally with an injection rate of 1 μl min^–1 (Hoffman and Mitchell, 2011 (link); Ni et al., 2019 (link)). Rats that exhibit signs of motor impairment are excluded from the experiment.

Way-100635 (5-HT1aR antagonist, Sigma-Aldrich), ketanserin (5-HT2aR antagonist, Sigma-Aldrich), ondansetron (5-HT3R antagonist, LKT Laboratories Inc., Saint Paul, MN, USA), and SB269970 (5-HT7R antagonist, Sigma-Aldrich) were used as 5-HTR antagonists, and SR57227A (Sigma-Aldrich) was used as a 5-HT3R agonist. Saline was used as a vehicle control. The infusion of each agent at a uniform rate was achieved using an osmotic pump (0.24 µL/h for 14 days; Alzet, Cupertino, CA, USA) implanted subcutaneously in the ventral part of the mice under inhalation anesthesia by isoflurane. The dosage of each antagonist and SR57227A was designed to be continuously administered at 120 and 72 µg/kg/day, respectively. Each antagonist was infused into mice kept on a standard diet, and SR57227A was infused 1 day before changing from a standard to a Trp-free diet. Each antagonist was infused for 7 days and SR57227A was infused for 8 days.
For determination of the inhibitory effect of ondansetron on 5-HT-stimulated secretion, 1 mg/kg 5-HT and 10 mg/kg of ondansetron were simultaneously injected i.p. The agonist SR57227A was injected at a dosage of 1 mg/kg i.p. Each experiment was performed after 2 days of feeding a Trp-free diet.