Naloxone hydrochloride

Sodium butyrate (SB; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) was dissolved in phosphate-buffered saline (PBS; 0.14 M NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ and 1.8 mM KH₂PO₄). LPS obtained from Escherichia coli with serotype 055:B5 (Sigma-Aldrich, St. Louis, MO, USA), a rat/human CRF (Peptide Institute, Inc., Asagi, Japan), N^G-nitro-L-arginine methyl ester (L-NAME), naloxone hydrochloride and domperidone (Fujifilm Wako Pure Chemical) were dissolved in normal saline. Compound C (dorsomorphin; LC Laboratories, Inc., Woburn, MA, USA), GW9662 (Focus Biomolecules, Plymouth Meeting, PA, USA) and sulpiride (Fujifilm Wako Pure Chemical) were dissolved in dimethyl sulfoxide (Fujifilm Wako Pure Chemical). The doses and routes of administration of the chemicals were determined according to previous publications^{5 (link),6 (link),17 (link)–20 (link)}.

Drugs were dissolved in Krebs solution and applied by perfusion via a three-way stopcock without any change in either the perfusion rate or the temperature. The drugs used in this study were Ultiva®, 2 mg (Janssen Pharmaceutical K.K., Tokyo, Japan), TTX (Wako, Osaka, Japan), glycine (Wako) and naloxone hydrochloride (Wako). All drugs were diluted to their final concentration in Krebs solution immediately before use. A 2-mg Ultiva® vial contains 15 mg glycine as an adjunct. Ultiva® containing 50 μM remifentanil hydrochloride with 2 mM glycine was superfused on to spinal cord slice preparations in vivo and ex vivo. The plasma concentration of remifentanil achieved by an intravenous infusion of Ultiva® at 0.05–0.20 μg·kg⁻¹ min⁻¹ is approximately 1–5 ng/ml [14 (link), 15 (link)]. Remifentanil has a rapid blood-brain equilibration half-time because of its rapid onset of action. Although the transit of remifentanil to the cerebrospinal fluid is not completely understood, the concentration of Ultiva® superfused was not determined from the plasma concentration, but from the effect obtained in a previous study of Ultiva® or pure remifentanil [5 (link), 16 (link)].