(A) SIA occurs in WT but not orexin/ataxin mice. From –30 to 0 minutes, mice (n = 8) were restrained; this was followed immediately by a hot-plate test at 0 minutes and again at 30 and 60 minutes following restraint. (B) SIA occurs in WT but not in orexin/ataxin-3 mice. Post-hoc tests revealed a significant increase in hot-plate latency in WT mice after 30 minutes of restraint stress (P < 0.05) but not in orexin/ataxin-3 mice. (C) Hcrt-1 administration produced acute analgesia. Hcrt-1 (1.5 nmol/mouse, i.c.v.) caused acute analgesia in both unrestrained and restrained animals compared with vehicle groups (P ≤ 0.05), mimicking SIA in orexin/ataxin-3 mice. (D) SIA occurs in WT mice subjected to i.c.v. injections. WT mice (n = 8 per group) were subjected to 30-minute restraint immediately after i.c.v. injection of either vehicle or Hcrt-1. (E) N/OFQ blocks SIA, and coapplication of Hcrt-1 with N/OFQ restores it in WT mice. WT mice (n = 8 per group) were subjected to 30-minute restraint immediately after i.c.v. injection of vehicle alone, vehicle plus N/OFQ, or a combination of the 2 neuropeptides. N/OFQ (1 nmol/mouse) inhibited SIA compared with vehicle restraint (P = 0.044) to a latency that was no different (P = 0.503) than in the unrestrained i.c.v. vehicle-injected WT mice shown in D. Coadministration of Hcrt-1 (1.5 nmol/mouse) with N/OFQ (1 nmol/mouse) restored SIA relative to vehicle/unrestrained mice (P = 0.026). Significance of differences compared with vehicle/unrestrained WT mice was determined by Fisher’s protected least significant difference test. *P < 0.05 compared with respective controls. Error bars represent SEM.