*5.2. Chemicals and DrugAadministration*

Crotalphine (<E-F-S-P-E-N-C-Q-G-E-S-Q-P-C, where <E is pyroglutamic acid and a disulfide bond between 7C-14C) was synthesized as described by Konno et al. (2008) [1] by the American Peptide Co. (Sunnyvale, CA, USA). The peptide was stored lyophilized

at −20 ◦C until use. For the stock solutions, the peptide was dissolved in sterile distilled water and then diluted in sterile saline immediately before the experiments. Crotalphine at doses of 8 pg/kg, 20 ng/kg or 1μg/kg (oral route) was administered 60 min before the first nociceptive evaluation. PGE2 was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and was prepared as previously described [7]. The PGE2 was injected (100 ng/paw) in a volume of 100 μL with 0.2% of ethanol [8]. ERK inhibitor (ERK-I), p38 inhibitor (SB20358; p38-I) and JNK inhibitor (SP660125; JNK-I), were dissolved in 5% dimethyl sulfoxide (DMSO) and administered intrathecally (i.t.) at the dose of 30 μg. PD98059, a MEK inhibitor, was dissolved in 5% DMSO and administered intraplantarly (i.pl.) (10 or 30 μg/paw) or intrathecally (30 μg) [33]. The peptide ζ-pseudosubstrate ([C]SIYRRGARRWRKLYRAN; amino acids 105-121 in ζ-PKC) was synthesized by Zhejiang Ontores Biotechnologies Co. (Hangzhou, Zhejiang, China) and fused to the cell permeable TAT peptide (YGRKKRRQRRR) transduction domain peptide. The control group received the TAT peptide (American Peptide Co., Sunnyvale, CA, USA). Stock solutions were made in sterile distilled water and diluted in sterile saline. This peptide was administered intrathecally at the dose of 3 μg. In summary, for these intrathecal injections, the animals were anesthetized with 2% isoflurane and the needle was placed in the subarachnoid space on the midline between L4 and L5 vertebrae [34], with a maximum volume of 50 μL. The animals recovered consciousness approximately 1 min after discontinuing the anesthetic.
