Endomorphin 1

Morphine sulfate and morphine alkaloid were obtained from the National Institute on Drug Abuse (NIDA), Neuroscience Center (Bethesda, MD). Naloxone was purchased from Abcam (Cambridge, MA), MK-801, from Hello Bio (Princeton, NJ), UK14304 tartrate and idazoxan (Ida) from Tocris (Bio-Techne Corp. Minneapolis, MN). Potassium methanesulfonate was from Alfa Aesar (Ward Hill, MA). [Met⁵] enkephalin (ME), endomorphin-1, muscarine, scopolamine, idazoxan and other reagents were from Sigma (St. Louis, MO). Caged-enkephalin (CYLE) and Caged-Naloxone (CNV-Nal) were gifts from Mathew Banghart.
Morphine alkaloid was converted to salt form with 0.1 M HCl and made up a stock solution in water. The working solution was diluted in artificial cerebrospinal fluid (ACSF) and applied during incubation or superfusion. Naloxone, endomorphin-1, muscarine, scopolamine, UK14304 tartrate and idazoxan were dissolved in water, diluted in ACSF and applied by bath superfusion. Bath perfusion of ME was with bestatin (10 mM) and thiorphan (1 mM) to limit breakdown of ME.

Endomorphin-2, Endomorphin-1, β-funaltrexamine (β-FNA, antagonist for mu-opioid receptor), phenyl N-tert-butylnitrone (PBN, scavenger for reactive oxygen species) and diprotin A (inhibitor of dipeptidylpeptidase IV) were purchased from Sigma-Aldrich.

Three weeks after SCI, a PE‐10 cannula was inserted into the subarachnoid space through L5‐L6 segments of the vertebral column under general anesthesia with a mixture of ketamine and xylazine. Muscimol and endomorphin‐1 (Sigma‐Aldrich), as well as normal saline, were injected with a Hamilton syringe in respective groups, three days after cannula fixation. The optimal dose of muscimol (0.01 µg/10 μl) and endomorphin‐1 (2/5 µg/10 μl) was administered for consecutive 7 days according to our pilot studies. Complete behavioral surveys were conducted to determination optimum dose of muscimol between 0.01, 0.1, and 1 µg also between doses of 2.5, 1, and 2 µg of endomorphine (Dose–response studies).