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3 protocols using idazoxan

1

Pharmacological Manipulation of Opioid Signaling

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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.
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2

Electrophysiological Opioid Receptor Assay

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Naloxone and MK-801 were purchased from Hello Bio (Princeton, NJ). [Met5]enkephalin and CTAP were purchased from Sigma (St. Louis, MO). UK14304 tartrate and idazoxan were from Tocris (R and D system, Minneapolis, MN). Morphine alkaloid was obtained from National Institute on Drug Abuse, Neuroscience Center (Bethesda, MD) and was converted to HCl salt with 0.1 M HCl as a stock solution. All drugs were diluted to the tested concentrations in artificial cerebrospinal fluid (ACSF) and applied during superfusion. All salts used in electrophysiological experiments were purchased from Sigma.
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3

Intrathecal Capsaicin and Adrenergic Receptor Modulation

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For studies involving CPM, capsaicin (8-methyl-n-vanillyl-6-nonenamide, Sigma Chemical Co, St. Louis, MO) was prepared fresh in a vehicle solution of 10% ethanol, 10% Tween-80, and 80% sterile saline to a concentration of 3 mg/ml. Idazoxan (Tocris Bioscience, Bristol, United Kingdom) was prepared in sterile saline and passed through a 0.2 μm filter. All intrathecal drug injections were made via percutaneous lumbar puncture. Successful puncture of the dura was confirmed by the presence of a tail flick. Anti-dopamine β hydroxylase conjugated to saporin (DβH-saporin) and control immunoglobulin G (IgG) -saporin were obtained from Advanced Targeting Systems, San Diego, CA and injected in a dose of 5 μg, intrathecally.
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