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11 protocols using dihydro β erythroidine hydrobromide

1

Pharmacological Modulation of Neuronal Signaling

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For in vitro experiments, SR 95531 hydrobromide (gabazine 5 μM, Tocris Bioscience, UK, Ref. 1262), NMDA (10 μM, Tocris Bioscience, UK, Ref. 0114), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (10 µM, NIH generous gift), GABA (10 μM, Sigma-Aldrich), isoguvacine (10 μM, Sigma-Aldrich, Ref. G002), DL -2-Amino-5-phosphonovaleric acid (40 µM, Sigma-Aldrich, Ref. A5282), and bumetanide (10 μM, Sigma-Aldrich, Ref. B3023) were directly added to the perfusion solutions. For ca experiments, slices were treated with bumetanide for 40 min before and during recordings. Cocktail of nicotinic receptor antagonists included mecamylamine hydrochloride (10 μM, Tocris Bioscience, Ref. 2843/10), methyllycaconitine citrate (0.1 μM, Tocris Bioscience, Ref. 1029/5), and dihydro-β-erythroidine hydrobromide (10 μM, Tocris Bioscience, Ref. 2349/10). For in vivo experiments, bumetanide pretreatment (3 mg kg−1) was given to mice in drinking water during 5 weeks.
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2

Modulatory Compounds for Oocyte Assays

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Compounds were purchased from Sigma-Aldrich (Buchs, Switzerland) except for Sazetidine A dihydrochloride, NS9283 and dihydro-β-erythroidine hydrobromide that came from Tocris (Tocris bioscience, a Bio-Techne brand, Switzerland). The modulatory compounds were prepared as 20 mM stock solutions in dimethyl sulfoxide (DMSO) and were dissolved in oocyte Ringer’s OR2 medium (see below), resulting in a maximal final DMSO concentration of 0.5% when compounds were tested at 100 μM. ACh was obtained from Sigma-Aldrich and prepared as stock solution at 100 mM in ddH2O.
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3

Modulation of Cortical Excitability Post-Stroke

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Ninety minutes after the start of photothrombosis, mice received a single injection (i.p.) of the GluN2B receptor antagonist Ro25-6981 (6 mg/kg, Tocris Bioscience), the α4β2 specific nAChR antagonist dihydro-β-erythroidine hydrobromide (3 mg/kg DHβE, Tocris Bioscience) or 0.9% saline (vehicle). An additional group of mice was injected with 3 mg/kg DHβE 180 min after stroke. Previous studies have shown that both drugs readily cross the blood brain barrier and alter cortical excitability (Liu et al., 2007 (link); Brown et al., 2012 (link)).
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4

Dihydro-β-erythroidine and Nicotine Protocol

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Dihydro-β-erythroidine hydrobromide ((2S,13bS)-2-Methoxy-2,3,5,6,8,9,10,13-octahydro-1H,12H-benzo[i]pyrano[3,4-g]indolizin-12-one hydrobromide; Tocris Bioscience, Bristol, UK) was dissolved in distilled water. 1 mM concentration aliquots were stored at −20°C or 4°C and diluted with oxygenated aCSF to final concentration before bath application on slices. (−)-Nicotine hydrogen tartrate was purchased from Sigma (St. Louis, MO), dissolved in sterile saline, and pH adjusted prior to subcutaneous injection.
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5

Nicotine Receptor Ligand Protocol

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Figure 1 shows the chemical structure of each drug, except nicotine, included in the study. (–)-Nicotine hydrogen tartrate salt, cytisine and epibatidine dihydrochloride were obtained from Sigma Chemical (St. Louis, MO). Varenicline dihydrochloride was obtained from the Research Technology Branch of the National Institute on Drug Abuse (Rockville, MD), mecamylamine from Waterstone Technology (Camel, IN) and dihydro-β-erythroidine hydrobromide from Tocris (Minneapolis, MN). 2′-Fluorodeschloroepibatidine (RTI-7527-36), 3′-(3″-dimethylaminophenyl) epibatidine (RTI-7527-76), and 2′-fluoro-(4-nitrophenyl) deschloroepibatidine (RTI-7527-102) were synthesized at the Center for Organic and Medicinal Chemistry, Research Triangle Institute (Research Triangle Park, NC) according to methods previously described (Carroll et al., 2004 (link), 2005 (link), 2010 (link)). Drugs were dissolved in 0.9% physiological saline and administered s.c. (except mecamylamine which was administered i.p.) in a volume of 10 ml/kg. Nicotine dose is expressed in terms of the weight of the free base; dose of other drugs is expressed as the weight of the base and salt.
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6

Pharmacological Evaluation of Psychoactive Compounds

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Drugs were delivered s.c. in the midscapular region of the back or i.v. via chronic indwelling catheter in a volume of 0.1-1 ml/kg. Drug doses are expressed as the weight, in mg/kg, of the forms listed below, except in the case of nicotine, which is expressed as the weight of the free base. (-)-Nicotine hydrogen tartrate salt (Sigma-Aldrich), epibatidine dihydrochloride (Sigma-Aldrich), varenicline dihydrochloride (Research Technology Branch of the National Institute on Drug Abuse, Rockville, MD), cocaine hydrochloride (Sigma-Aldrich), midazolam hydrochloride (Bedford Laboratories, Bedford, OH), morphine sulfate (Research Technology Branch of the National Institute on Drug Abuse), mecamylamine (Waterstone Technology, Carmel, IN), dihydro-β-erythroidine hydrobromide (Tocris Bioscience, Bristol, UK), and methyllycaconitine citrate (Research Technology Branch of the National Institute on Drug Abuse) were dissolved in physiological saline. For nicotine, the amount of sodium hydroxide (Sigma-Aldrich) needed to adjust the pH of the solution to 7 was determined and figured into the calculation of volume for a desired concentration.
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7

Pharmacological Modulation of Neuronal Signaling

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Picrotoxin and MK-801 were from Abcam. CGP55845, DNQX, Dihydro-β-erythroidine hydrobromide, SCH 23390 hydrochloride, and sulpiride were obtained from Tocris Bioscience. BAPTA was obtained from Invitrogen. ω-Conotoxin GVIA and ω-Agatoxin TK were from Alomone Labs. All the other chemicals (α-methyl-DL-tyrosine, DEAB, TTX, 4-Aminopyridine, EGTA-AM, BAPTA-AM, scopolamine hydrobromide) were from Sigma-Aldrich.
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8

Perineural Injection of Neuroactive Agents for Pain Management

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TC-2559 difumarate (4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine difumarate), sazetidine A dihydrochloride (6-[5-[(2S)-2-azetidinylmethoxy]-3-pyridinyl]-5-hexyn-1-ol dihydrochloride), and dihydro-β-erythroidine hydrobromide ((2S,13bS)-2-methoxy-2,3,5,6,8,9,10,13-octahydro-1H,12H-benzo[i]pyrano[3,4-g]indolizin-12-one hydrobromide; DHβE) were purchased from Tocris Biosciences (Bristol, UK). These agents were dissolved in sterile water at a higher concentration and diluted with sterile PBS for use. Perineural injection was performed according to a method described previously [9 (link), 37 (link)]. In brief, under isoflurane anesthesia, the agents (10 μl) were injected without a skin incision into the region surrounding the SCN, using a microsyringe fitted with a 30-gauge needle connected to a cannula. Injections of TC-2559, sazatidine A, or DHβE were administered consecutively for 4 days during the first week (days 0, 1, 2, and 3; early phase), second week (days 7, 8, 9, and 10; middle/late phase), or fourth week (days 21, 22, 23, and 24; much later phase) after PSL. Bupivacaine hydrochloride (Sigma-Aldrich) was dissolved in sterile saline at 0.5% concentration, which were administered for consecutive 6 days after PSL (days 0–5).
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9

Nicotinic Receptor Ligands Pharmacology

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(−)-Nicotine hydrogen tartrate salt was obtained from Sigma Chemical (St. Louis, MO). Varenicline dihydrochloride was obtained from the National Institute on Drug Abuse drug supply program (Rockville, MD). Mecamylamine was obtained from Waterstone Technology (Camel, IN). The epibatidine derivatives 2′-Fluorodeschloroepibatidine (RTI-7527-36), 3′-(3″-dimethylaminophenyl) epibatidine (RTI-7527-76), and 2′-fluoro-3′-(4-nitrophenyl) deschloroepibatidine (RTI-7527-102) were synthesized at the Center for Organic and Medicinal Chemistry, Research Triangle Institute (Research Triangle Park, NC) as previously described [16 (link)–18 (link)]. Dihydro-β-erythroidine hydrobromide was obtained from Tocris (Minneapolis, MN). Drugs were dissolved in 0.9% physiological saline and administered s.c. (except mecamylamine which was administered intraperitoneally) in a volume of 10 mL/kg. The dose of nicotine is expressed as the weight of the free base. Other drug doses are expressed as the weight of the base plus the salt.
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10

Pharmacological Modulation of Neuronal Signaling

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Picrotoxin and MK-801 were from Abcam. CGP55845, DNQX, Dihydro-β-erythroidine hydrobromide, SCH 23390 hydrochloride, and sulpiride were obtained from Tocris Bioscience. BAPTA was obtained from Invitrogen. ω-Conotoxin GVIA and ω-Agatoxin TK were from Alomone Labs. All the other chemicals (α-methyl-DL-tyrosine, DEAB, TTX, 4-Aminopyridine, EGTA-AM, BAPTA-AM, scopolamine hydrobromide) were from Sigma-Aldrich.
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