Epibatidine

Nicotine hydrogen tartrate salt (free base nicotine concentrations of 0.25⁻¹.5 mg·kg^-1) (Sigma-Aldrich, St. Louis, MO, USA), mec (2.5 mg·kg^-1) (Abcam, Cambridge, MA, USA), DHβE (3.0 mg·kg^-1) (Tocris Bioscience, Bristol, UK), epibatidine (5 μg·kg^-1) (Tocris Bioscience, Bristol, UK), and Ibuprofen sodium (20 mg·kg^-1) (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in 0.9% saline. The volume of liquid injected into the mice was calculated based on animal weight of ~10 ml·kg^-1 [52 (link)]. The control animals were injected with 0.9% saline.

20-methyl spirolide G (≥97% purity), obtained from laboratory cultures of the dinoflagellate Alexandrium ostenfeldii, was purchased from Cifga (Lugo, Spain). [¹²⁵I]α−Bungarotoxin ([¹²⁵I]α−BTX) (210–250 Ci·mmol−1), and (±)[³H]epibatidine (55 Ci·mmol⁻¹), were purchased from PerkinElmer (Courtaboeuf, France). Isoflurane (AErrane^®) was purchased from Baxter S.A. (Lessines, Belgium). Other chemicals were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France), or other standard sources. The α-toxin from Naja nigricollis was synthesized, refolded, and purified, as previously described [30 (link)]. Methyllycaconitine (MLA), and (±)epibatidine were purchased from Tocris Bioscience (Bristol, UK).

All chemicals to prepare the recording and Barth’s solutions, as well as dimethyl sulfoxide, acetylcholine chloride (ACh), carbamylcholine chloride (carbachol) and choline chloride were obtained from Sigma-Aldrich (Poole, U.K.). Dihydro-β-erythroidine hydrobromide (DHβE), methyllycaconitine citrate (MLA), (±)-epibatidine, and PNU120596 were purchased from Tocris (Bristol, U.K.). Compound B ([R-N-(1-azabicyclo[2 (link),2 (link),2 (link)]oct-3-yl)(2-pyridyl)thiophene-2-carboxamide, previously also named as compound A) was synthesized at Eli Lilly & Co and had a purity greater than 95% [19 (link)].

For measurements of intracellular calcium concentration [Ca²⁺]_i changes, transfected Neuro2a cells plated on glass coverslips were perfused at room temperature with the buffer containing 140 mM NaCl, 2 mM CaCl₂, 2.8 mM KCl, 4 mM MgCl₂, 20 mM HEPES, 10 mM glucose; pH 7.4. Expression of Case12, a fluorescent genetically encoded sensor of calcium ions (ex/em = 491/516 nm), allowed direct monitoring of changes in [Ca²⁺]_i using an epifluorescence microscope with an appropriate filter combination and a CAM-XM10 cooled CCD camera (Olympus, Japan). Videos were made and processed using CellA Imaging Software (Olympus Soft Imaging Solutions GmbH, Germany), Image J, CellX, and OriginPro 7.5 software (OriginLab, MA, USA, for statistical analysis). The cells were exposed to acetylcholine iodide (Sigma, Germany), epibatidine (Tocris, UK), and α-cobratoxin purified from Naja kaouthia venom, and changes in Case12 fluorescence were recorded from each cell independently. To increase the registered changes, all ligand solutions contained the α7 nAChR positive allosteric modulator PNU120596 (10 μM, Tocris, UK). To allow recovery of the cells, the washing steps lasted 5–10 minutes. All recordings were made at room temperature.