Galanthamine

Anhydrous ethyl alcohol was purchased from Commercial Alcohols (Brampton, ON, Canada). Ammonium acetate (anhydrous, ACS reagent grade) was obtained from MP Biomedicals (Solon, OH, USA). Ammonia (ca. 7N solution in methanol) was ordered from ACROS Organics (www.acros.com). Agarose (protein electrophoresis grade, high gelling temperature); Tris base (for molecular biology); EDTA, disodium salt dihydrate; sulfuric acid; acetic acid, glacial (certified ACS); methanol (HPLC grade); chloroform (HPLC grade) and acetonitrile (HPLC grade) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). The alkaloids narciclasine and galanthamine were purchased from Tocris Bioscience (Bristol, UK), whereas lycorine and papaverine were obtained from Sigma-Aldrich (Oakville, ON, Canada).

In all experiments, recording aCSF contained atropine (400 nM; Sigma-Aldrich) to block the actions of muscarinic ACh receptors when ACh was applied, or in case of nicotine application, to prevent muscarinic receptor activation by nicotine-induced endogenous ACh release68 (link). This ensured only the actions of nAChRs were measured and provided standardized background experimental conditions for both nicotine and ACh experiments. Other bath-applied drugs were similarly dissolved in aCSF at the desired concentration (nicotine (300 nM in tLTP experiments, 10 μM in two-photon imaging experiments; Sigma-Aldrich), ACh (1 mM; Sigma-Aldrich), Galanthamine (0.1 or 1 μM; Tocris Bioscience), DHβE (10 μM; Tocris Bioscience)). All experiments were performed in the absence of synaptic blockers, except the experiments with light-evoked endogenous ACh release on L2/3 interneurons (Fig. 2a, top traces), the DHβE pharmacology on L6 pyramidal neurons (Fig. 2b), the experiments investigating dendritic expression of nAChRs (Fig. 4) and the DHβE pharmacology experiments on human L6 pyramidal neurons (Fig. 6d), where GABAzine (10 μM; Tocris Bioscience) and DNQX (10 μM; Tocris Bioscience) were included in the aCSF. In the BAPTA-tLTP experiments (Fig. 5a,b), BAPTA (1 mM; Sigma-Aldrich) was added to the intracellular solution.