4.6. nAChR α7 Activity Testing

Human α7 nAChR receptor subunits were cloned and inserted into expression vectors as described previously [53]. Plasmid cDNAs were linearized using a downstream Not I restriction site and purified. cRNA was prepared and capped from the linearized cDNA using the mMessage mMachine T7 transcription kit according to the manufactures protocol. Purified cRNA was aliquoted and stored at a concentration of 0.5 µg·µL⁻¹ at −80 °C until further use.

Xenopus laevis oocytes were obtained as described previously [54], briefly, ovary lobes were removed by surgical incision, sliced into small pieces and defolliculated by collagenase treatment. The protocol for this specific study was approved by the Animal Ethics Committee of the University of Sydney (Protocol number: 2013/5915) and carried out according to these guidelines. Stage V and VI oocytes were injected with a total of ~25 ng of cRNA encoding human α7 nAChR with RIC3 (in 5:1 ratio), a protein enhancing the expression of the receptor. Injected oocytes were incubated for 3–5 days at 18 °C in a saline solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES (hemisodium, pH 7.4)) supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, and 50 µM gentamycin.

Electrophysiological recordings from Xenopus laevis oocytes were performed using the two-electrode voltage-clamp technique as described previously [54,55]. Briefly, oocytes were placed in a custom-built recording chamber and continuously perfused with a saline solution. The saline solution contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES (hemisodium, pH 7.4). Pipettes were backfilled with 3 M KCl and open-pipette resistances ranged from 0.3 to 1.5 MΩ when submerged in the saline solution. Oocytes were voltage clamped at a holding potential of −60 mV using an Axon Geneclamp 500B amplifier (Molecular Devices, LLC, San Jose, CA, USA). Rapid solution exchange in the oocyte vicinity (order of a few seconds) was ensured by application through a 1.5 mm diameter capillary tube placed approximately 2 mm from the oocyte as described previously [54]. The solution flow rate through the capillary was 2.0 mL/min. Experiments were performed as follows: nAChR currents were initially evoked with three ACh_control applications (~EC₂₀, 30 µM), a maximum efficacious concentration of ACh_max (EC₁₀₀, 3 mM) followed by three additional ACh_control applications. Thereafter, test compounds in increasing concentration were applied (25 s), the maximal tested concentration was 200 µM. A wash period of at least 2 min was kept between each application. After the agonist test, new ACh controls were applied (ACh_max followed by three additional ACh_control applications) and compounds that displayed <1% activation were tested for their ability to modulate the effect of ACh. In these experiments, 10 and 100 µM concentration of a test compound was co-applied with 30 µM ACh. Peak current amplitudes were normalized with respect to the amplitude of current elicited by 3 mM or 30 µM ACh for the agonist and antagonist test, respectively. All experiments were conducted at least in triplicate.

ACh was initially dissolved in milliQ water as 10 mM stock solution. Screened compounds were dissolved as a 50 mM stock solution in DMSO, except for Ýmir-2 and compound 12 which were kept as 10 mM stock solutions. The maximal DMSO concentration in the final dilution did not exceed 2%. This DMSO concentration did not evoke any current from the receptors. Compound dilutions were prepared in a saline solution on the day of the experiment.