Nicotinic agonist-induced responses in iPSC-derived neurons examined by FLIPR.

<p>Representative examples of changes in fluorescence detected in iPSC-derived neurons with a range of concentrations of compound B (10 nM—3 μM; A) and epibatidine (10 nM—3 μM; B). C) and D) Averaged data for agonist induced responses in experiments performed at 4 and 28 days in culture. Data represent average of replicates ± SEM. Co-application of the α7-selective PAM PNU-120596 (3 μM; pre-applied for 60 s) with either compound B (1 μM; E) or epibatidine (1 μM; F) resulted in large fluorescence responses. Data are means ± SEMs from 3 experiments. All values are normalised to a control response to application of KCl (30 mM).</p

Agonist activity of 4BP-TQS in iPSC-derived neurons examined by FLIPR.

<p>(A) Representative examples of FLIPR traces with the α7-selective allosteric agonist 4BP-TQS (10 μM; closed circles). Responses to 4BP-TQS are inhibited by the α7-selective antagonist MLA (1 μM; open circles). (B) Concentration-response curve for 4BP-TQS CRC (circles) and 4BP-TQS in the presence of the α7-selective antagonist MLA (1 μM; triangles). Data are means ± SEM of 3 independent experiments.</p

Pharmacological properties of nAChR ligands on iPSC-derived neurons.

<p>Data are means ± SEM of 3–5 independent experiments.</p><p>Pharmacological properties of nAChR ligands on iPSC-derived neurons.</p

Potentiation and antagonism of nAChR agonist responses in iPSC neurons.

<p>(A) Representative of FLIPR traces produced with a range of compound B concentrations (0.3 nM—1 μM) in the presence of PNU-120596 (3 μM). Also shown are concentration-response curves for the agonists compound B (circles), epibatidine (triangles) and choline (squares), in the presence of PNU-120596 (3 μM) (B). Responses to compound B (1 μM) in the presence of PNU-120596 (3 μM) were blocked completely in a concentration-dependent manner by the α7-selective antagonist MLA (C). Data are means ± SEM of 3–5 independent experiments.</p

Agonist and voltage induced responses in iPSC-derived neurons examined by patch-clamp electrophysiology.

<p>A)Representative recording showing no detectable currents with acetylcholine (1 mM) (Left) and a slowly desensitising current in response to co-application of acetylcholine (1 mM; black bar) and PNU-120596 (3 μM; blue bar) (Right). PNU-120596 was pre-applied for 20 s before co-application with acetylcholine. B) The average response data from n = 5 cells was 225.2 +/- 85.9 pA. Representative voltage step current responses with Nav and Kv currents are shown in C). The fast component of the inward current was analyzed and plotted for the Na⁺ current (first 5 ms) and (n = 5 cells) in D. The steady-state current (last 5 ms) corresponding to currents through K⁺ channels is shown in E (n = 5 cells).</p

Influence of temperature on potentiation of Compound B responses by PNU-120596.

<p>A) Representative FLIPR traces showing the change in fluorescence observed when compound B (1 μM) and PNU-120596 (100 μM) were co-applied to iPSC-derived neurons at room temperature (RT) (closed circles) and at 37°C (open circles). B) Concentration-response relationship of PNU-120596 in the presence of compound B (1 μM) at room temperature (RT) and at 37°C. Data points are means of 4 independent experiments each of which generated paired data from the same batch of cells incubated at two temperatures.</p

Relative Expression of nAChR subunits examined by RT-PCR.

<p>A) Levels of gene expression relative to TBP, using data generated from 10 ng RNA input. The following transcripts were either undetectable or detected only at very low levels at 10 ng input RNA: <i>CHRNA1</i> (encoding the α1 nAChR subunit), <i>CHRNA2</i> (α2), <i>CHRNA9</i> (α9), <i>CHRNA10</i> (α10), <i>CHRNB3</i> (β3), <i>CHRNG</i> (γ), CHRND (δ) and CHRNE (ε). Note, all of the CT values at lower input levels were near 35 or not detected. In contrast, the following transcripts were detected at relatively high levels: <i>CHRNA3-CHRNA7</i> (α3-α7), <i>CHRNB1</i> (β1), <i>CHRNB2</i> (β2), <i>CHRNB4</i> (β4) and the partially duplicated gene B) Agarose gel electrophoresis using PCR products from the 50 ng input.</p

Characterisation of nAChRs in iPSC-derived neurons, examined by single-cell intracellular calcium imaging.

<p>A) Pseudocolour images of human iPSC-derived neurons corresponding to low initial resting calcium levels (Left panel) and higher calcium levels after co-application of compound B (1 μM) with PNU-120596 (3 μM) (Right panel). B) Single-cell traces for neurons present in the optical field, showing the effects of application of compound B (1 μM) followed by co-application of compound B (1 μM) with PNU-120596 (3 μM). C) Single-cell traces in response to two consecutive applications of epibatidine (1 μM). D) Single-cell traces in response to two consecutive applications of 5-Iodo-A-85380 (1 μM). In B-D, individual single-cell traces are displayed in cyan, whereas a mean response (average of multiple cells) is shown in red.</p