Subtype selectivity of compound B, TQS and 4BP-TQS examined with human recombinant nAChR subtypes expressed in <i>Xenopus</i> oocytes.

<p>A) Responses to ACh (3 mM on α7 nAChRs and 100 µM on other nAChR subtypes; black bars) and to compound B (30 µM; grey bars). B) Responses to ACh (100 µM) and to ACh (100 µM; black bars) pre- and co-applied with TQS (100 µM; grey bars). C) Responses to ACh (3 mM on α7 nAChRs and 100 µM on other nAChR subtypes; black bars) and to 4BP-TQS (60 µM on α7 nAChRs and 100 µM on other nAChR subtypes; grey bars).</p

Characterisation of TQS and 4BP-TQS in human induced pluripotent stem cell-derived neurons examined by fluorescence-based intracellular calcium imaging.

<p>A) Pre- and co-application of TQS (30 µM) with compound B (30 nM) potentiates the amplitude of compound B -evoked calcium-responses (almost undetectable in the absence of TQS) (Left). The trace represents the average response of 35 individual traces ± SEM. Responses to compound B (30 µM) co-applied with TQS (30 µM) are blocked when pre- and co-applied with MLA (1 µM; Right). The trace represents the average response of 137 individual traces ± SEM. B) Agonist-evoked calcium-responses were detectable in response to the allosteric agonist 4BP-TQS (30 µM; Left). The trace represents the average response of 122 individual traces ± SEM. Responses to 4BP-TQS (30 µM) were blocked by pre- and co-application of MLA (1 µM; Right). The trace represents the average response of 118 individual traces 5± SEM.</p

Positive allosteric modulation of α7 nAChRs by TQS examined by patch-clamp in rat primary hippocampal cells.

<p>A) Representative recordings showing responses to the application of compound B (1 µM; Left; black bar) and of TQS (10 µM; grey bar) pre-applied for 5 s and then co-applied with compound B (1 µM; Right; black bar). B) Co-application of compound B (1 µM; black bar) with TQS (10 µM; grey bar) results in a two-component response.</p

Characterisation of native nAChRs in rat primary hippocampal cells and human induced pluripotent stem cell-derived neurons, examined by fluorescence-based intracellular calcium imaging.

<p>A) Pseudocolour images of rat hippocampal neurons corresponding to low initial resting calcium levels (Left) and higher calcium levels, after application of 30 µM 4BP-TQS (Right). B) Single cell traces (cyan) for all neurons present in the optical field. The average response is shown in red (n = 89 cells). C) Histogram illustrating the percentage of cells that responded to compound B (1 µM), compound B co-applied with TQS (1 µM and 10 µM, respectively) and 4BP-TQS (30 µM) in rat primary hippocampal cells (blue) and in iCell neurons (red). Data were normalised to the total number of cells that responded to KCl (50 mM) (n = 3–31).</p

Pharmacological properties of TQS and 4BP-TQS on nAChR subtypes.

*<p>Fold potentiation of response to ACh (100 µM) by TQS (100 µM).</p>**<p>Percentage inhibition of response to ACh (100 µM) by TQS (100 µM).</p>†<p>Agonist response of 4BP-TQS (100 µM) expressed as a fold response normalised to maximal concentration of ACh (3 mM).</p>††<p>Percentage inhibition of response to ACh (100 µM) by 4BP-TQS (100 µM). Data are means ± SEM.</p

Block of 4BP-TQS responses by TQS in rat primary hippocampal cells examined by fluorescence-based intracellular calcium imaging.

<p>Dose response data are presented for a range of concentrations of TQS (0.3 to 30 µM) in the presence of 4BP-TQS (10 µM). In all cases TQS was pre-applied for 30 s and then co-applied with 4BP-TQS. Responses were normalised to 4BP-TQS (10 µM). Data are means ± SEM of 3 independent experiments.</p

Characterisation of 4BP-TQS on α7 nAChRs examined by patch-clamp recording of rat primary hippocampal cells.

<p>A) Representative recordings showing responses to the application of compound B (30 µM; Left; black bar) and of 4BP-TQS (30 µM; Right; grey bar). B) Prolonged exposure of α7 nAChRs to compound B (1 µM; black bar) results in receptor activation, followed by rapid desensitisation. In continued presence of compound B (1 µM; black bar), co-application of either TQS (1 µM; Left; grey bar) or 4BP-TQS (30 µM; Right; black bar) resulted in reactivation of desensitised receptors. C) Responses to 4BP-TQS (30 µM; Left; black bar) are blocked by MLA (10 nM; grey bar) when MLA was pre-applied for 15 s and then co-applied with 4BP-TQS (30 µM; black bar).</p

Chemical structure of α7 nAChR orthosteric and allosteric ligands examined in the present study.

<p>Chemical structure of α7 nAChR orthosteric and allosteric ligands examined in the present study.</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