The GABA_B1 subunit expresses at the membrane in transfected AtT-20 cells.

<p>AtT-20 cells were transfected with both subunits for the GABA_B receptor along with GFP (shown in green). Immunohistochemistry was performed for the GABA_B1 subunit (red) and cell nuclei were stained with DAPI (blue). <b>a)</b> Magnified image of an isolated cell demonstrating GABA_B1 subunit protein at the cell membrane in a GFP positive cell. <b>b)</b> Negative control (no primary antibody for GABA_B1). A merged image is shown at the bottom of each column. Scale bars are 5 μm.</p

R-isovaline does not activate GABA_B receptors in cultured hippocampal neurons.

<p><b>a</b>) Current at a holding potential of -80 mV in a high extracellular K⁺ solution at baseline (open bars) and on application of R-baclofen (n = 7), R-baclofen + CGP 52432 (n = 7) and R-isovaline at low (1 μM; n = 3) and high (50 μM; n = 8) concentrations (shaded bars). <b>b</b>) Example shows R-baclofen-mediated current <b>c</b>) Example shows lack of effect of low concentration of R-isovaline in a cell that responded to R-baclofen both before and after R-isovaline application. <b>d</b>) Example shows the effect of R-baclofen after 5 min pretreatment with 50 μM R-isovaline. <b>e</b>) Example shows the effects of two applications of R-baclofen after prolonged incubation (> 1 hour) with R-isovaline. <b>f</b>) Graph shows the magnitude of the current evoked by 5 μM R-baclofen in control extracellular solution (n = 8), after 5 minutes pretreatment with R-isovaline (n = 4) or after > 1 hour pretreatment with R-isovaline (n = 8). Data represent mean ± SEM. ** = P < 0.01.</p

GABA, R-baclofen and R-isovaline do not evoke currents in untransfected AtT-20 cells.

<p><b>a)</b> Baseline subtracted IV curve for GABA (n = 4), R-baclofen (n = 4) and R-isovaline (n = 5). <b>b</b>) Example currents at the maximum hyperpolarising step (-112.5 mV, voltage step depicted above currents) at baseline (black) and on application (grey) of GABA, R-baclofen or R-isovaline. <b>c</b>) Summary graph showing currents recorded at -112.5 mV at baseline (open bars) and on application of GABA, R-baclofen, or R-isovaline (shaded bars). Data are represented as mean ± SEM.</p

GABA and R-baclofen, but not isovaline, evoke inwardly rectifying currents in transfected AtT-20 cells.

<p><b>a)</b> Baseline subtracted IV curve for GABA and GABA + CGP 52432 (n = 7). Inset shows example currents at baseline (black), in the presence of GABA (dark grey) or in the presence of GABA + CGP 52432 (light grey). <b>b</b>) Baseline subtracted IV curve for R-baclofen (n = 6). Inset shows example currents at baseline (black) or in the presence of R-baclofen (dark grey). <b>c</b>) Baseline subtracted IV curves for R-isovaline at three concentration (50 μM n = 6; 250 μM n = 8; 1 mM n = 4). <b>d</b>) Example currents at baseline (black) and on application of three concentrations of R-isovaline (dark grey). <b>e</b>) Baseline subtracted IV curve for S-isovaline (n = 4). Inset shows example currents at baseline (black) or in the presence of S-isovaline (dark grey). <b>f</b>) Summary graph showing the currents recorded at -112.5 mV at baseline (open bars) or on application of GABA, R-baclofen, R-isovaline or S-isovaline (shaded bars). Data are represented as mean ± SEM. *** = p < 0.001.</p

R-isovaline does not modulate GABA-evoked currents in transfected AtT20 cells.

<p><b>a</b>) Baseline subtracted IV curves for GABA (1 μM; n = 3) and co-application of R-isovaline with GABA (1 μM n = 3; 10 nM n = 6). <b>b</b>) Graph shows that R-isovaline does not alter the current evoked by a high concentration of GABA. Inset shows example currents at baseline (black), on application of GABA (1 μM; dark grey) and on co-application of 1 μM GABA with 250 μM R-isovaline (light grey). <b>c</b>) Graph shows that R-isovaline does not alter the current when co-applied with a low concentration of GABA. Inset shows example currents at baseline (black), on application of 1 μM GABA (dark grey), and on co-application of 10 nM GABA with 250 μM R-isovaline (light grey). Data are expressed as mean ± SEM. * = p < 0.05, ** = p < 0.01.</p