Determination of Dissociation Constants for GABAA Receptor Antagonists using Spontaneously Active Neuronal Networks in vitro

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Changes in spontaneous spike activities recorded from murine frontal cortex networks grown on substrate-integrated microelectrodes were used to determine the dissociation constant (KB) of three GABAA antagonists. Neuronal networks were treated with fixed concentrations of GABAA antagonists and titrated with muscimol, a GABAA receptor agonist. Muscimol decreased spike activity in a concentration dependent manner with full efficacy (100% spike inhibition) and a 50% inhibitory concentration (IC50) of 0.14 ± 0.05 µM (mean ± SD, n=6). At 10, 20, 40 and 80 µM bicuculline, the muscimol IC50 values were shifted to 4.3 ± 1.8 µM (n=6), 6.8 ± 1.7 µM (n=6), ... continued below

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Oli-Rijal, Sabnam December 2005.

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  • Oli-Rijal, Sabnam

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Changes in spontaneous spike activities recorded from murine frontal cortex networks grown on substrate-integrated microelectrodes were used to determine the dissociation constant (KB) of three GABAA antagonists. Neuronal networks were treated with fixed concentrations of GABAA antagonists and titrated with muscimol, a GABAA receptor agonist. Muscimol decreased spike activity in a concentration dependent manner with full efficacy (100% spike inhibition) and a 50% inhibitory concentration (IC50) of 0.14 ± 0.05 µM (mean ± SD, n=6). At 10, 20, 40 and 80 µM bicuculline, the muscimol IC50 values were shifted to 4.3 ± 1.8 µM (n=6), 6.8 ± 1.7 µM (n=6), 19.3 ± 3.54 µM (n=10) and 43.5 µM (n=2), respectively (mean ± SD). Muscimol titration in the presence of 10, 20, 40 µM of gabazine resulted in IC50s values of 20.1 (n=2), 37.17 (n=4), and 120.45 (n=2), respectively. In the presence of 20, 80, and 160 µM of TMPP (trimethylolpropane phosphate) the IC50s were 0.86 (n=2), 3.07 (n=3), 6.67 (n=2) µM, respectively. Increasing concentrations of GABAA antagonists shifted agonist log concentration-response curves to the right with identical efficacies, indicating direct competition for the GABAA receptor. A Schild plot analysis with linear regression resulted in slopes of 1.18 ± 0.18, 1.29 ± 0.23 and 1.05 ± 0.03 for bicuculline, gabazine and TMPP, respectively. The potency of antagonists was determined in terms of pA2 values. The pA2 values were 6.63 (gabazine), 6.21 (bicuculline), and 5.4 (TMPP). This suggests that gabazine has a higher binding affinity to the GABAA receptor than bicuculline and TMPP. Hence, using spike rate data obtained from population responses of spontaneously active neuronal networks, it is possible to determine key pharmacological properties of drug-receptor interactions.

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  • December 2005

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  • Feb. 15, 2008, 4:38 p.m.

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  • Dec. 15, 2008, 2:34 p.m.

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Oli-Rijal, Sabnam. Determination of Dissociation Constants for GABAA Receptor Antagonists using Spontaneously Active Neuronal Networks in vitro, thesis, December 2005; Denton, Texas. (digital.library.unt.edu/ark:/67531/metadc4911/: accessed February 25, 2017), University of North Texas Libraries, Digital Library, digital.library.unt.edu; .