Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones.
Bottom Line: Taken together our data support the notion that hECN GABAARs have an α2/3β3γ2 subunit composition - a composition that also predominates in immature rodent cortex.GlyRs expressed by hECNs were activated by glycine with an EC50 of 167 μm.RNA-seq indicates GlyRs are likely to be composed of α2 and β subunits.
Affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, National Centre for Biological Sciences, Bangalore, 560065, India Euan MacDonald Centre for MND Research, University of Edinburgh, Edinburgh, EH16 4SB, UK.Show MeSH
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Mentions: We next performed a series of pharmacological assays to assess the presence of γ and/or δ subunit-containing GABAARs. Applications of γ-selective allosteric potentiator diazepam (30 nm and 3 μm) to GABA (EC10; 35 μm)-mediated currents potentiated the control GABA response by 10 ± 6 % (P = 0.1 vs. control) and 46 ± 10 % (P < 0.001 vs. control, Welch's t test, n = 17, N = 3), respectively, indicating the presence of the γ subunit (Fig. 2A). In contrast, applications of Zn2+ (10 μm and 300 μm), which selectively inhibits GABAARs composed of α and β subunits only (Draguhn et al. 1990), did not inhibit GABA (EC50)-evoked currents (10 μm, 6 ± 3 %, P = 0.053 vs. control; 300 μm, 11 ± 5 %, P = 0.052 vs. control; unpaired t tests; n = 9, N = 1; Fig. 2B). Furthermore, the potent δ-containing GABAAR-selective agonist gaboxadol (3 μm and 300 μm; Storustovu & Ebert, 2006) gave only nominal currents (6.0 ± 2.3% and 14.6 ± 3.7%; both data P < 0.001 vs. GABA (3 mm); unpaired t tests; n = 6–7, N = 1, respectively) compared to the maximum response that could be elicited by GABA (3 mm; Fig. 2C), confirming that a population of GABAARs that contain δ subunits is negligibly expressed. We confirmed that the low potency of GABA we observed was not a consequence of the specific culture conditions that we employed. Indeed GABA potency was not influenced by the culture of hECNs in atmospheric O2 48 h prior to recording (222 ± 13 μm, n = 3, N = 1), the absence of brain-derived neurotrophic factor and glial cell-derived neurotrophic factor media supplements (222 ± 36 μm, n = 5, N = 2), or maintaining hECNs for extended (49–56 DIV) culture periods (204 ± 17 μm, n = 5, N = 2). Moreover, even for hECNs maintained for extended culture periods gaboxadol (300 μm)-evoked currents remained very low (9.7 ± 4.1 %, n = 4, N = 1) with respect to GABA-evoked currents and indicated that hECNs maintained in culture for prolonged time periods (49–56 DIV) did not begin to express a δ-containing receptor population.
Affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, National Centre for Biological Sciences, Bangalore, 560065, India Euan MacDonald Centre for MND Research, University of Edinburgh, Edinburgh, EH16 4SB, UK.