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Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones.

James OT, Livesey MR, Qiu J, Dando O, Bilican B, Haghi G, Rajan R, Burr K, Hardingham GE, Chandran S, Kind PC, Wyllie DJ - J. Physiol. (Lond.) (2014)

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.

View Article: PubMed Central - PubMed

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.

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Modulation of hECN GABAARs by diazepam, Zn2+ and gaboxadolA, left panel: representative whole-cell recording depicting the co-application of diazepam (30 nm and 3 μm, as indicated by bars) to control GABA-evoked responses. A, right panel: modulation of GABAAR-mediated currents by diazepam (30 nm and 3 μm, n = 17, N = 3). Data are presented as mean percentage modulation with respect to control recordings. No difference was observed between percentage modulation and the batch from which cells were prepared. Calibration bar 50 pA, 50 s. B, left panel: example whole-cell recording depicting the co-application of Zn2+ (10 μm and 300 μm, as indicated by bars) to control GABA-evoked responses. B, right panel: mean percentage modulation of control GABAAR-mediated currents by Zn2+ (n = 9, N = 1). Calibration bar 100 pA, 50 s. C, left panel: example whole-cell recording of GABA (3 mm)-evoked currents and gaboxadol (3 μm and 300 μm)-induced currents. C, right panel: mean percentage gaboxadol-induced activation of GABAAR currents with respect to (w.r.t.) maximum GABA-evoked currents (n = 6–7, N = 1). Calibration bar 500 pA, 50 s.
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fig02: Modulation of hECN GABAARs by diazepam, Zn2+ and gaboxadolA, left panel: representative whole-cell recording depicting the co-application of diazepam (30 nm and 3 μm, as indicated by bars) to control GABA-evoked responses. A, right panel: modulation of GABAAR-mediated currents by diazepam (30 nm and 3 μm, n = 17, N = 3). Data are presented as mean percentage modulation with respect to control recordings. No difference was observed between percentage modulation and the batch from which cells were prepared. Calibration bar 50 pA, 50 s. B, left panel: example whole-cell recording depicting the co-application of Zn2+ (10 μm and 300 μm, as indicated by bars) to control GABA-evoked responses. B, right panel: mean percentage modulation of control GABAAR-mediated currents by Zn2+ (n = 9, N = 1). Calibration bar 100 pA, 50 s. C, left panel: example whole-cell recording of GABA (3 mm)-evoked currents and gaboxadol (3 μm and 300 μm)-induced currents. C, right panel: mean percentage gaboxadol-induced activation of GABAAR currents with respect to (w.r.t.) maximum GABA-evoked currents (n = 6–7, N = 1). Calibration bar 500 pA, 50 s.

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.


Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones.

James OT, Livesey MR, Qiu J, Dando O, Bilican B, Haghi G, Rajan R, Burr K, Hardingham GE, Chandran S, Kind PC, Wyllie DJ - J. Physiol. (Lond.) (2014)

Modulation of hECN GABAARs by diazepam, Zn2+ and gaboxadolA, left panel: representative whole-cell recording depicting the co-application of diazepam (30 nm and 3 μm, as indicated by bars) to control GABA-evoked responses. A, right panel: modulation of GABAAR-mediated currents by diazepam (30 nm and 3 μm, n = 17, N = 3). Data are presented as mean percentage modulation with respect to control recordings. No difference was observed between percentage modulation and the batch from which cells were prepared. Calibration bar 50 pA, 50 s. B, left panel: example whole-cell recording depicting the co-application of Zn2+ (10 μm and 300 μm, as indicated by bars) to control GABA-evoked responses. B, right panel: mean percentage modulation of control GABAAR-mediated currents by Zn2+ (n = 9, N = 1). Calibration bar 100 pA, 50 s. C, left panel: example whole-cell recording of GABA (3 mm)-evoked currents and gaboxadol (3 μm and 300 μm)-induced currents. C, right panel: mean percentage gaboxadol-induced activation of GABAAR currents with respect to (w.r.t.) maximum GABA-evoked currents (n = 6–7, N = 1). Calibration bar 500 pA, 50 s.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215781&req=5

fig02: Modulation of hECN GABAARs by diazepam, Zn2+ and gaboxadolA, left panel: representative whole-cell recording depicting the co-application of diazepam (30 nm and 3 μm, as indicated by bars) to control GABA-evoked responses. A, right panel: modulation of GABAAR-mediated currents by diazepam (30 nm and 3 μm, n = 17, N = 3). Data are presented as mean percentage modulation with respect to control recordings. No difference was observed between percentage modulation and the batch from which cells were prepared. Calibration bar 50 pA, 50 s. B, left panel: example whole-cell recording depicting the co-application of Zn2+ (10 μm and 300 μm, as indicated by bars) to control GABA-evoked responses. B, right panel: mean percentage modulation of control GABAAR-mediated currents by Zn2+ (n = 9, N = 1). Calibration bar 100 pA, 50 s. C, left panel: example whole-cell recording of GABA (3 mm)-evoked currents and gaboxadol (3 μm and 300 μm)-induced currents. C, right panel: mean percentage gaboxadol-induced activation of GABAAR currents with respect to (w.r.t.) maximum GABA-evoked currents (n = 6–7, N = 1). Calibration bar 500 pA, 50 s.
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus