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. 2017 May 31;37(22):5447–5462. doi: 10.1523/JNEUROSCI.2164-16.2017

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Copyright © 2017 Wright, Newey et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License Creative Commons Attribution 4.0 International, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 7. — Synaptically driven GABA_BR activation can shift E_GABAA. A, Diagram of the experimental setup for synaptically activating postsynaptic GABA_ARs and GABA_BRs. Presynaptic GABAergic interneurons were stimulated in rat organotypic hippocampal slices via a bipolar tungsten electrode positioned at the stratum radiatum/pyramidale border, 50–100 μm from the recorded cell. B, Isolating GABA_AR and GABA_BR responses. Representative traces show monosynaptic GABAergic postsynaptic currents in a CA3 pyramidal neuron recorded in response to single presynaptic stimuli (left) or trains of 6 stimuli applied at 20 Hz (middle). GABA_AR and GABA_BR responses could be pharmacologically isolated by application of the selective GABA_AR antagonist SR95531 (10 μm) and then the GABA_BR antagonist CGP55845 (5 μm). GABA_BR responses were not evoked by single stimuli but were evident for the multiple-stimuli condition. In the absence of these receptor blockers (right), the flux of chloride through GABA_ARs could be minimized by clamping the postsynaptic neuron close to its E_GABAA. Calibration: 100 pA, 500 ms. C, The amplitude of the postsynaptic GABA_BR response is sensitive to presynaptic stimulus frequency. Whereas GABA_AR conductances (gGABA_A) were detected across the range of stimulus frequencies, GABA_BR-mediated conductances (gGABA_B) were largest for high-frequency stimuli of ∼20 Hz and were minimal at lower frequencies (n = 9). D, Resting E_GABAA values measured by muscimol activation of the GABA_AR (n = 25) and by synaptic activation of the GABA_AR (n = 22). Synaptic E_GABAA exhibited a greater range of values and had a mean value of −76.7 ± 2.1 mV, compared with −81.1 ± 1.1 mV for the muscimol-evoked recordings (p = 0.06, t test). E, Example GABA_AR I–V plots for a CA3 pyramidal neuron before (black data) and after (gray data) delivering a conditioning protocol designed to strongly activate postsynaptic GABA_BRs (90 stimuli delivered as 15 bursts of 6 stimuli at 20 Hz, at 5 s intervals). Insets, Raw traces. Calibration: 50 pA, 1 s. F, Change in E_GABAA in a population of CA3 pyramidal neurons (n = 6) following delivery of the GABA_BR conditioning protocol (vertical arrow). G, CA3 pyramidal neurons that underwent the GABA_BR synaptic conditioning protocol (n = 6) showed a significantly larger positive shift in E_GABAA than neurons that experienced a control stimulation protocol (90 stimuli delivered at 1 Hz) designed to generate minimal GABA_BR activation (n = 6, *p = 0.017, ANOVA followed by post hoc Dunnett's correction). The change in E_GABAA induced by the GABA_BR synaptic conditioning protocol was also prevented by blocking GABA_BRs with the selective antagonist CGP55845 (n = 5, *p = 0.022) or by blocking KCC2 activity with VU0240551 (25 μm; n = 7, ***p = 0.001.