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Engineering a light-regulated GABAA receptor for optical control of neural inhibition.

Lin WC, Davenport CM, Mourot A, Vytla D, Smith CM, Medeiros KA, Chambers JJ, Kramer RH - ACS Chem. Biol. (2014)

Bottom Line: The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism.In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents.LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABAAR-mediated neural inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.

ABSTRACT
Optogenetics has become an emerging technique for neuroscience investigations owing to the great spatiotemporal precision and the target selectivity it provides. Here we extend the optogenetic strategy to GABAA receptors (GABAARs), the major mediators of inhibitory neurotransmission in the brain. We generated a light-regulated GABAA receptor (LiGABAR) by conjugating a photoswitchable tethered ligand (PTL) onto a mutant receptor containing the cysteine-substituted α1-subunit. The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism. In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents. Moreover, it allowed reversible photocontrol over neuron excitation in response to presynaptic stimulation. LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABAAR-mediated neural inhibition.

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α1-LiGABAR enables photocontrol over miniature inhibitorypostsynaptic currents (mIPSCs) and epileptiform formation in hippocampalneurons. (a) A representative continuous trace from a cultured hippocampalneuron containing α1-LiGABAR. The cell was held at −60mV and was treated with inhibitors of voltage-gated sodium channelsand ionotropic glutamate receptors. (b) Average mIPSC traces fromthe same cell shown in panel a. The green and purple traces representaverage mIPSCs when MAB-0 was in the trans (500 nm)and cis (380 nm + dark) configuration, respectively.(c) Quantification of mIPSC photoregulation (mean ± SEM) as thepercent decrease when MAB-0 was switched from cis to trans. The total charge transfer was measuredby integrating the area of average mIPSC.17 (d) Photocontrol over neuronal excitation in a hippocampal slice.Current-clamp recording was carried out in a LiGABAR-containing CA1pyramidal neuron. Illumination by 500 nm light resulted in an “epileptic”plateau potential that was subsequently eliminated by 380 nm light.
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fig4: α1-LiGABAR enables photocontrol over miniature inhibitorypostsynaptic currents (mIPSCs) and epileptiform formation in hippocampalneurons. (a) A representative continuous trace from a cultured hippocampalneuron containing α1-LiGABAR. The cell was held at −60mV and was treated with inhibitors of voltage-gated sodium channelsand ionotropic glutamate receptors. (b) Average mIPSC traces fromthe same cell shown in panel a. The green and purple traces representaverage mIPSCs when MAB-0 was in the trans (500 nm)and cis (380 nm + dark) configuration, respectively.(c) Quantification of mIPSC photoregulation (mean ± SEM) as thepercent decrease when MAB-0 was switched from cis to trans. The total charge transfer was measuredby integrating the area of average mIPSC.17 (d) Photocontrol over neuronal excitation in a hippocampal slice.Current-clamp recording was carried out in a LiGABAR-containing CA1pyramidal neuron. Illumination by 500 nm light resulted in an “epileptic”plateau potential that was subsequently eliminated by 380 nm light.

Mentions: The α1-GABAARis known to cluster at the inhibitorysynapse,7 where it detects GABA releasedfrom the presynaptic axon terminal and mediates transient postsynapticresponses.17,18 GABA is released into the synapticcleft through vesicle exocytosis either spontaneously (which elicitsminiature inhibitory postsynaptic current, mIPSC) or driven by actionpotential.17 To test whether LiGABAR enablesphotocontrol over synaptic inhibition, we measured the photosensitivityof mIPSCs in LiGABAR-containing neurons. Cultured hippocampal neuronswere transfected with a bicistronic construct encoding α1(T125C)and an expression marker (eGFP). The cells were subsequently treatedwith MAB-0 to generate LiGABARs in situ. As shownin Figure 4a, the amplitude of mIPSCs in aLiGABAR-containing neuron increased within 5 s of exposure to 380nm light and remained elevated in darkness for at least 2 min, consistentwith the slow thermal relaxation of cis MAB-0 measuredin Figure 3. The amplitude of mIPSCs decreasedagain upon exposure to 500 nm light. To quantify photosensitivity,we calculated the average mIPSC when MAB-0 was in either trans or cis configuration (Figure 4b). Cis-to-trans photoisomerizationcaused a 38 ± 2% decrease in the peak amplitude and a 57 ±4% decrease in the total charge transfer (n = 6;Figure 4b and c). The robust photocontrol overmIPSCs validates the applicability of LiGABAR for neurophysiologicalinvestigations.


Engineering a light-regulated GABAA receptor for optical control of neural inhibition.

Lin WC, Davenport CM, Mourot A, Vytla D, Smith CM, Medeiros KA, Chambers JJ, Kramer RH - ACS Chem. Biol. (2014)

α1-LiGABAR enables photocontrol over miniature inhibitorypostsynaptic currents (mIPSCs) and epileptiform formation in hippocampalneurons. (a) A representative continuous trace from a cultured hippocampalneuron containing α1-LiGABAR. The cell was held at −60mV and was treated with inhibitors of voltage-gated sodium channelsand ionotropic glutamate receptors. (b) Average mIPSC traces fromthe same cell shown in panel a. The green and purple traces representaverage mIPSCs when MAB-0 was in the trans (500 nm)and cis (380 nm + dark) configuration, respectively.(c) Quantification of mIPSC photoregulation (mean ± SEM) as thepercent decrease when MAB-0 was switched from cis to trans. The total charge transfer was measuredby integrating the area of average mIPSC.17 (d) Photocontrol over neuronal excitation in a hippocampal slice.Current-clamp recording was carried out in a LiGABAR-containing CA1pyramidal neuron. Illumination by 500 nm light resulted in an “epileptic”plateau potential that was subsequently eliminated by 380 nm light.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215903&req=5

fig4: α1-LiGABAR enables photocontrol over miniature inhibitorypostsynaptic currents (mIPSCs) and epileptiform formation in hippocampalneurons. (a) A representative continuous trace from a cultured hippocampalneuron containing α1-LiGABAR. The cell was held at −60mV and was treated with inhibitors of voltage-gated sodium channelsand ionotropic glutamate receptors. (b) Average mIPSC traces fromthe same cell shown in panel a. The green and purple traces representaverage mIPSCs when MAB-0 was in the trans (500 nm)and cis (380 nm + dark) configuration, respectively.(c) Quantification of mIPSC photoregulation (mean ± SEM) as thepercent decrease when MAB-0 was switched from cis to trans. The total charge transfer was measuredby integrating the area of average mIPSC.17 (d) Photocontrol over neuronal excitation in a hippocampal slice.Current-clamp recording was carried out in a LiGABAR-containing CA1pyramidal neuron. Illumination by 500 nm light resulted in an “epileptic”plateau potential that was subsequently eliminated by 380 nm light.
Mentions: The α1-GABAARis known to cluster at the inhibitorysynapse,7 where it detects GABA releasedfrom the presynaptic axon terminal and mediates transient postsynapticresponses.17,18 GABA is released into the synapticcleft through vesicle exocytosis either spontaneously (which elicitsminiature inhibitory postsynaptic current, mIPSC) or driven by actionpotential.17 To test whether LiGABAR enablesphotocontrol over synaptic inhibition, we measured the photosensitivityof mIPSCs in LiGABAR-containing neurons. Cultured hippocampal neuronswere transfected with a bicistronic construct encoding α1(T125C)and an expression marker (eGFP). The cells were subsequently treatedwith MAB-0 to generate LiGABARs in situ. As shownin Figure 4a, the amplitude of mIPSCs in aLiGABAR-containing neuron increased within 5 s of exposure to 380nm light and remained elevated in darkness for at least 2 min, consistentwith the slow thermal relaxation of cis MAB-0 measuredin Figure 3. The amplitude of mIPSCs decreasedagain upon exposure to 500 nm light. To quantify photosensitivity,we calculated the average mIPSC when MAB-0 was in either trans or cis configuration (Figure 4b). Cis-to-trans photoisomerizationcaused a 38 ± 2% decrease in the peak amplitude and a 57 ±4% decrease in the total charge transfer (n = 6;Figure 4b and c). The robust photocontrol overmIPSCs validates the applicability of LiGABAR for neurophysiologicalinvestigations.

Bottom Line: The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism.In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents.LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABAAR-mediated neural inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.

ABSTRACT
Optogenetics has become an emerging technique for neuroscience investigations owing to the great spatiotemporal precision and the target selectivity it provides. Here we extend the optogenetic strategy to GABAA receptors (GABAARs), the major mediators of inhibitory neurotransmission in the brain. We generated a light-regulated GABAA receptor (LiGABAR) by conjugating a photoswitchable tethered ligand (PTL) onto a mutant receptor containing the cysteine-substituted α1-subunit. The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism. In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents. Moreover, it allowed reversible photocontrol over neuron excitation in response to presynaptic stimulation. LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABAAR-mediated neural inhibition.

Show MeSH
Related in: MedlinePlus