Fig. 1: Specific activation of NG2 glia by optogenetic stimulation.
From: NG2 glia-derived GABA release tunes inhibitory synapses and contributes to stress-induced anxiety
a Cartoon illustrating the procedure for generating transgenic mouse line by breeding Pdgfrα-creERTM with ChR2(H134R)-eYFP (Ai32) mice to induce ChR2 expression in NG2 glia after consecutive 5-day tamoxifen injections. The immunohistochemistry images below show the ChR2-eYFP labeling (in green) is specifically expressed in NG2 antibody-labeled NG2 glia (in red) in adult hippocampus from Pdgfrα-creERTM;ChR2(H134R)-eYFP mice at postnatal 4–6 weeks. The colocalization is indicated by arrows. Scale bar, 20 μm. b Representative images show a whole-cell patched NG2 glia expressing ChR2-eYFP loaded with Alexa Fluor 568 in the patch pipette in the stratum radiatum region of the adult hippocampus at P30 and identified with post-immunostaining of NG2 antibody. The colocalization is indicated by arrows. Scale bar, 20 μm. c Whole-cell voltage-clamp and current-clamp recordings from NG2 glia showing a typical sodium channel current depolarization but not generating action potentials as the arrows indicate (voltage steps: –160–70 mV, 10 mV/step; current steps: −200–1200 pA, 100 pA/step). The summary bar graphs show there is no change of the cell membrane properties after ChR2 expression in NG2 glia compared with its mGFP control (resting membrane potentials for mGFP, −93.0 ± 1.7 mV, n = 14 cells; ChR2, −89.9 ± 1.4 mV, n = 10 cells, Mann–Whitney test, P = 0.1721; membrane input resistances for mGFP, 105.5 ± 10.3 MΩ, n = 14 cells; ChR2, 112.1 ± 20.8 MΩ, n = 11 cells, two-tailed unpaired t test, P = 0.7645). d Representative traces show that a 100 ms blue light pulse or 10 ms 15 Hz light pulses stimulation at 5 mW/mm2 illumination intensity induces one or a series of non-degrading photocurrents in NG2 glia from Pdgfrα-creERTM; ChR2 mouse hippocampus at postnatal 4–6 weeks. e Bar graph summarizing the peak and steady-state ChR2-evoked photocurrents in NG2 glia. n = 11 cells. f Representative images of cFos staining in NG2 glia before and after ChR2 photoactivation. The arrows indicate a significant increase of cFos-positive NG2 glia after blue light stimuli in acute hippocampal slices at postnatal 4–6 weeks. Scale bars, 20 μm. g Summary graph shows the percentage increase of cFos-positive NG2 cells in a whole population of NG2 glia after photostimulation. No blue light stimuli: 24.5 ± 7.23%; After blue light stimuli: 73.8 ± 6.14%, n = 5 mice, two-tailed unpaired t test, P = 0.0008. Data are presented as mean values ± SEM and the error bar represents SEM.
