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. 2020 Sep 16;23(10):101570. doi: 10.1016/j.isci.2020.101570

Figure 3.

Figure 3

PD-1 Modulates GABA-Mediated Currents in Brain Slices and Dissociated Cortical Neurons

(A) Left: in situ hybridization for Pd1 mRNA expression on somatosensory cortical neurons (co-stained with Nissl in gray) in WT mice. The box is enlarged in three middle panels (labeled with 1). Boxes 2 and 3 are enlarged on the right panels.

(B–D) GABA-induced currents in S1 cortex of brain slices. (B) Experimental setup used for local perfusion of GABA in the layer IV of somatosensory cortex. (C) Left: exemplary traces of currents irritated by transient GABA (1 mM) perfusion from WT and Pd1−/− mice. Right: unit currents. n = 8 and 9 from 3 animals/group. (D) Left: exemplary traces of currents produced by GABA (1 mM) perfusion after incubation with IgG or Nivo (300 ng/mL, 2 h). Right: unit currents. n = 6 and 8 from 5 and 3 animals, respectively.

(E–G) Light-evoked GABA currents in brain slices produced from vGATAi32 mice. (E) Experimental setup showing vGAT+ neurons stimulated with blue light (473 nm, 4 ms) and postsynaptic current recorded from vGAT-negative neurons in the layer IV of somatosensory cortex. (F) Left: exemplary traces of light-induced currents from vGAT-negative neurons treated with IgG or Nivo (300 ng/mL, 2 h). Right, unit currents of light-induced currents. n = 9 and 8, from 3 animals/group. (G) Percentile reduction of light-induced unit currents by Nivolumab (300 ng/mL, 2 h) in different regions of spinal cord and brain.

(H–J) Inhibition of GABA-induced currents by Nivolumab in cultured cortical neurons. (H) Left: double staining of PD-1 and NeuN in cultured cortical neurons. Right: absence of PD-1 immunostaining upon treatment of a blocking peptide. (I) Top: bright field images of cultured cortical neurons. Middle and bottom: immunostaining for Nivo or IgG4 after Nivo or IgG4 (300 ng/mL, 2 h). (J) Upper: exemplary traces of GABA (1 mM)-induced currents in cortical neurons treated with IgG or Nivo (300 ng/mL, 2 h). Lower: unit currents of GABA currents. n = 19 and 26 from 3 animals per group. The cortical neurons were cultured for 7–8 days before experiment. Gray arrowheads indicate the application of GABA. Blue arrowheads indicate the illumination of blue light. VH = 0 mV. Unpaired two-tailed t test. All error bars indicate the mean ± SEM.