Fig. 5: Optogenetic activation of PrL GABAergic projections in the Cg1/2 enhances vF-evoked responses in L5.

a Schematic of the approach for Ca²⁺ imaging of Cg1/2 neurons and optogenetic stimulation of axon terminals of PrL GABAergic projection neurons in head-fixed GAD^Cre mice that received GRIN lens and optic fiber implantation. The animals were placed on a grid and received von Frey (vF) stimulation to the hind paw contralateral to the implantation site. b AAV-FLEX-tdTomato (control, N = 13 mice) or AAV-Syn-FLEX-ChrimsonR-tdTomato (ChrimsonR, N = 15 mice) was injected into the PrL, and AAV-CaMKIIa-GCaMP6s was injected into the Cg1/2 of GAD^Cre mice. c Virus-induced expression of tdTomato (control) or tdTomato-tagged ChrimsonR in GABAergic cells of the PrL. Virus-induced expression of GCaMP6s in excitatory cells, implantation of GRIN lens and illumination with red light from an optic fiber tip implanted in the Cg1/2. d Representative field of view through the implanted GRIN lens using the two-photon microscope showing GCaMP6 expressing neurons. Scale bar, 50 μm. e Schematic of the procedure used for Ca²⁺ imaging experiments, indicating stimulation details and temporal profile of vF and red-light stimulation in the three sessions. f Average neuronal responses to vF stimuli in Cg1/2 neurons during session 1 (stippled line), 2 (solid line) and 3 (dotted line) in control mice expressing only td-Tomato (N = 13 mice, n = 307 cells) and in ChrimsonR-expressing mice (N = 15 mice, n = 397 cells). ΔF/F0 traces were smoothed with a Gaussian filter (s = 100 ms) for visualization purpose. Data are shown as means ± S.E.M. Black vertical line indicates the onset of the vF stimulus. Black stippled vertical line indicates the end of the stimulus. g Area under the curves (AUCs) of the vF-evoked responses in control (p = 0.00872, Friedman test, session 1 vs. 2, p = 0.00239, session 1 vs. 3, p = 0.533, DF = 306, two-sided Wilcoxon signed-rank test followed by Bonferroni correction) and h in ChrimsonR mice (p = 6.05e−05, Friedman test, session 1 vs. 2, p = 1.07e−05, session 1 vs. 3, p = 0.00348, DF = 396, two-sided Wilcoxon signed-rank test followed by Bonferroni correction). i Fold change of AUC for session 2 and 3 relative to session 1 in all layers (session 2, p = 3.94e−07, session 3, p = 0.00378, DF = 306, two-sided Mann–Whitney U test followed by Bonferroni correction), j Fold change of AUC for session 2 and 3 relative to session 1 in L2/3 (control, n = 38 neurons, ChrimsonR, n = 94 cells, session 2, p = 1.00, session 3, p = 0.132, DF = 37, two-sided Mann–Whitney U test followed by Bonferroni correction) and k Fold change of AUC for session 2 and 3 relative to session 1 in L5 neurons (control, n = 127 cells, ChrimsonR, n = 112 neurons, session 2, p = 2.44e−06, session 3, p = 0.0538, DF = 111, two-sided Mann–Whitney U test followed by Bonferroni correction). Box and whisker plots in (g–k) indicate median, interquartile range and 10th to 90th percentiles of the distribution. n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001, DF degree of freedom.