Fig. 5.

Optogenetic excitation of orexin^LH→LC-induced freezing behavior in Context A′. a Experimental procedure. b Schematic representation showing sites of AAV ₁₀ -FLEX-ChR2-EYFP delivery and optic fiber implantation in Orexin-Cre mice. c Focal expression of Cre-dependent ChR2 in orexin^LH neurons (as shown with white arrow heads) and their axon terminals in the LC. Scale bars: upper panel, 100 μm; lower panel, 50 μm; right panel, 100 μm. d, e After cued fear conditioning, optogenetic stimulation of orexin ^LH to LC was applied in context A′ for 150 s. f Control experiments indicating that laser stimulation did not show any effect on freezing without fear conditioning. g Optogenetic stimulation of orexin fibers induced robust Fos expression in NA^LC neurons (white arrow heads). Scale bars: right panel, 100 μm. h Fos⁺ /TH+cells were markedly increased by optogenetic manipulation as compared to the control group (Orexin-Cre-;ChR2) and the WT (CS+) groups. i, j After cued fear conditioning, SB334867 administration 1 h before test session blocked optogenetically induced increase of freezing. k Laser stimulation itself did not show any effect on freezing without prior fear learning. *p < 0.05, **p < 0.01, ***p < 0.001. Values are presented as mean ± SEM