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. 2021 Jun 24;46(11):1918–1926. doi: 10.1038/s41386-021-01071-w

Fig. 1. aINS inputs to brainstem regulated CLAD but not AOD or saccharin intake.

Fig. 1

(A) Schematic of an alcohol-drinking study: 3mo intermittent intake, then 5d/week 20-min access; test sessions for “Opto” (optogenetics) involved “laser” or “no-las” (no laser) tests during alcohol-only (AOD) and compulsion-like alcohol-drinking (CLAD) sessions; “Pharm” (pharmacology) experiments were similar except with “drug” or “veh” (vehicle) (details in “Materials and Methods”). (B) Cartoon of optogenetic methods targeting AINS to LC Area (LCA). (C) Horizontal section showing aINS inputs (green) overlap LC (red for TH + staining) but are primarily lateral to LC. (D, E) Examples of fiber-optic placement in (F) eYFP rat and (G) halorhodopsin rat (TH + LC is torn in E). Note that eYFP expression is less strong in brainstem relative to halorhodopsin–eYFP, which likely reflects that the latter has targeting to the surface membrane. (F) 488 nanometer laser light in brainstem significantly reduced CLAD in halorhodopsin- but not eYFP-expressing rats. * indicates p < 0.05 difference between CLAD/Halorh/Laser and both CLAD/eYFP/Laser and AOD/Halorh/Laser. For the 3-way ANOVA, there was a significant interaction for drinkXlaser (F1,52 = 8.982, p = 0.004) but no interaction for drinkXvirus (F1,52 = 2.306, p = 0.135) or laserXvirus (F1,52 = 0.011, p = 0.917), and significant main effect of drink (F1,52 = 7.766, p = 0.007) but not laser (F1,52 = 3.538, p = 0.066) or virus (F1,52 = 0.171, p = 0.681). Two-way ANOVAs showed a significant effect of laser stimulation with halorhodopsin [F(drinking-condition;1,6) = 8.653, p = 0.007; F(laser;1,6) = 1.841, p = 0.187; F(interaction;1,6) = 11.811, p = 0.002] but not eYFP [F(drinking-condition;1,7) = 1.952, p = 0.205; F(laser;1,7) = 4.559, p = 0.070; F(interaction;1,7) = 1.060, p = 0.337]. Two-way ANOVAs determined post hocs. (G) No effect of halorhodopsin inhibition of aINS–brainstem on saccharin intake. Scale bars indicate 1, 2, and 0.6 mm in (C), (D), and (E). PB parabrachium, TH tyrosine hydroxylase.