Fig. 3: Chemogenetic inhibition of LHb-DRN projection neurons and instigation-heightened aggression.

a Schematics and timeline of this experiment. Retrograde AAVretro-EF1α-mCherry-IRES-Cre was injected into the DRN, and Cre-dependent AAV2-CAG-Flex-rev-hM4D-2A-GFP (or control AAV2-EF1α-DIO-ChR2-EYFP) was injected into bilateral LHb. Aggression tests were conducted every other day, and the RI test and Inst test were conducted alternately. CNO was injected only on Day 7, and saline (SAL) was injected all other days. Then, animals were tested in the RI test with SAL and CNO injections. b, c Expression of hM4D-EYFP (green) was observed in bilateral LHb (b scale bar 500 μm). Higher magnification of white-dotted square is indicated in c (scale bar 50 μm). Inserted picture shows an enlarged picture of the LHb (outlined in white lines, scale bar 10 μm). d Duration of total aggressive behaviors in each session. Black line (yellow circles) indicates average of EYFP control (n = 11 biologically independent animals), and green line (green squares) indicates average of hM4D group (n = 11 biologically independent animals, Mean ± SEM). e Summarized data for the duration of total aggressive behavior in each condition. Social instigation increased total aggressive behavior regardless of SAL or CNO injection compared to RI in EYFP controls, but the CNO + Inst group did not show significant differences from the SAL + RI session of the LHb-DRN hM4D group (two-way repeated measures ANOVA, Cont n = 11, hM4D n = 11 biologically independent animals, main effect of test-type: F(2,40) = 9.599, p = 0.0004, post hoc t test with Tukey’s multiple comparisons tests (two-sided)). f–i, Detailed behavioral analysis showed that CNO blocked pro-aggressive effects of social instigation, specifically in the hM4D group, on the frequency of attack bites (f two-way repeated measures ANOVA, Cont n = 11, hM4D n = 11 biologically independent animals, Group × Test-type interaction: F(2,40) = 3.877, p = 0.0289, post hoc t test with Tukey’s multiple comparisons tests (two-sided)). No Group × Test-type interaction was observed in sideways threats (g), locomotion (h) or social contacts (i) (two-way repeated-measures ANOVA, Cont n = 11, hM4D n = 11 biologically independent animals, Group × Test-type interaction: all n.s., main effect of test-type: sideways F(2,40) = 12.04, p < 0.0001 (g), locomotion and social contact n.s. (h, i), post hoc t test with Tukey’s multiple comparisons tests (two-sided)). No effect of CNO was observed in the RI test on the duration of total aggressive behavior (j), frequency of attack bites (k), duration of sideways threats (l), locomotion (m), and social contacts (n) (two-way repeated-measures ANOVA, Cont n = 11, hM4D n = 11 biologically independent animals, all n.s.). Each bar represents mean value ± SEM, and gray line indicates each individual’s data. *p < 0.05, **p < 0.01. Source data are provided as a Source Data file.