Fig. 5: Optogenetic activation of LHb-DRN projection neurons and aggressive behavior.

a Schematic of viral targeting and optic stimulation strategy. AAV2-hSyn-hChR2(H134R)-EYFP virus was injected into the LHb, and an optic fiber was inserted into the DRN. On the test day, optic stimulation was applied to the DRN (473 nm, 20 Hz, 10 ms pulse, 3 mW). b Expression of ChR2-EYFP (green) in bilateral LHb. Inset shows an enlarged picture of the LHb (scale bar 200 μm). Blue: DAPI. c ChR2-EYFP-expressing LHb projections (green) were observed in the DRN (outlined in white lines). Putative fiber insertion site was indicated as yellow dotted line. Red: Tph2 antibody immunohistochemistry (scale bar 200 μm). d c-Fos expression in the DRN after 1 min light stimulation. Cyan: Tph2 antibody, Magenta: c-Fos antibody immunohistochemistry (scale bar 200 μm). Inset shows enlarged picture of c-Fos expression in Tph2+ neurons (filled arrowhead) and Tph2- cells (arrowhead without fill). Light stimulation increased c-Fos expression in both Tph2+ 5-HT neurons (e unpaired t test (two-sided), OFF n = 6, ON n = 8 biologically independent animals, t(12) = 4.842, p = 0.0004) and Tph2- non-5-HT neurons (f unpaired t test (two-sided), OFF n = 6, ON n = 8 biologically independent animals, t(12) = 4.297, p = 0.0010). g Each animal was tested with 4 types of condition; OFF: No light stimulation, ON: Light stimulation during aggressive encounter, ON/OFF: Light stimulation 1 min before aggressive encounter, but no light stimulation during aggressive encounter, ON/ON: Light stimulation both 1 min before aggressive encounter and during the aggressive encounter. h Timeline of this experiment. Aggression test was conducted once a day, and OFF sessions were conducted between any type of stimulation sessions. i Duration of total aggressive behaviors in each session. Black line indicates average data of 9 animals (Mean ± SEM), and gray line indicates each individual’s data. j Summarized data for the duration of total aggressive behavior in each condition. Value of OFF session was calculated as average data from day 1, 3, 6, and 8. Both ON and ON/ON sessions showed significantly longer duration of aggressive behavior than the OFF session (one-way repeated measures ANOVA, n = 9 biologically independent animals, F(3,24) = 9.8265, p = 0.0002, post hoc t test with Tukey’s multiple comparisons test (two-sided)). k–n Detailed behavioral analysis showed that both ON and ON/ON sessions increased aggressive behaviors (one-way repeated measures ANOVA, n = 9 biologically independent animals, attack bites: F(3,24) = 14.320, p < 0.0001 (k), sideways treats: F(3,24) = 6.754, p = 0.0018 (l), post hoc t test with Tukey’s multiple comparisons test (two-sided)), but not non-aggressive behaviors (m locomotion) compared to OFF sessions. Social interaction was increased in ON compared to OFF sessions (n one-way repeated measures ANOVA, n = 9 biologically independent animals, F(3,24) = 6.173, p = 0.0029, post hoc t test with Tukey’s multiple comparisons test (two-sided)). o Timeline of subthreshold social instigation (S-Inst) test. S-Inst: A caged-instigator male was presented for 1 min before 5-min aggressive encounter. ON/OFF: 1 min light stimulation was delivered during S-Inst, but no light stimulation during aggressive encounter. p Duration of total aggressive behaviors in each session. Black line indicates average data of 9 animals (Mean ± SEM), and gray line indicates each individual’s data. q Summarized data for the duration of total aggressive behaviors in each condition. Value of OFF session was calculated as average data from Day 1 and 3. Combination of S-Inst and light stimulation (ON/OFF), but not S-Inst without light stimulation (OFF), showed significant increase of the duration of total aggressive behavior (Friedman test (two-sided), n = 9 biologically independent animals, Friedman statistic = 14.00, p = 0.0002, post hoc test with Dunn’s multiple comparison test). r Optogenetic stimulation of LHb-DRN projection (ON/ON) did not induce female-directed aggressive behavior. Each bar represents mean value ± SEM, and gray line indicates each individual’s data. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.