Article Figures & Data
Figures
- Figure 1.
CaMKIIα+ neurons of the DR are activated by attack. A–F, Mice (10 weeks of age) were examined for aggression using the RI assay. Mice were perfused 60 min after the assay for immunostaining. A, Representative images of brain sections stained for c-Fos and CaMKIIα in the DR from resident or control animals 1 h after the RI test. B, Representative high-magnification images of CaMKIIα+ cells in the DR that were positive for c-Fos. C, D, Percentage of c-Fos+ cells that colocalize with CaMKIIα+ and DAPI+ cells in resident and control mice for A. E, F, Quantification of CaMKIIα+ (E) and DAPI+ (F) cells in the DR in resident and control mice. G, Representative images of brain sections stained for CaMKIIα and TpH2 throughout the DR (bregma −4.34 to −4.84 mm). High-magnification image of the DR is taken from a brain slice at bregma position −4.84, as this is where the majority of TpH2+ cells can be found. H, Quantification of total number of CaMKIIα+ and TpH2+ cells in the DR. Asterisks indicate statistical significance of CaMKIIα (green) or TpH2 (red) from merged set at the specified coordinate. One slice was quantified per area per animal. Animal number is indicated in parentheses. I, Percentage of DAPI+ cells in the DR that co-label for CaMKIIα and TpH2. Asterisks indicate statistical significance of CaMKIIα (green) or TpH2 (red) from merged set at the specified coordinate. Only cells within the DR were counted. Scale bars: 200 μm (A, G) and 25 μm (B). Data are presented as mean ± SEM; *p < 0.05, **p < 0.01. Statistics can be found in Table 2.
- Figure 2.
Inhibition of CaMKIIα+ DR neurons shortens attack. A, Representative high-magnification and low-magnification images of ChR2-mCherry and ArchT-EYFP expression in the DR three weeks after viral injection. B, Representative images of brain sections stained for c-Fos in the DR of mice injected with ChR2-EYFP or GFP and photostimulated with 473 nm light (pulsed at 10 Hz for 10 s). C, D, Percentage of YFP+ or GFP+ cells that colocalize with c-Fos (C) and quantification of total DAPI+ cells (D) in the DR after photostimulation for B. Only cells within the DR were counted. E, F, Representative images of brain sections expressing ChR2 (E) or ArchT (F) under the CaMKIIα promoter co-stained for TpH2 in the DR. G, Quantification of ChR2 or ArchT (opsin) expressing cells that were stained positive for TpH2. Only cells within the DR were counted. One slice was quantified per area per animal. Animal number is indicated in parentheses. H, L, P, Schematic drawing of viral injection (AAV expressing ChR2 and ArchT or GFP control virus), placement of optic fiber, and stimulation procedure. Opsin expressing mice in H–K and opsin stimulated mice in L-S were injected with ChR2 virus and ArchT virus. The same mice were stimulated with 473 or 561 nm light on separate days. I, Raster plots of attack events during each interaction episode (rows in the raster plot, defined as the period from 20 s before to 20 s after the onset of a spontaneous attack) for each mouse. All attack events during the testing period are shown. J, % of episodes (rows) in which mice attacked at each time point in I; red and dark lines indicate the mean, pink and gray areas indicate SEM. K, Quantification of average attack time per mouse during the 10 s after the onset of a spontaneous attack, as represented in the boxed area in J. M, Raster plots of trials (rows) of mice photostimulated with 473 nm light at the DR when the mouse was not attacking. All trials are aligned to the onset of light. N, % of trials in which mice attacked at each time point in M; red and dark lines indicate the mean, pink and gray areas indicate SEM. O, Quantification of attack time per mouse before and during light stimulation for M. Q, Raster plots of trials (rows) of mice photostimulated with 561 nm light at the DR during an attack. All trials are aligned to onset of light. R, % of trials in which mice attacked at each time point in Q; red and dark lines indicate the mean, pink and gray areas indicate SEM. S, Quantification of attack time per mouse before and during light stimulation for Q. Scale bars: 200 μm (low-magnification image; top; A, E, F); 50 μm (high-magnification image; bottom; A, E, F); 10 μm (B). Data are presented as mean ± SEM; ***p < 0.001. Statistics can be found in Table 2.
- Figure 3.
DR neurons project to and activate the MeOC. A–E, Schematic drawing of ChR2-EYFP viral injection and representative images of ChR2 expression in DR neurons (B) and DR projections at the MeOC (C–E) three weeks later. F–K, c-Fos labeling of mice photostimulated at the DR-MeOC. L, M, Quantification of total number of cells (L) and percentage (M) of c-Fos+ cells in the MeOC for D–K. Only cells within the MeOC were counted. One slice was quantified per area per animal. Animal number is indicated in parentheses. Scale bars: 200 μm (A, B; low-magnification images in C, D) and 50 μm (high-magnification images in C, D). Data are presented as mean ± SEM; **p < 0.01, ***p < 0.001. Statistics can be found in Table 2.
- Figure 4.
DR neurons project to and activate the MeA. A–E, Schematic drawing of ChR2-EYPF injections into the DR and representative images of ChR2 expression in DR neurons (B) and axons at the MeA (C–E) three weeks later. F–K, c-Fos labeling of mice photostimulated at the DR-MeA. L–U, Quantification of total number of cells (L–M) and percentage (Q–U) of c-Fos+ cells in the MeA (MeAa, anterior MeA; MeApd, posteriordorsal MeA; MeApv, posteriorventral MeA) for D–K. Only cells within the MeA were counted. One slice was quantified per area per animal. Animal number is indicated in bars of bar graphs. Scale bars: 200 μm (A); 200 μm (low-magnification images of B–D, low-magnification images of F–K); 50 μm (high-magnification images of F–K). Data are presented as mean ± SEM; *p < 0.05, **p < 0.01. Statistics can be found in Table 2.
- Figure 5.
Inhibition of CaMKIIα+ DR input to the MeOC shortens attack. A, E, I, Schematic drawing of viral injection (AAV expressing ChR2 and ArchT or GFP control virus), placement of optic fiber, and stimulation procedure. Opsin expressing mice in A–D and opsin stimulated mice in E–L were injected with ChR2 virus and ArchT virus. The same mice were stimulated with 473 or 561 nm light on separate days. B, Raster plots of attack events during each interaction episode (rows in the raster plot, defined as the period from 20 s before to 20 s after the onset of a spontaneous attack) for each mouse. All attack events during the testing period are shown. C, % of episodes (rows) in which mice attacked at each time point in B; red and dark lines indicate the mean, pink and gray areas indicate SEM. D, Quantification of average attack time per mouse during the 10 s after the onset of a spontaneous attack, as represented in the boxed area in C. F, Raster plots of trials (rows) of mice photostimulated with 473 nm light at the DR-MeOC when the mouse was not attacking. G, % of trials in which mice attacked at each time point in F; red and dark lines indicate the mean, pink and gray areas indicate SEM. H, Quantification of attack time per mouse before and during light stimulation for F. J, Raster plots of trials (rows) of mice photostimulated with 561 nm light at the DR-MeOC during an attack. K, % of trials in which mice attacked at each time point in J; red and dark lines indicate the mean, pink and gray areas indicate SEM. L, Quantification of attack time per mouse before and during light stimulation for J. Data are presented as mean ± SEM; **p < 0.01. Statistics can be found in Table 2.
- Figure 6.
Effects on attack of CaMKIIα+ DR input to the MeA. A, E, I, Schematic drawing of viral injection (AAV expressing ChR2 and ArchT or GFP control virus), placement of optic fiber, and stimulation procedure. Opsin expressing mice in A–D and opsin stimulated mice in E–L were injected with ChR2 virus and ArchT virus. The same mice were stimulated with 473 or 561 nm light on separate days. B, Raster plots of attack events during each interaction episode (rows in the raster plot, defined as the period from 20 s before to 20 s after the onset of a spontaneous attack) for each mouse. All attack events during the testing period are shown. C, % of episodes (rows) in which mice attacked at each time point in B; red and dark lines indicate the mean, pink and gray areas indicate SEM. D, Quantification of average attack time per mouse for the 10 s after the onset of a spontaneous attack, as represented in the boxed area in C. F, Raster plots of trials (rows) of mice photostimulated with 473 nm light at the DR-MeA when the mouse was not attacking. G, % of trials in which mice attacked at each time point in F; red and dark lines indicate the mean, pink and gray areas indicate SEM. H, Quantification of attack time per mouse before and during light stimulation for F. J, Raster plots of trials (rows) of mice photostimulated with 561 nm light at the DR-MeA during an attack. K, % of trials in which mice attacked at each time point in J; red and dark lines indicate the mean, pink and gray areas indicate SEM. L, Quantification of attack time per mouse before and during light stimulation for J. Data are presented as mean ± SEM. Statistics can be found in Table 2.
- Figure 7.
Activation of the DR-MeOC prolongs attack and activation of the DR-MeA shortens attack. Mice were injected with ChR2 virus or GFP virus into the DR and optical fibers were placed into the MeOC (A, E) or the MeA (I, M). RI tests were performed eight weeks later. Separate groups of mice were used for A–H and I–P. B, Raster plots of attack events during each interaction episode (rows in the raster plot, defined as the period from 20 s before to 20 s after the onset of a spontaneous attack) for each mouse. All attack events during the testing period are shown. C, % of episodes (rows) in which mice attacked at each time point in B; red and dark lines indicate the mean, pink and gray areas indicate SEM. D, Quantification of average attack time per mouse for the 10 s after the onset of a spontaneous attack, as represented in the boxed area in C. F, Raster plots of trials (rows) of mice photostimulated with 473 nm light at the DR-MeOC during an attack. G, % of trials in which mice attacked at each time point in F; red and dark lines indicate the mean, pink and gray areas indicate SEM. H, Quantification of attack time per mouse before and during light stimulation for F. J, Raster plots of attack events during each interaction episode (rows in the raster plot, defined as the period from 20 s before to 20 s after the onset of a spontaneous attack) for each mouse. All attack events during the testing period are shown. K, % of episodes (rows) in which mice attacked at each time point in J; red and dark lines indicate the mean, pink and gray areas indicate SEM. L, Quantification of average attack time per mouse for the 10 s after the onset of a spontaneous attack, as represented in the boxed area in J. N, Raster plots of trials (rows) of mice photostimulated with 473 nm light at the DR-MeA during an attack. O, % of trials in which mice attacked at each time point in N; red and dark lines indicate the mean, pink and gray areas indicate SEM. P, Quantification of attack time per mouse before and during light stimulation for N. Data are presented as mean ± SEM; ***p < 0.001. Statistics can be found in Table 2.
Tables
Resource type Specific reagent or resource Source or reference Identifiers Dilution or concentration Organism/strain C57BL/6J Charles River Strain code: 556 Antibody CaMKIIa (Cba-2) mouse monoclonal antibody Abcam Catalog #137300 1:250 dilution Antibody c-Fos rabbit polyclonal antibody Abcam Catalog #ab190289 1:2000 dilution Antibody GFP polyclonal antibody MBL Catalog #598 1:1000 dilution Antibody Anti-TpH2 antibody Abcam Catalog #ab184505 1:500 dilution Antibody Alexa Fluor 488 goat anti-mouse IgG ThermoFisher Catalog #A-10680 1:200 dilution Antibody Alexa Fluor 555 goat anti-mouse IgG ThermoFisher Catalog #A-21422 1:200 dilution Antibody Alexa Fluor 488 goat anti-rabbit IgG ThermoFisher Catalog #A-11034 1:200 dilution Antibody Alexa Fluor 555 goat anti-rabbit IgG ThermoFisher Catalog #A-21428 1:200 dilution Bacterial or viral strain AAV2/9.CaMKIIa (1.3 kb). hChR2 (E123A)-mCherry.WPRE. hGH Addgene Catalog #35505-AAV9 ≥1 × 10¹³ vg/ml, 500 nl injected Bacterial or viral strain AAV2/9.CaMKIIa (1.3 kb). hChR2 (E123A)-eYFP.WPRE. hGH Addgene Catalog #35506-AAV9 ≥1 × 10¹³ vg/ml, 500 nl injected Bacterial or viral strain AAV2/9.CaMKIIa (1.3 kb).ArchT 3.0-eYFP.WPRE. hGH Addgene Catalog #99039-AAV9 ≥1 × 10¹³ vg/ml, 500 nl injected Bacterial or viral strain pRRlsin.eGFP In house Commercial assay or kit Metabond Parkell Catalog #S380 Commercial assay or kit Dental cement DuraLay Catalog #602-7395 Commercial assay or kit Vectashield HardSet Antifade Mounting Medium with DAPI Vector Laboratories Catalog #H-1500 Software; algorithm SigmaPlot IBM https://www.ibm.com Data Method Factor n T, U, or F stat p value Post hoc correction Fig. 1C Mann–Whitney Resident vs control 6, 7 U = 2 0.008 Fig. 1D Mann–Whitney Resident vs control 6, 7 U = 5 0.027 Fig. 1E Student’s t test Resident vs control 6, 7 T(11) = 0.164 0.872 Fig. 1F Student’s t test Resident vs control 6, 7 T(11) = 0.062 0.952 Fig. 1H One-way ANOVA # of cells at bregma −4.84 5, 5 F(2,14) = 13.339 <0.001 Tukey’s One-way ANOVA # of cells at bregma −4.60 5, 5 F(2,14) = 6.357 0.013 Tukey’s One-way ANOVA # of cells at bregma −4.34 5, 5 F(2,14) = 168.490 <0.001 Tukey’s Fig. 1I One-way ANOVA % of DAPI at bregma −4.84 5, 5 F(2,14) = 13.859 <0.001 Tukey’s One-way ANOVA % of cells at bregma −4.60 5, 5 F(2,14) = 4.493 0.035 Tukey’s One-way ANOVA % of cells at bregma −4.34 5, 5 F(2,14) = 20.455 <0.001 Tukey’s Fig. 2C Mann–Whitney ChR2 vs GFP 6, 6 U = 0 0.005 Fig. 2D Student’s t test ChR2 vs GFP 6, 6 T(10) = 0.203 0.843 Fig. 2G One-way ANOVA TpH2 vs ChR2 5, 5 F(2,17) = 13.714 <0.001 Tukey’s One-way ANOVA TpH2 vs ArchT 5, 5 F(2,17) = 20.868 <0.001 Tukey’s Fig. 2K Student’s t test Opsin vs GFP 5, 4 T(7) = 0.793 0.454 Fig. 2O Student’s t test Before light onset (10 s) 5, 4 T(7) = −0.607 0.563 Student’s t test During light (10 s) 5, 4 T(7) = 0.882 0.407 Fig. 2S Student’s t test Before light onset (10 s) 5, 3 T(6) = 0.136 0.896 Student’s t test During light (10 s) 5, 3 T(6) = −10.784 <0.001 Fig. 3L Student’s t test ChR2 vs GFP at bregma 2.68 6, 6 T(10) = 0.325 0.752 Student’s t test ChR2 vs GFP at bregma 2.68 6, 6 T(10) = 0.890 0.346 Student’s t test ChR2 vs GFP at bregma 2.34 6, 6 T(10) = 0.396 0.701 Fig. 3M Student’s t test ChR2 vs GFP at bregma 2.34 6, 6 T(10) = 8.033 <0.001 Student’s t test ChR2 vs GFP at bregma 2.10 6, 6 T(10) = 3.850 0.003 Student’s t test ChR2 vs GFP at bregma 2.10 6, 6 T(10) = 7.965 <0.001 Fig. 4L Student’s t test ChR2 vs GFP 6, 6 T(10) = 0.474 0.646 Fig. 4M Student’s t test ChR2 vs GFP 6, 6 T(10) = 0.558 0.589 Fig. 4N Student’s t test ChR2 vs GFP 6, 6 T(10) = 0.258 0.801 Fig. 4O Student’s t test ChR2 vs GFP 6, 6 T(10) = 1.100 0.297 Fig. 4P Student’s t test ChR2 vs GFP 6, 6 T(10) = 1.203 0.257 Fig. 4Q Student’s t test ChR2 vs GFP 6, 6 T(10) = 4.693 <0.001 Fig. 4R Student’s t test ChR2 vs GFP 6, 6 T(10) = 2.745 0.021 Fig. 4S Student’s t test ChR2 vs GFP 6, 6 T(10) = 3.104 0.011 Fig. 4T Student’s t test ChR2 vs GFP 6, 6 T(10) = 1.597 0.141 Fig. 4U Student’s t test ChR2 vs GFP 6, 6 T(10) = 2.525 0.030 Fig. 5D Student’s t test Opsin vs GFP 4, 3 T(5) = 1.101 0.321 Fig. 5H Student’s t test Before light onset (10 s) 4, 3 T(5) = 0.845 0.437 Mann–Whitney During light (10 s) 4, 3 U = 28 0.952 Fig. 5L Student’s t test Before light onset (10 s) 4, 3 T(5) = 0.632 0.555 Student’s t test During light (10 s) 4, 3 T(5) = −6.606 <0.001 Fig. 6D Student’s t test Opsin vs GFP 5, 4 T(7) = 0.137 0.895 Fig. 6H Student’s t test Before light onset (10 s) 4, 3 T(5) = 0.845 0.437 Mann–Whitney During light (10 s) 4, 3 T(5) = 0 1.000 Fig. 6L Student’s t test Before light onset (10 s) 6, 4 T(8) = −1.217 0.278 Student’s t test During light (10 s) 6, 4 T(8) = 0.743 0.491 Fig. 7D Student’s t test Opsin vs GFP 4, 3 T(5) = 1.130 0.310 Fig. 7H Student’s t test Before light onset (10 s) 4, 3 T(5) = 1.425 0.214 Student’s t test During light (10 s) 4, 3 T(5) = 8.871 <0.001 Fig. 7L Student’s t test Opsin vs GFP 6, 4 T(8) = 1.010 0.342 Fig. 7P Student’s t test Before light onset (10 s) 6, 4 T(8) = 0.229 0.826 Student’s t test During light (10 s) 6, 4 T(8) = −12.538 <0.001
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