Figures and data
Predator cues in cat saliva induce context-dependent defensive behavior in mice
(A) A schematic diagram illustrating the elicitation of "freeze" or "flight" defensive behaviors in relation to distance. (Adapted with permission from (Blanchard and Blanchard, 1989))
(B) A schematic illustration depicting a test chamber for evaluating defensive behaviors.
(C) Raster plots displaying freezing episodes of individual mice exposed to a clean control swab (gray), fresh saliva (green), or old saliva (yellow). The introduction of a clean, fresh, or old saliva swab into a mouse’s cage is denoted as time 0. A red line indicates the first interaction with a swab for each mouse.
(D) Latency to the first episode of interaction with a swab.
(E) The percentage of total freezing episodes after the first contact.
(F) Latency to the first freezing episode in mice exhibited freezing after the first interaction.
(G) The percentage of total interaction episodes.
(H) Plasma ACTH concentrations following exposure to clean, fresh, or old saliva swabs.
In (D) - (H), the values are presented as means ± S.E.M., with individual dots representing individual mice. Statistical analysis was performed using One-Way ANOVA with Tukey’s multiple comparisons. *, **, and **** denote significance levels of p < 0.05, 0.01, and 0.0001, respectively.
Cat saliva activates VNO neurons and modulates freezing behavior
(A) The percentage of total freezing episodes directed towards control or fresh saliva swabs in cassettes that prevent direct interaction with the swabs.
(B) The percentage of total interaction episodes with cassettes containing control or fresh saliva swabs.
(C) The percentage of total freezing episodes directed toward control or fresh saliva swabs in Trpc2-WT or -KO mice.
(D) The percentage of total interaction episodes with control or fresh saliva swabs in Trpc2-WT or -KO mice.
(E) Representative images illustrating the expression of pS6 (green) and Gαo (magenta) in the VNO of mice exposed to control, fresh, or old saliva swabs. DAPI was used as a nuclear counterstain. Scale bar: 50 μm.
(F, G) The number of pS6-IR positive cells in 1000 VNO cells in the apical (F) and basal (G) regions of the VNO neuroepithelium.
(H) The percentage of total freezing episodes in relation to the number of pS6-positive cells in the VNO of individual mice. Correlation analysis was conducted using Pearson’s Correlation.
(A - D, F, G) the values are presented as means ± S.E.M., with individual dots representing individual mice. Statistical analysis was performed T-tests (A, B), Two-Way ANOVA (C, D), and One-Way ANOVA (F, G) with Tukey’s multiple comparisons. *, **, ***, and **** denote significance levels of p < 0.05, 0.01, 0.001, and 0.0001, respectively.
Cat saliva activates the V2R-A4 subfamily of the vomeronasal receptors
(A) Phylogenetic tree of the V2R family and its subfamilies used for V2R cRNA probe design.
(B) Representative images displaying the expression of V2RA4 (magenta) and pS6 (green) in the VNO of a mouse exposed to a fresh saliva swab. DAPI was utilized as a nuclear counterstain. Scale bar: 50 μm.
(C) The percentage of cells co-labeled with V2R probes and anti-pS6 antibody among the total pS6-positive cells in the VNO of mice exposed to fresh saliva (top) or old saliva (bottom).
Cat saliva activates neurons in the AOB and VMH
(A) Schematic diagrams illustrating the VNO sensory pathway to the VMH.
(B) Representative images displaying the expression of cFos (magenta) in the mitral cell (MCL) and granule cell (GCL) layers of the AOB of mice exposed to control, fresh, or old saliva swabs. Gαi2 (green) visualizes the anterior region of the glomerular layer (GL). DAPI was utilized as a nuclear counterstain. Scale bar: 200 μm.
(C, D) The number of cFos-IR positive mitral cells per 1000 mitral cells in the anterior (C) and posterior (D) regions of the AOB.
(E, F) The number of cFos-IR positive granule cells per 1000 granule cells in the anterior (E) and posterior (F) regions of the AOB.
(G) Representative images displaying the expression of cFos (red) in the VMH of mice exposed to control, fresh, or old saliva swabs. The VMH is delineated by a dashed white line, defined by counterstaining with DAPI. Scale bar: 200 μm.
(H) The number of cFos-IR positive neurons per 1000 cells in the VMH.
In (C-F, H), the values are presented as means ± S.E.M., with individual dots representing individual mice. Statistical analysis was performed using One-Way ANOVA with Tukey’s multiple comparisons. * and ** denote significance levels of p < 0.05 and 0.01, respectively.
Fresh and old cat saliva differentially activate VMH neurons to control defensive behavior
(A-C) Percentage of the total freezing episodes as a function of the number of cFos-positive posterior AOB mitral cells (A) and granule cells (B), as well as VMH neurons (C). Green, and yellow symbols represent values from mice exposed to control, fresh saliva, and old saliva, respectively. Correlation was analyzed using Pearson’s Correlation. * denotes statistical significance with p < 0.05.
A population of VMH neurons is more sensitive to fresh saliva than old saliva
(A) Schematic diagram illustrating the cFos-IR distribution analysis. Each VMH region in five consecutive sections (140 μm apart) was prepared along the dorsomedial (dm)-ventrolateral (vl) axis and divided into 10 bins that are vertical to the dm-vl axis. cFos-IR signals in each bin were counted.
(B) The number of cFos-IR-positive cells in each bin in the VMH sections. The values are presented as means + S.E.M., with individual dots representing values from individual VMH. Statistical analysis was performed using Two-Way ANOVA with Tukey’s multiple comparisons. Black * and ** denote significance levels of p < 0.05 and 0.01, respectively, between control and fresh or old saliva conditions. Red * and ** denote significance levels of p < 0.05 and 0.01, respectively, between fresh and old saliva conditions.
A model of the VMH-to-VMH pathway processing imminence of predator threat
The circuitry is selectively activated based on the varying imminence of predator cues. Fresh saliva activates more V2R-A4-expressing VNO neurons (blue), which results in stronger activation of the mitral cells innervating V2R-A4 glomeruli. This eventually triggers freezing-correlated neural activity in the VMH. In contrast, old saliva activates fewer V2R-A4-expressing VNO neurons (blue), which results in weaker activation of the mitral cells innervating V2R-A4 glomeruli, leading to freezing-non-correlated activity in the VMH.
