Figures and data
Increase in CRHPVN activity during conspecific intruder appraisal.
A. Coronal hemi-section illustrating the paraventricular nucleus (PVN) of the hypothalamus (right) and corresponding confocal image (left) demonstrating GCaMP6f expression in CRH-expressing PVN neurons (scale bar = 50 µm). B. Schematic of single-fiber photometry recordings from CRHPVN neurons during social investigation. Confocal image (right) shows the ferrule position above the PVN (scale bar = 100 µm), with the social interaction paradigm illustrated below. C. Mean Ca2+ activity traces from CRHPVN neurons during social assessment of a familiar conspecific (n = 10). D. Schematic depicting a resident mouse in the home cage (HC) approaching familiar (green) and unfamiliar (brown) intruders, with approach direction indicated by a dashed red arrow. E. Approach-aligned heatmaps of CRHPVN Ca2+ responses, with each row representing an individual animal and the corresponding mean z-scored activity trace shown below (F). G. Mean z-scored CRHPVN Ca2+ activity across 5-second time windows (−5 to 0, 0 to 5, 5 to 10 s), with time 0 indicating resident approach towards intruder (n = 10). Left: familiar intruder (repeated-measures one-way ANOVA, F(1.653,14.87) = 11.55, p = 0.001; Bonferroni’s multiple comparisons: −5 to 0 vs 0 to 5, p < 0.0001, t = 6.316; −5 to 0 vs 5 to 10, p=0.0626, t=2.795; 0 to 5 vs 5 to 10, p = 0.4272, t = 1.606). Right: unfamiliar intruder (repeated-measures one-way ANOVA, F(1.445,13.00) = 58.81, p < 0.0001; Bonferroni’s multiple comparisons: −5 to 0 vs 0 to 5, p < 0.0001, t = 8.511; −5 to 0 vs 5 to 10, p = 0.008, t = 4.116; 0 to 5 vs 5 to 10, p < 0.0001, t = 9.405). H-J. Comparison of the first post-approach calcium transients between familiar and unfamiliar intruder exposures (n = 10 mice per group). Peak amplitude: p = 0.0004, t = 4.393 (H); peak area under the curve (AUC): p = 0.0013, t = 3.803 (I); time to baseline: p = 0.04, t = 2.200 (J; unpaired t-tests).
Differential CRHPVN neuron activity persists in novel context and across conspecific strains.
A. Schematic illustrating a resident mouse exposed to familiar (top) and unfamiliar intruders (bottom) in a novel context. B. Group-averaged z-scored CRHPVN Ca2+ responses aligned to approach, with shaded area representing s.e.m. C. Comparison of individual peak z-scores for the first Ca2+ transient following approach (p = 0.0274, t = 2.776; n = 8 mice per group; unpaired t-test). D. Comparison of peak area under the curve (AUC) for the first Ca2+ transient following approach (n = 8 mice per group; p = 0.0118, t = 3.376; unpaired t-test). E. Schematic depicting resident interactions with familiar and unfamiliar same-strain (C57) intruders and a non–same-strain (CD1) intruder in the home cage. F. Group-averaged z-scored CRHPVN Ca2+ responses aligned to approach, with shaded area representing s.e.m. G. Individual peak z-scores of the first calcium transient following approach (ordinary one-way ANOVA, F(2,33) = 2.154, p = 0.0056; Tukey’s multiple comparisons: Fam vs Unfam-C57 p = 0.0271, q = 3.849; Fam vs Unfam-CD1 p = 0.0073, q = 4.597; Unfam-C57 vs Unfam-CD1 p = 0.8575, q = 0.7487; n = 12 mice per group). H. Individual peak area under the curve (AUC) of the first calcium transient following approach (ordinary one-way ANOVA, F(2,33) = 5.743, p = 0.0072; Tukey’s multiple comparisons: Fam vs Unfam-C57 p = 0.0289, q = 3.809; Fam vs Unfam-CD1 p = 0.01, q = 4.424; Unfam-C57 vs Unfam-CD1 p = 0.9014, q = 0.6149; n = 12 mice per group).
CRHPVN activity is similar during exposure to unfamiliar juvenile intruders and objects
A. Schematic depicting exposure of resident to familiar, unfamiliar, and juvenile intruders in home cage. B. Group average z-score of CRHPVN calcium responses aligned at approach, shade represents s.e.m. C. Individual peak z-scores of the first peak after approach (Ordinary 1-way ANOVA, F (2,35) = 10.02, p = 0.0059, Tukey’s multiple comparison test, Fam-Adult vs. Unfam-Adult p = 0.0095, q=5.185, Unfam-Adult vs. Unfam-Juvenile p = 0.0378, q=4.051, and Fam-Adult vs. Unfam-Juvenile, p = 0.4343, q=1.811, n = 12 mice per group). D. Individual peak area under the curve for the calcium activity for the first peak after approach (Ordinary 1-way ANOVA, F (2,35) = 11.87, p = 0.0041, Tukey’s multiple comparison test, Fam-Adult vs. Unfam-Adult p = 0.0083, q=5.298, Unfam-Adult vs. Unfam-Juvenile p = 0.0178, q=4.669, and Fam-Adult vs. Unfam-Juvenile, p = 0.9124, q=0.5792, n = 12 mice per group). E. Schematic depicting resident interactions with familiar and unfamiliar objects in homecage. F. Group average z-score of CRHPVN calcium responses aligned at approach, shade represents s.e.m. G. Individual peak z-scores of the first peak after approach (p = 0.4284, t = 0.8404, n = 12 mice per group, unpaired, t-test). H. Individual peak area under the curve for the calcium activity for the first peak after approach (p = 0.4256, t = 0.8458, n = 12 mice per group, unpaired, t-test).
Increased social investigation during exposure to unfamiliar intruder.
A. Raster plots illustrating the temporal distribution of behavioural events following exposure to familiar (left) and unfamiliar (right) intruders. Each row corresponds to an individual animal, with event markers denoting discrete behavioural events over time. B. Vertical bars showing individual sniffing durations for resident-to-intruder (bottom) and intruder-to-resident (top) interactions under familiar (Bi) and unfamiliar (Bii) conditions. Total sniffing duration during unfamiliar investigations is higher compared to familiar investigations (Biii) (p = 0.0015, t = 3.550, n = 14 mice per group, unpaired, t-test). C. Temporal dynamics of sniffing behavior during social investigation, quantified as sliding sums of anogenital (Ci), head (Cii), torso (Ciii), and tail (Civ) sniffing time, averaged in 60-second bins with a 3-second sliding window. Cumulative sum of sniffing events during social investigations, showing total time spent in anogenital (Di), head (Dii), torso (Diii), and tail (Div) sniffing. Differential sniffing behavior during familiar and unfamiliar intruder investigation, quantified across anogenital (Ei), head (Eii), torso (Eiii), and tail (Eiv) regions. (Anogenital: p = 0.0006, t = 3.896, n = 14 mice per group, unpaired t-test; Head: p = 0.0028, t = 3.299, n = 14 mice per group, unpaired t-test; Torso: p = 0.1459, t = 1.499, n = 14 mice per group, unpaired t-test; Tail: p = 0.1604, t = 1.445, n = 14 mice per group, unpaired t-test). (F). Transition probability plots depicting the dynamics of switching between investigative behaviours during interaction with familiar (i) and unfamiliar (ii) intruders during social investigation. Nodes correspond to behavioural states, and directed edges are weighted by transition probabilities.
Increase in CRHPVN activity is essential for targeted anogenital investigation
A. Top: Viral construct for optogenetic inhibition experiment, bottom: Strategy of virus injection and ferrule implantation for optogenetic inhibition of CRHPVN neurons, right: Confocal image depicting Cre-dependent localization of aav2/9-EF1a-DIO-Arch3.0 mcherry virus in the CRHPVN neurons (Scale bar = 50 μm). B. Experimental timeline depicting photo-inhibition of CRHPVN neurons in the resident mouse during the first 20s of the social investigation of an intruder; and representative whole-cell recording from a CRHPVN neuron in a brain slice, demonstrating spike suppression in response to 561-nm light. C. Percentage of total sniffing behavior during social investigation, comparing control (mCherry) and optogenetically inhibited (Arch) residents during investigation of familiar and unfamiliar intruders (n=14-20 per group, 1-way ANOVA, F(4,68) = 11.18, p < 0.0001, Holm-Sidak’s multiple comparison test, Fam-mCherry vs Unfam-mCherry p= 0.0007, t=4.003; Fam-mCherry vs Fam-Arch p = 0.1492, t=1.794, Unfam-mCherry vs. Unfam-Arch p = 0.0016, t=3.505; Fam-Arch vs Unfam-Arch p=0.1492, t= 1.634). D. Pie charts showing the distribution of sniffing behavior in control (mCherry) and optogenetically inhibited (Arch) residents. (i) Similar distribution during investigation of a familiar intruder. (ii) Altered distribution during unfamiliar intruder investigation. E. Temporal profile of anogenital sniffing during social investigation, shown as mean sniffing time averaged in 60-second bins with a 3-second sliding window, comparing mCherry controls and Arch-inhibited groups during (i) familiar and (ii) unfamiliar intruder investigation. F. Example raster plots of anogenital sniffing events for (i) familiar and (ii) unfamiliar intruders (top: mCherry-control; bottom: Arch-inhibited). G. Cumulative anogenital sniffing dynamics during investigation of (i) familiar and (ii) unfamiliar intruders, shown as group-averaged duration in mCherry controls and Arch-inhibited animals. H. Comparison of anogenital sniffing time between mCherry controls and Arch-inhibited residents, shown as mean ± s.e.m., during investigation of (i) familiar intruders (p = 0.1353, t = 1.532) and (ii) unfamiliar intruders (p = 0.0023, t = 3.307; unpaired t-tests). I. Transition matrices illustrating the probability of switching between investigative behaviours during interaction with familiar (i) and unfamiliar (ii) intruders, comparing mCherry controls (left) and Arch-inhibited residents (right).
