Abstract
Social encounters are associated with varying degrees of emotional arousal and stress. The mechanisms underlying adequate socioemotional balance are unknown. The medial amygdala (MeA) is a brain region associated with social behavior in mice. Corticotropin-releasing factor receptor type-2 (CRF-R2) and its specific ligand urocortin-3 (Ucn3), known components of the behavioral stress response system, are highly expressed in the MeA. Here we show that mice deficient in CRF-R2 or Ucn3 exhibit abnormally low preference for novel conspecifics. MeA-specific knockdown of Crfr2 (Crhr2) in adulthood recapitulated this phenotype. In contrast, pharmacological activation of MeA CRF-R2 or optogenetic activation of MeA Ucn3 neurons increased preference for novel mice. Furthermore, chemogenetic inhibition of MeA Ucn3 neurons elicited pro-social behavior in freely behaving groups of mice without affecting their hierarchal structure. These findings collectively suggest that the MeA Ucn3–CRF-R2 system modulates the ability of mice to cope with social challenges.
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Acknowledgements
We thank S. Ovadia for his devoted assistance with animal care. We thank J. Keverne for professional English editing, formatting and scientific input. This work is supported by an FP7 grant from the European Research Council (260463; A.C.); research grants from the Israel Science Foundation (1565/15) (A.C.); research support from Roberto and Renata Ruhman (A.C.); research support from Bruno and Simone Licht; I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation (grant no. 1916/12 to A.C.); the Nella and Leon Benoziyo Center for Neurological Diseases (A.C.); the Henry Chanoch Krenter Institute for Biomedical Imaging and Genomics (A.C.); the Perlman Family Foundation, founded by Louis L. and Anita M. Perlman (A.C.); the Adelis Foundation (A.C.); the Irving I. Moskowitz Foundation (A.C.); grants from the Israel Science Foundation (1351/12) and the European Commission (ERC StG #337637 and Marie Curie CIG #321919) (O.Y.) and a Human Frontier Program career development award (O.Y.); a Human Frontier Science Program grant (E.S.); European Research Council grant # 311238 (E.S.); an Israel Science Foundation grant #1629/12 (E.S.); research support from Martin Kushner Schnur (E.S.); and Mr. and Mrs. Lawrence Feis (E.S.).
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Contributions
Y. Shemesh and O.F. designed and performed most of the experiments. M.M., M.E., J.D., and J.M.D. designed and performed electrophysiological studies. S.A., S.M., T.S., E.E., L.T., G.E., E.S.A., Y.J.B.-E., S.G., Y.K., and S.H. assisted in experiments. Y. Shemesh, Y. Sztainberg, and O.F. wrote the code for analyzing behavior and validated it. E.S., O.Y., and A.C. conceived, designed, and supervised the project. Y. Shemesh, O.F., and A.C. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Crfr2 KO and Ucn3 KO mice were similar to WT mice in their nonsocial exploratory behavior
(a) Schematic illustration of the social approach test. (b) An illustration of the novel object preference test. (c) Exploratory behavior (number of visits to each chamber in the social approach test) of Crfr2 KO mice (n=7) and WT littermate controls (n=8) did not differ (two-tailed t-test, p=0.1907, t=1.380, df=13). Exploratory behavior (number of visits to each chamber) of Ucn3 KO mice (n=6) and WT littermate controls (n=7) did not differ (two-tailed t-test, p=0.7824, t=0.283, df=11). (d) Average speed of Crfr2 KO mice (n=7) and WT littermate controls (n=8) in the social maze did not differ (two-tailed t-test, p=0.3394, t=0.9918, df=13). Average speed of Ucn3 KO mice (n=6) and WT littermate controls (n=7) in the social maze did not differ (two-tailed t-test, p=0.1611, t=1.502, df=11). Bars represent mean ± s.e.m.
Supplementary Figure 2 Validation of the Crfr2::Cre line
Mice expressing Cre-recombinase under the control of the Crfr2 promoter were crossed with a line carrying a reporter allele encoding the tdTomato red fluorescent protein upon Cre-mediated recombination (Rosa26-CAG-lsl-tdtomato, Ai9). Red cells represent cells that expressed CRF-R2 at any point of time from early development (n=4 mice).
Supplementary Figure 3 Validation of the Ucn3::Cre line
Mice expressing Cre-recombinase under the control of the Ucn3 promoter were crossed with a line carrying a reporter allele encoding the tdTomato red fluorescent protein upon Cre-mediated recombination (Rosa26-CAG-lsl-tdtomato, Ai9). To obtain the best immunostaining we used colchicine-injected mice. Images depict the co-localization of anti-Ucn3 antibody (green) with Ai9 fluorescence (red) in regions that typically express Ucn3 (PeF, BNST, MeA). As expected, the anti-Ucn3 is highly specific to the Ai9 cells, 90±5 % of the green cells are red (averaged over all 3 areas in 3 different mice). Since not all Ai9 expressing cells produced Ucn3 at the time of sacrifice, many of the red cells are not green.
Supplementary Figure 4 The MeA Ucn3–CRF-R2 circuit is embedded within the social brain network
(a) Injection site (MeA) of the rabies, herpes and conditional synaptophysin viruses. (b) Monosynaptic inputs (green cells) to MeA Ucn3 neurons. Ucn3::Cre mice were injected with conditional helpers (AAV5-FLEX-TVA-mCherry, AAV8-FLEX-RG, and a conditional rabies virus SADΔG-GFP (EnvA)). (c) Synaptic outputs from MeA Ucn3 neurons. Conditional Cre dependent anterograde transneuronal tracer based on the H129 strain of herpes simplex virus (H129 TK-TT) was delivered to MeA Ucn3 cells. Red cells indicate the tdTomato marker expressed in cells anterograde to MeA Ucn3::Cre expressing cells. (d) Efferent projections from MeA CRF-R2 cells. An AAV9-CMV-Flex-synaptophysin-mCherry viral vector was injected bilaterally into the MeA of Crfr2::Cre mice. Red dots represent synapses that originated from MeA CRF-R2 neurons.
Supplementary Figure 5 Crfr2 KD mice were similar to control mice in their nonsocial exploratory behavior
(a) Exploratory behavior (number of visits to each chamber in the social approach test) of control mice (n=10), MeA Crfr2 KD mice (n=10) and LS Crfr2 KD mice (n=6) did not differ (two-tailed t-test, p=0.6891, F(2,23)=0.3785). (b) Average speed of control mice (n=10), MeA Crfr2 KD mice (n=10) and LS Crfr2 KD mice (n=6) did not differ (two-tailed t-test, p=0.3109, F(2,23)=1.230). (c,d) MeA Crfr2 KD mice did not display any changes in anxiety-like behavior compared to controls. (c) Open-field test. Left panel shows a schematic representation of the setup (n=7,8 respectively, number of rearing, two-tailed t-test, p=0.99, t=0.0127, df=13; distance- two tailed t-test, p=0.983, t=0.021, df=13). (d) Light/dark transfer test. Left panel shows a schematic representation of the setup (n=9,9 respectively, distance - two tailed t-test, p=0.693, t=0.4013, df=16; latency- two-tailed t-test, p=0.8212, t=0.229, df=16). Bars represent mean ± s.e.m.
Supplementary Figure 6 CRF-R2 neurons in the MeA express GAD65/GAD67
Double in situ hybridization of tdTomato (silver grains), and GAD65/67 mRNA (red staining) in the MeA (Bregma -1.7 mm) of the CRF-R2 tdTomato+ mouse brain. Filled arrows indicate cells coexpressing tdTomato and GAD65/67.
Supplementary Figure 7 mUcn3 administration to the MeA does not alter nonsocial exploratory behavior
(a) Exploratory behavior (number of visits to each chamber in the social approach test) of MeA saline administered mice (n=13, control) and MeA mUcn3 administered mice (n=11) did not differ (two-tailed t-test, p=0.7165, t=0.3678, df=22). (b) Average speed of MeA saline administered mice (n=13, control) and MeA mUcn3 administered mice (n=11) did not differ (two-tailed t-test, p=0.5484, t=0.6096, df=22). (c) Exploratory behavior (number of visits to each chamber in the social approach test) of MeA saline+mUcn3 administered mice (n=12, control) and MeA CRF-R2 antagonist + mUcn3 administered mice (n=8) did not differ (two-tailed t-test, p=0.5699, t=0.5788, df=18). (d) Average speed of MeA saline+mUcn3 administered mice (n=12, control) and MeA CRF-R2 antagonist+mUcn3 administered mice (n=8) did not differ (two-tailed t-test, p=0.6956, t=0.3975, df=18). Bars represent mean ± s.e.m.
Supplementary Figure 8 Optogenetic activation of MeA Ucn3 neurons does not alter nonsocial exploratory behavior
(a) Illustration of the social maze with the optogenetic setup. (b) Transgenic mice expressing Cre-recombinase specifically in Ucn3 expressing neurons (Ucn3::Cre+) were injected with AAV5-EF1α-DIO-ChR2-eYFP virus bilaterally into the MeA, resulting in site specific (MeA) and cell specific (Ucn3) expression of Channelrhodopsin-2. (c) Exploratory behavior (number of visits to each chamber in the social approach test) of Ucn3::Cre+ mice (n=3, treatment) and Ucn3::Cre− control mice (n=4) did not differ (two-tailed Mann Whitney U test, p=0.4571, U=3.5, df=6). (d) Average speed of Ucn3::Cre+ mice (n=3, control) and Ucn3::Cre− control mice (n=4) did not differ (Two-tailed Mann Whitney U test, p=0.40, U=3, df=6). Bars represent mean ± s.e.m.
Supplementary Figure 9 Mouse models of social avoidance
(a) Chronic Social Defeat - a model of environmentally-induced social avoidance where a test mouse is subjected to an aggressive bully mouse for ten consecutive days. (b) Shank3 KO, a genetic model of social avoidance and autism-like behavior.
Supplementary Figure 10 Tracking group behavior before and after DREADD-based MeA-Ucn3 inhibition
(a) Groups of four mice were introduced into the system and recorded for 12 hours each day while under low light conditions that mimic a bright night (2 lux). Each mouse was marked by a different hair dye, which enabled automatic tracking of its location. The arena consisted of an open 70×50 cm box which contained nests, feeders, water, elevated areas, and barriers. Starting from the 4th day CNO was added to the drinking water for three days, resulting in a sub-chronic inhibition of the MeA-Ucn3 cells. (b) Dyadic interactions were automatically classified based on the distance between the mice (d), and the angle of movement of one of the mice with respect to the other (θ). The algorithm used these parameters to determine if a mouse was moving toward another mouse, away from it, or neither. (c) We defined contacts as times in which the distance between the mice was less than 10 cm. We used a hidden Markov model (HMM) in order to determine the events leading up to the contact (like who initiated the contact), and how it concluded. In cases where the contact ended with one mouse following the other, we used an additional classifier to determine if the interaction was an aggressive chase-escape. (d) Functional validation of DREADD based MeA Ucn3 inhibition. cFos expression in the MeA is reduced in MeA Ucn3::Cre::hM4D injected mice following CNO administration. (e) Quantification of the cFos staining. We found significantly reduced number of cFos positive cells in the MeA of Ucn3::Cre::hM4D mice injected with CNO compared to control mice (one-tailed Mann Whitney test, p=0.0079, U=0, df=8). Bars represent mean ± s.e.m.
Supplementary Figure 11 Basal behavior in the social maze of the control groups in the different experiments
(a, b) The time WT siblings of Crfr2 KO (a) and Ucn3 KO (b) spent in each chamber of the social maze. (c, d) The time MeA-shame injected siblings of MeA Crfr2 KD (c) and MeA-sham injected siblings of LS KD mice (d) spent in each chamber of the social maze. (e) The time MeA saline administered mice spent in each chamber of the social maze. These mice were littermates of mice that were injected with mUcn3 to the MeA. (f) The time MeA saline+mUcn3 administered mice spent in each chamber of the social maze. These mice were littermates and controls to mice that were injected with mUcn3+assteressin2b (CRF-R2 antagonist). (g, h) Basal behavior in the social maze of groups served as a control for MeA Ucn3 optogenetic activation. (g) The time Ucn3::Cre− mice (light on and light off) spent in each chamber of the social maze. (h) The time Ucn3::Cre− mice (light on and light off) treated with CRF-R2 antagonist spent in each chamber of the social maze. Bars represent mean ± s.e.m.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–11 (PDF 1725 kb)
Optogenetic activation of MeA Ucn3 neurons
MeA Ucn3-expressing neurons were optogenetically activated while the mouse was tested in the social maze. The maze comprised three arms: one with a mouse familiar to the actor, another with a novel mouse, and the third empty (non-social). The arm that contained the familiar mouse can be recognized in the movie by the note "F" below it. (MOV 5029 kb)
Tracking a freely behaving group of mice in a semi-natural environment
The video shows the tracked position of each of four mice. As mice are nocturnal, we recorded them in low light conditions that mimic a bright night (2 lux). Each mouse was marked using a different hair dye, which due to the use of sensitive cameras was distinguishable even under these light levels. The arena contained nests, feeders, water, and additional enrichments which are highlighted in the video. (MOV 11304 kb)
Aggressive chase-escape interactions
Each contact between the mice was analyzed to determine which behavior preceded it (who initiated the interaction), and how it concluded. Interactions that ended with one mouse going after the other while the other moves away were automatically classified as an aggressive chase-escape or as non-aggressive. (MOV 31834 kb)
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Shemesh, Y., Forkosh, O., Mahn, M. et al. Ucn3 and CRF-R2 in the medial amygdala regulate complex social dynamics. Nat Neurosci 19, 1489–1496 (2016). https://doi.org/10.1038/nn.4346
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DOI: https://doi.org/10.1038/nn.4346
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