1. Volume 28, Issue 7, Pages 1066-1078.e6 (April 2018)
Oxytocin Signaling in the Lateral Septum Prevents Social Fear during Lactation 
Rohit Menon, Thomas Grund, Iulia Zoicas, Ferdinand Althammer, Dominik Fiedler, Verena Biermeier, Oliver J. Bosch, Yuichi Hiraoka, Katsuhiko Nishimori, Marina Eliava, Valery Grinevich, Inga D. Neumann 
Current Biology 
Volume 28, Issue 7, Pages e6 (April 2018)
DOI: /j.cub
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2. Figure 1
Lactating Mice Do Not Express SFC-Induced Social Fear compared to Virgin Mice
(A–D) Schematic representation of the SFC paradigm (A). Behavioral responses of virgin (Vir; blue) and lactating (Lac; lactation day [LD] 5–7; red) mice during the social fear conditioning (SFC) paradigm. Vir and Lac mice were social fear conditioned (SFC+) or left unconditioned (SFC−) during social fear acquisition (day 1; B) (n = 9–13 mice/group). Percentage investigation of three non-social (empty cage) and six social (cage with a conspecific) stimuli was monitored during social fear extinction training (day 2; C), and this was followed by social fear extinction recall with six social stimuli (day 3; D). Data represent mean ± SEM. ∗p < 0.05 versus Vir SFC+ using one-way ANOVA with repeated-measures followed by Bonferroni’s post hoc analysis (C).
(E) Percentage time spent investigating a non-social stimulus (empty cage) and a social stimulus (cage with a conspecific) was calculated for Vir (blue) and Lac (LD 4; red) mice (n = 18 mice/group) as a measure of naturally occurring social preference in the social preference test. Data represent mean ± SEM. ∗p < 0.05 versus non-social stimulus using two-way ANOVA followed by Bonferroni’s post hoc analysis.
(F and G) Anxiety-like behavior was measured for Vir (blue) and Lac (LD 4; red) mice (n = 18 mice/group); general anxiety was indicated by the time spent on the open arm (F), and locomotion was indicated by the number of closed-arm entries (G) on the elevated plus maze. Data represent mean ± SEM.
(H) Effect of suckling on social fear extinction was measured for Lac mice. Lac mice were social fear conditioned (SFC+) and then either separated from their pups after acquisition (Lac SFC+/pups−; n = 6) or left overnight with their pups until 2 hr before extinction (Lac SFC+/pups+; n = 6). Percentage investigation of 3 non-social (empty cage) and six social (cage with a con-specific) stimuli was monitored during social fear extinction training. Data represent mean ± SEM.
(I) Thermal pain sensitivity of Vir (blue) and Lac (LD 5; red) mice (n = 6 mice/group) was measured as paw-withdrawal latency during Hargreaves’ plantar test. Data represent mean ± SEM.
See also Figure S1.
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3. Figure 2 Neuronal Plasticity of the LS during Lactation
(A–H) Number of cFos-immunopositive neurons indicating neuronal activation within the LS of virgin (Vir; blue) and lactating (Lac; lactation day; LD 5; red) mice (A; bregma + 0.3 mm). Social fear conditioned (SFC+) or unconditioned (SFC−) mice were sacrificed 90 min after either social fear acquisition (B, Vir SFC−; C, Vir SFC+; E, Lac SFC−; F, Lac SFC+; day 1; n = 6/group) or social fear extinction (D, Vir SFC+/Ext; G, Lac SFC+/Ext; n = 6/group; day 2). Data in (H) represent mean number of cFos-immunopositive neurons/0.5 mm2 ± SEM. #p < 0.05 versus Vir SFC− and ∗p < 0.05 versus Lac SFC+; using two-way ANOVA with Bonferroni’s post hoc analysis.
(I–Q) Co-stained LS brain slices from virgin OXTR reporter mice for green fluoroscent protein (GFP; I) and calbindin (CB; J) were examined. Merged image shows GFP- and CB-positive neurons (K; arrows indicate co-stained neurons; scale bar, 200 μm; L, higher magnification of K). Number of OXT-immunoreactive axons innervating the LS of Vir (M; n = 5) and Lac (LD 5; N; n = 5) mice is shown. OXT-positive (red) fibers were found to be in proximity to OXTR-positive neuronal cell bodies (green; the staining was performed in OXTR reporter mouse brains) (O, scale bar, 500 μm; P, scale bar, 25 μm). LSD, LSI, and LSV, indicate dorsal, intermediate, and ventral subdivisions of LS nucleus, respectively (M–O); lv, lateral ventricle; MS, medial septal nucleus. ∗p < 0.05 versus Vir using Student’s t test (Q).
(R–T) OXT release within the LS in response to social fear extinction. Vir (blue) and Lac (LD 5; red) mice were exposed to SFC on day 1 (Vir SFC+: n = 6; Vir SFC−: n = 6; Lac SFC+: n = 6) and to social fear extinction training 24 hr later. Microdialysates were collected before (dialysates Bs1 and Bs2; basal conditions), during (dialysate Ex1 during exposure to social stimuli 1–3; dialysate Ex2 during exposure to social stimuli 4–6), and after (dialysate Bs3; basal conditions) the extinction training. (R) represents microphotographs of microdialysis probe placement in coronal brain slices. Data represent the percentage of mean basal values ± SEM. ∗p < 0.05 versus Vir SFC+ using one-way ANOVA with repeated measures followed by Bonferroni’s post hoc analysis versus respective basal groups (S). Data show absolute OXT concentrations in 100 μL microdialysates from Vir SFC+, Vir SFC−, and Lac SFC+ mice; ∗p < 0.05 versus Bs2 using one-way ANOVA with repeated measures followed by Bonferroni’s post hoc analysis (T).
See also Figure S2.
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4. Figure 3
Pharmacological Manipulation of OXTR Signaling within the LS Modulates Social Fear Expression in Female Mice
(A) Scheme of the septum, including the LS, and the pharmacological interventions in lactating (Lac) and virgin (Vir) mice. Infusions located within the gray areas were considered as correct placements.
(B–D) Revival of SFC-induced social fear in Lac mice. Lac mice were social fear conditioned (SFC+) or left unconditioned (SFC−) during social fear acquisition (day 1; B; n = 6–9 mice/group). Percentage investigation of three non-social (empty cage) and six social (cage with a conspecific) stimuli during social fear extinction (day 2; C) and social fear extinction recall (day 3; D) was measured. SFC+ and SFC− mice were bilaterally infused with Veh (Ringer/0.2 μL/side) or OXT receptor antagonist (OXTR-A; 20 ng/0.2 μL/side) into the LS 30 min prior to extinction training. One animal was removed from statistical analysis due to misplaced cannula (i.e., outside the gray area; A). Data represent mean ± SEM. ∗p < 0.05: SFC+/OXTR-A versus SFC+/Veh; #p < 0.05: SFC+/OXTR-A versus SFC−/OXTR-A and SFC−/Veh using one-way ANOVA with repeated measures followed by Bonferroni’s post hoc analysis (C).
(E–G) Reversal of SFC-induced social fear in Vir mice. Vir mice were social fear conditioned (SFC+) or left unconditioned (SFC−) during social fear acquisition (day 1; E; n = 5–7 mice/group). Percentage investigation of three non-social (empty cage) and six social (cage with a conspecific) stimuli during social fear extinction (day 2; F) and social fear extinction recall (day 3; G) was measured. SFC+ and SFC− mice were bilaterally infused with Veh (Ringer/0.2 μL/side) or OXT (0.1 ng/0.2 μL/side) into the LS 10 min prior to extinction training. Three animals were removed from statistical analysis due to misplaced cannula (i.e., outside the gray area; A). Data represent mean ± SEM. ∗p < 0.05: SFC+/Veh versus SFC+/OXT; #p < 0.05: SFC+/Veh versus SFC−/OXT and SFC−/Veh using one-way ANOVA with repeated measures followed by Bonferroni’s post hoc analysis (F).
See also Figure S3.
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5. Figure 4
Genetic Manipulation of OXTR Expression in the LS Modulates Social Fear Expression in Female Mice
(A–F) Enhancement of OXTR expression within the GABAergic neurons in the LS of virgin mice by local infusion of an EF1-OXTR-IRES:EYFP (AAV-OXTR; green) construct or vehicle (Veh; blue) (scheme of infusion site, A and B). Co-staining for OXTR-EYFP (green) with calbindin (red) (C) is shown. AAV-OXTR- and Veh-treated mice were either social fear conditioned (SFC+) or left unconditioned (SFC−) (day 1; D; n = 7–13/group). Percentage investigation of three non-social (empty cage) and six social (cage with a conspecific) stimuli during social fear extinction (day 2; E) and social fear extinction recall (day 3; F) were measured. Data represent mean ± SEM. ∗p < 0.05: SFC+/AAV-OXTR versus SFC+/Veh; #p < 0.05: SFC+/AAV-OXTR versus all SFC− groups; one-way ANOVA with repeated measures followed by Bonferroni’s post hoc analysis (E).
(G–I) Downregulation of OXTR with constitutive knockout (KO; red) in virgin mice. Wild-type (WT; blue) and KO mice were social fear conditioned (SFC+) or left unconditioned (SFC−) (day 1; G; n = 9–10/group). Percentage investigation of three non-social (empty cage) and six social (cage with a conspecific) stimuli during social fear extinction (day 2; H) and social fear extinction recall (day 3; I) were assessed. Data represent mean ± SEM. ∗p < 0.05: SFC+/KO versus SFC+/WT using one-way ANOVA with repeated measures followed by Bonferroni’s post hoc analysis (H).
See also Figure S4.
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6. Figure 5
Chemogenetic Silencing of Hypothalamic OXT Neurons Projecting to the LS Leads to Blockade of Social Investigation in Lactating Mice
(A) Schematic representation of viral infusions and intraperitoneal (i.p.) drug administration. CNO, clozapine N-oxide; OXT, oxytocin; PVN, paraventricular nucleus; SON, supraoptic nucleus; V1, vehicle 1 (Ringer); V2, vehicle 2 (distilled water).
(B–D) Chemogenetic blockade of social investigation in lactation (Lac) mice. Lac mice were infused with V1 or AAV (CAV2-Cre and OXTpr-DIO-hM4Di:mCherry). Three weeks later, they were social fear conditioned (day 1; B; n = 6–7/group). Percentage investigation of three non-social (empty cage) and six social (cage with a conspecific) stimuli during social fear extinction (day 2; C) and social fear extinction recall (day 3; D) was measured. SFC+ mice were i.p. injected with vehicle V2 or CNO (15 mg/kg) 30 min before extinction training. Data represent mean ± SEM. ∗p < 0.05: AAV/CNO versus V1/V2 and V1/CNO using one-way ANOVA with repeated measures followed by Bonferroni’s post hoc analysis (C and D).
(E and F) Immunohistochemical staining for Fluorogold (FG; blue), mCherry (red), and OXT (green) within the SON and PVN hypothalamic nuclei (E) of virus-infused Lac mice. Mean number of back-labeled OXT-immunoreactive neurons ± SEM in the PVN and SON per mouse (n = 7; F) is shown.
See also Figure S5.
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7. Figure 6
Role of OXT Neurons of the SON Projecting to the LS in Attenuating Social Fear during Lactation
We hypothesize that the enhanced activity of SON-LS OXTergic neurons resulting in increased release of OXT in LS during social fear extinction activates local OXTR-expressing GABAergic neurons and, consequently, elevates social motivation, possibly via downstream regions, such as the ventromedial hypothalamus (VMH).
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