Journal: bioRxiv

Article Title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

doi: 10.1101/2022.03.15.484224

Figure Lengend Snippet: a. CRH-Cre mice injected in ILA with AAV2/DJ hSyn.FLEX.mGFP.2A.Synatophysin-mRuby. b-e. Immunohistochemistry images of an ILA (b) and LS (c-e) section labelled for GFP (b-d) or mRuby (e). Scale bars: 100 µm (b), 500 µm (c) and 200 µm (d-e). f. CRH-Cre;Ai9 mice injected in rdLS with CtB-488. g. Image of a coronal brain section containing the injection site. Scale bar: 400 µm. h-j. Images of coronal brain sections containing the mPFC. Scale bar: 400 µm (h), 200 µm (i) and 50 µm (j). k. Distribution of CtB + cells in ILA. l. Number of CtB + /CRH + cells per ILA layer. Each point is from a different section. N = 3 mice. Bar graph represent mean ± S.E.M. One-way ANOVA, p < 0.0001. m. CRH-Cre mice injected in ILA with AAV2/8 syn.DIO.TVA-2A-GFP-2A-B19G and rabies SAD-B19.EnvA.ΔG.mCherry. n. Immunohistochemistry images of an ILA section showing GFP-expressing “starter” cells. Scale bar: 200 µm. o. Relative distribution of presynaptic Rabies-infected mCherry + cells in the brain. dLS: dorsal lateral septum. vCA1: ventral CA1 region. PLA: peri-limbic area of the PFC. DPA: dorsal peduncular area of the PFC. ACA: anterior cingulate area. ILA: infra-limbic area of the PFC. N = 3 mice.

Article Snippet: We injected AAV2/DJ hSyn.FLEX.mGFP.2A.Synatophysin-mRuby (Addgene #71760 prepared by the Stanford University vector core #1930), AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (Addgene #44362-AAV8), AAV2/8 hSyn.DIO.mCherry (Addgene #50459-AAV8), AAV2/5-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, #44361-AAV5), AAV2/5 hsyn.DIO.mCherry (Addgene #50459-AAV5), AAV2/1 syn.FLEX.GCaMP6f.WPRE.SV40 (Addgene #100833-AAV1), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA anti -Crh ), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled), AAV2/2 CAG.FLEX.ArchT-TdTomato (Addgene #28305 prepared by the University of North Carolina vector core) and AAV2/2 CAG.FLEX.TdTomato (Addgene #28306 prepared by the University of North Carolina vector core) into the ILA of CRH-Cre mice.

Techniques: Injection, Immunohistochemistry, Expressing, Infection

Journal: bioRxiv

Article Title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

doi: 10.1101/2022.03.15.484224

Figure Lengend Snippet: a. CRH-Cre mice injected in ILA with AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (iDREADD) or AAV2/8 hSyn.DIO.mCherry. b. Immunohistochemistry image showing viral expression in the ILA. Scale bar: 500 µm. c. Schematic of the social novelty preference test. d. Interaction time with novel (red) or familiar (blue) mouse during trial 2 in mice expressing mCherry (mC) or hM4Di (iD). Both groups were injected with saline (left groups) or 5 mg/kg of the DREADD agonist CNO (right groups). Grey dots are different mice. Paired t tests: p = 0.04, p = 0.02, p = 0.03 and p = 0.2. e. Discrimination indexes for social novelty preference of the four groups during recall trial. One-sample t tests compared to 0: mC + saline, p = 0.04; iD + saline, p = 0.02; mC + CNO, p = 0.006; iD + CNO, p = 0.2. Two-way ANOVA: trial x injection F 1, 54 = 4,795, p =0,03; virus F 1, 54 = 7,068, p = 0,01; injection F 1, 54 = 1,535, p = 0,2. Tukey’s multiple comparisons tests compared to the iD + CNO group: mC + saline, p = 0.04; iD + saline, p = 0.008; mC + CNO, p = 0.04. f. Schematic of the repetitive social presentation test. g. Normalized interaction times during social presentations (inhibitory DREADD-expressing mice and controls injected with CNO). 8 mice per group. Two-way ANOVA: trial x virus F 12,139 = 2.09, p = 0.02; trial F 4,139 = 17.21, p < 0.0001; virus F 3,139 = 15.76, p < 0.0001. h. Normalized interaction times during repetitive social presentation test in CRH-Cre mice injected in ILA with AAV5 hSyn.DIO.hM3D(Gq)-mCherry (excitatory DREADD) or with AAV5 hSyn.DIO.mCherry as a control. 8 mice per group. Two-way ANOVA: trial x virus F 12,140 = 0.96, p = 0.5; trial F 4,140 = 34.21, p < 0.0001; virus F 3,140 = 3.01, p = 0.03. i. CRH-Cre mice injected in ILA with AAV2/1 syn.FLEX.GCaMP6f and implanted with an optical ferrule over ILA. j. Schematic of the fiber-photometry recording experiment. k. Example trace of recording during presentation of a familiar mouse. l. Average peak amplitude of the z-score during social presentations. Each dot is a different recording session using 2 mice. Paired t test: p = 0.005. m. Frequency of calcium events during presentation of a novel or familiar mouse. Paired t test: p = 0.8. n. Average peak amplitudes during each type of presentation. Kruskal-Wallis test: F 4,36 = 4.991, p = 0.07. Multiple comparisons tests: cage vs. familiar mouse, p = 0.3; novel vs. familiar mouse, p = 0.02; novel object vs. familiar mouse, p = 0.3; familiar object vs. familiar mouse, p = 0.3. o. Discrimination indexes for familiarity preference calculated from z-scores during mouse or object presentation. Each point is one recording session. N = 4 mice. One-sample t tests compared to 0: p = 0.001 and p = 0.3 respectively. Unpaired t test between groups: p = 0.002. p. Peri-stimulus time histogram during social interaction with novel or familiar mouse. q. Area under the curve during familiar and novel mouse interaction. Each point is an interaction. N = 4 mice. Unpaired t test between groups: p = 0.0003. r. Fiber-photometry recording during repetitive social presentation test (10 sessions in 5 mice). One-sample t test compared to: p = 0.06 (trial 2), p =. 0.002 (trial 3) and p = 0.04 (trial 4). s. Frequency of calcium events during repetitive social presentation test (10 sessions in 5 mice). t. CRH-Cre;Ai9 mice were presented with novel or familiar mice after overnight isolation before being processed for immunohistochemistry. u. Interaction times following 2 min social presentation. v. Immunohistochemistry images of c-Fos labelling in ILA. Scale bars: 200 µm. w. Percentage of ILA CRH cells positive for c-Fos per layer. Each point corresponds to each side of 2 sections. 4 mice per group. Unpaired t test, p < 0.0001. x. Percentage of ILA CRH cells positive for c-Fos in layer 2/3 vs. interaction time during social interaction with novel (red) or familiar (blue) mouse. Each point represents one mouse. For the entire figure, bar graphs represent mean ± S.E.M.

Article Snippet: We injected AAV2/DJ hSyn.FLEX.mGFP.2A.Synatophysin-mRuby (Addgene #71760 prepared by the Stanford University vector core #1930), AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (Addgene #44362-AAV8), AAV2/8 hSyn.DIO.mCherry (Addgene #50459-AAV8), AAV2/5-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, #44361-AAV5), AAV2/5 hsyn.DIO.mCherry (Addgene #50459-AAV5), AAV2/1 syn.FLEX.GCaMP6f.WPRE.SV40 (Addgene #100833-AAV1), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA anti -Crh ), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled), AAV2/2 CAG.FLEX.ArchT-TdTomato (Addgene #28305 prepared by the University of North Carolina vector core) and AAV2/2 CAG.FLEX.TdTomato (Addgene #28306 prepared by the University of North Carolina vector core) into the ILA of CRH-Cre mice.

Techniques: Injection, Immunohistochemistry, Expressing, Isolation

Journal: bioRxiv

Article Title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

doi: 10.1101/2022.03.15.484224

Figure Lengend Snippet: CRH-Cre mice injected in ILA with AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (iD) or AAV2/8 hSyn.DIO.mCherry (mC). a. Schematic of the open-arena test. b. Total distance travelled during open-field test. c. Time spent in the center or surround of the open-field. d. Ratio of the time spent in the center/surround. e. Schematic of the elevated-plus maze test. f. Time spent in the open or closed arms. g. Discrimination indexes for closed arm preference using the time spent in the arms. h. Number of entries in the open or closed arms. i. Discrimination indexes for closed arm preference using the number of arm entries. j. Schematic of the novelty suppressed feeding test. k. Latency to feed. l. Feeding duration. m. Number of entries in the feeding zone. For the entire figure, bar graphs represent mean ± S.E.M. Grey dots are different mice.

Article Snippet: We injected AAV2/DJ hSyn.FLEX.mGFP.2A.Synatophysin-mRuby (Addgene #71760 prepared by the Stanford University vector core #1930), AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (Addgene #44362-AAV8), AAV2/8 hSyn.DIO.mCherry (Addgene #50459-AAV8), AAV2/5-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, #44361-AAV5), AAV2/5 hsyn.DIO.mCherry (Addgene #50459-AAV5), AAV2/1 syn.FLEX.GCaMP6f.WPRE.SV40 (Addgene #100833-AAV1), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA anti -Crh ), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled), AAV2/2 CAG.FLEX.ArchT-TdTomato (Addgene #28305 prepared by the University of North Carolina vector core) and AAV2/2 CAG.FLEX.TdTomato (Addgene #28306 prepared by the University of North Carolina vector core) into the ILA of CRH-Cre mice.

Techniques: Injection

Journal: bioRxiv

Article Title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

doi: 10.1101/2022.03.15.484224

Figure Lengend Snippet: CRH-Cre mice injected in ILA with AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (iD) or AAV2/8 hSyn.DIO.mCherry (mC). a. Schematic of the sociability test. b. Interaction times with mouse (purple) or object (orange). Paired t tests: p = 0.01, p = 0.009. c. Discrimination indexes for social preference. One-sample t tests compared to 0: p = 0.03 and p = 0.02. d-e. Total interaction times during learning (d) or recall trial of the social novelty preference test. f. Interaction time with each novel mouse during the learning trial of the social novelty preference test. One-way ANOVA, F 3,114 = 0.8694, p = 0.5. g. Normalized interaction times during social presentations (inhibitory DREADD-expressing mice and controls injected with saline). 8 mice per group. h. Normalized interaction times during social presentations (excitatory DREADD-expressing mice and controls injected with saline). 8 mice per group. For the entire figure, bar graphs represent mean ± S.E.M. Grey dots are different mice expect for f. where two observation per mice could be made since the mice interacted with two novel mice during the learning trial.

Article Snippet: We injected AAV2/DJ hSyn.FLEX.mGFP.2A.Synatophysin-mRuby (Addgene #71760 prepared by the Stanford University vector core #1930), AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (Addgene #44362-AAV8), AAV2/8 hSyn.DIO.mCherry (Addgene #50459-AAV8), AAV2/5-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, #44361-AAV5), AAV2/5 hsyn.DIO.mCherry (Addgene #50459-AAV5), AAV2/1 syn.FLEX.GCaMP6f.WPRE.SV40 (Addgene #100833-AAV1), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA anti -Crh ), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled), AAV2/2 CAG.FLEX.ArchT-TdTomato (Addgene #28305 prepared by the University of North Carolina vector core) and AAV2/2 CAG.FLEX.TdTomato (Addgene #28306 prepared by the University of North Carolina vector core) into the ILA of CRH-Cre mice.

Techniques: Injection, Expressing

Journal: bioRxiv

Article Title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

doi: 10.1101/2022.03.15.484224

Figure Lengend Snippet: CRH-Cre mice injected in ILA with AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (iD) or AAV5 hSyn.DIO.hM3D(Gq)-mCherry (excitatory DREADD). Mice received CNO or saline i.p. injection 30 min before being presented to a familiar animal for 2 min. Mice were thereafter perfused and processed for c-Fos labelling. Scale bars: 100 µm. a. Immunohistochemistry images of the ILA labelled for c-Fos. b. Interaction times. Each point is one mouse. c. Percentage of mCherry + cells in ILA expressing c-Fos. Unpaired t tests: p = 0.0005 and p = 0.0009.

Article Snippet: We injected AAV2/DJ hSyn.FLEX.mGFP.2A.Synatophysin-mRuby (Addgene #71760 prepared by the Stanford University vector core #1930), AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (Addgene #44362-AAV8), AAV2/8 hSyn.DIO.mCherry (Addgene #50459-AAV8), AAV2/5-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, #44361-AAV5), AAV2/5 hsyn.DIO.mCherry (Addgene #50459-AAV5), AAV2/1 syn.FLEX.GCaMP6f.WPRE.SV40 (Addgene #100833-AAV1), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA anti -Crh ), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled), AAV2/2 CAG.FLEX.ArchT-TdTomato (Addgene #28305 prepared by the University of North Carolina vector core) and AAV2/2 CAG.FLEX.TdTomato (Addgene #28306 prepared by the University of North Carolina vector core) into the ILA of CRH-Cre mice.

Techniques: Injection, Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

doi: 10.1101/2022.03.15.484224

Figure Lengend Snippet: CRH-Cre mice injected in ILA with AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (iD) or AAV2/8 hSyn.DIO.mCherry (mC). a. Schematic of the object recognition memory test. b-c. Total interaction times during learning (d) or recall trial of the object memory test. Grey dots are different mice. d. Interaction times with familiar (blue) or novel object (red). Grey dots are different mice. Paired t tests: p = 0.05 and p = 0.03. e. Discrimination indexes for novel object preference. Grey dots are different mice. One-sample t tests compared to 0: p = 0.02 and p = 0.02. Unpaired t test between groups: p = 0.5. f. Schematic of the repetitive object presentation test. g. Normalized interaction times during the repetitive object presentation test (inhibitory DREADD-expressing mice and control mice injected with 0.5 mg/kg CNO). 4 mice per group, 8 mice total. Two-way ANOVA: trial x virus F 4,30 = 0.233, p = 0.91; trial F 4,30 = 14.02, p < 0.0001; virus F 3,40 = 0.36, p = 0.56. For the entire figure, bar graphs represent mean ± S.E.M.

Article Snippet: We injected AAV2/DJ hSyn.FLEX.mGFP.2A.Synatophysin-mRuby (Addgene #71760 prepared by the Stanford University vector core #1930), AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (Addgene #44362-AAV8), AAV2/8 hSyn.DIO.mCherry (Addgene #50459-AAV8), AAV2/5-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, #44361-AAV5), AAV2/5 hsyn.DIO.mCherry (Addgene #50459-AAV5), AAV2/1 syn.FLEX.GCaMP6f.WPRE.SV40 (Addgene #100833-AAV1), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA anti -Crh ), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled), AAV2/2 CAG.FLEX.ArchT-TdTomato (Addgene #28305 prepared by the University of North Carolina vector core) and AAV2/2 CAG.FLEX.TdTomato (Addgene #28306 prepared by the University of North Carolina vector core) into the ILA of CRH-Cre mice.

Techniques: Injection, Expressing

Journal: bioRxiv

Article Title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

doi: 10.1101/2022.03.15.484224

Figure Lengend Snippet: a. DIC image of rdLS during patch-clamp recording. Scale bar: 500 µm. b. Example traces of IPSCs before or 15 min after application of 300 nM stressin-1. c. Frequency of IPSCs. d. Amplitude of IPSCs. e. IPSCs area under the curve. For c-e, points are obtained from individual cells recorded from separate slices in 6 mice. f. C57BL/6J wild-type mice injected in rdLS with AAV2/1 Syn.GCaMP6f and implanted with an optical ferrule above rdLS. Implanted mice were presented with novel and familiar mice. g. Interaction time during social presentation. Dots are from 9 recording sessions using 5 mice. h. Average peak amplitude of the z-score during presentation of a novel or familiar mouse. Paired t test: p = 0.007. i. Frequency of events during presentation of a novel or a familiar mouse. Paired t test: p = 0.6. j. Discrimination index for social familiarity preference calculated from z-scores. One-sample t tests compared to 0: p = 0.006. k. Immunohistochemistry images of c-Fos labelling in rdLS following social presentation with a novel or familiar mouse (same experiment than ). Scale bars: 500 µm. l. Density of rdLS cells positive for c-Fos. We made one observation on each side of a rLS section. 4 mice per group. Unpaired t test, p = 0.0003. m. Density of rdLS cells positive for c-Fos vs. interaction time during social interaction. Each point represents one mouse. n. Percentage of layer 2/3 ILA CRH cells positive for c-Fos (cf. ) vs. density of rdLS cells positive for c-Fos following social interaction. Each point represents a mouse. o-p. Immunohistochemistry images of c-Fos labelling in ILA (o) and rdLS (p). CRH-Cre mice injected in ILA with AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(anti- Crh ) or AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled) were presented with a familiar mouse for 2 min before being processed for immunohistochemistry. Scale bars: 300 µm. q. Duration of interaction during familiar presentation. Each point is one mouse. Unpaired t test, p = 0.001. r. Percentage of layer 2/3 ILA CRH cells positive for c-Fos in layer 2/3 of ILA. Each point corresponds to each side of 2 sections. 9 mice per group. s. Density of rdLS cells positive for c-Fos. We made one observation on each side of a rLS section. 9 mice per group. Unpaired t test, p < 0.0001. t. Percentage of layer 2/3 ILA CRH cells positive for c-Fos vs. density of rdLS cells positive for c-Fos following social interaction with a familiar mouse. Each point represents one mouse. u. C57BL/6J wild-type mice were injected with AA2/2 hSyn1.hChR2(H134R)-mCherry or AA2/2 hSyn1.mCherry as control and an optical fiber was implanted above the injection site. Mice were then presented to a familiar mouse for 2 min meanwhile 450 nm light was applied (20 Hz, 1 ms). Mice were also run without light as additional controls. Scale bar: 1 mm. v. Total interaction time with familiar mouse. Each point represents one mouse. One-way ANOVA: F 3,32 = 7.05, p = 0.0009. Dunnett’s multiple comparisons tests: ChR-light vs. YFP-no light p = 0.0005, ChR-light vs. ChR-no light p = 0.006, ChR-light vs. YFP-light p = 0.01. w. Average duration of each bout of social interaction. Each point represents one mouse. One-way ANOVA: F 3,31 = 10.62, p < 0.0001. Dunnett’s multiple comparisons tests: ChR-light vs. YFP-no light p = 0.0001, ChR-light vs. ChR-no light p = 0.0001, ChR-light vs. YFP-light p = 0.002. x. Total distance travelled. Each point represents one mouse. y. Schematic of the vCA1-ILA CRH -rdLS circuit. For the entire figure, bar graphs represent mean ± S.E.M.

Article Snippet: We injected AAV2/DJ hSyn.FLEX.mGFP.2A.Synatophysin-mRuby (Addgene #71760 prepared by the Stanford University vector core #1930), AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (Addgene #44362-AAV8), AAV2/8 hSyn.DIO.mCherry (Addgene #50459-AAV8), AAV2/5-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, #44361-AAV5), AAV2/5 hsyn.DIO.mCherry (Addgene #50459-AAV5), AAV2/1 syn.FLEX.GCaMP6f.WPRE.SV40 (Addgene #100833-AAV1), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA anti -Crh ), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled), AAV2/2 CAG.FLEX.ArchT-TdTomato (Addgene #28305 prepared by the University of North Carolina vector core) and AAV2/2 CAG.FLEX.TdTomato (Addgene #28306 prepared by the University of North Carolina vector core) into the ILA of CRH-Cre mice.

Techniques: Patch Clamp, Injection, Immunohistochemistry, shRNA

Journal: bioRxiv

Article Title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

doi: 10.1101/2022.03.15.484224

Figure Lengend Snippet: a. CRH-Cre mice injected in ILA with AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(anti- Crh ) to downregulate Crh or control AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled) (top). In situ hybridization images of ILA slices expressing shRNA against crh (left) or a scrambled shRNA (right) labelled for mCherry and Crh . Yellow arrowhead denotes a Cre-, mCherry- and anti- Crh shRNA-expressing neuron with reduced levels of Crh . White arrowheads denote Crh + neurons that express the scrambled shRNA and Crh. White arrows denote neurons that do not express the virus ( mCherry - ) with intact levels of Crh . Scale bars: 50 µm. b. Quantification of Crh expression in cells using in situ hybridization images in ILA slices from mice injected with AAV expressing scrambled or anti- Crh shRNAs. In each slice neurons were classified as to whether they were uninfected or infected with virus based on mCherry expression (3 mice per group; each point is from a different neuron). Note reduction in Crh expression in neurons infected with anti- Crh shRNA. 2-way ANOVA; shRNA x viral expression F 1, 144 = 61.34, p < 0.0001; shRNA F 1, 144 = 24.80, p < 0.0001; viral expression F 1, 144 = 26.75, p < 0.0001 followed by Tukey’s post-hoc test; anti- Crh + mCherry + vs. anti- Crh + mCherry - , p < 0.0001. c. Normalized interaction time during the repetitive social presentation test in mice expressing scrambled or anti- Crh shRNAs. 4 mice per group. Two-way ANOVA; trial x virus F 4,30 = 2.00, p = 0.1; trial F 4,30 = 6.07, p = 0.001; virus F 1,30 = 14.62, p = 0.0006. d. Interaction time with familiar (blue) or novel (red) mouse during the recall trial of the social novelty preference test in mice expressing scrambled or anti- Crh shRNAs. Grey dots are different mice. Paired t test: p = 0.0095, p = 0.6. e. Discrimination indexes for social novelty preference during recall trial. Grey dots are different mice. Unpaired t test: p = 0.03. f. Images of mGFP expression following the injection of AAV DIO.mGFP in rdLS of CRHR1-Cre mice. Scale bars: 400 µm (left), 100 µm (right). g. C57BL/6J wild-type mice infused in rdLS with 2 µg of antalarmin dissolved in 0.6 µL of DMSO or DMSO as a control. h. Interaction time with familiar (blue) or novel (red) mouse during the recall trial of the social novelty preference test in mice infused with antalarmin or DMSO. Grey dots are different mice. Paired t tests: p = 0.05, p = 0.3. i. Discrimination index for social novelty preference during recall trial. Grey dots are different mice. Unpaired t test: p = 0.01. j. CRH-Cre mice injected in ILA with AAV2/2 CAG.FLEX.ArchT-TdTomato or control AAV2/2 CAG.FLEX.TdTomato. Optical ferrule implant is above rdLS. Scale bar: 500 µm. k. Interaction time with familiar (blue) or novel (red) mouse during the recall trial of the social novelty preference test in the same mice. Laser was on during the learning or recall trial. Grey dots are different mice. Paired t test: p = 0.045, p = 0.009, p = 0.04 and p = 0.8. l. Discrimination index for social novelty preference during recall trial of the social novelty preference test in the same mice. Grey dots are different mice. One-way ANOVA: F 3,27 = 3.61, p = 0.03. Dunnett’s multiple comparisons tests: mC + light on learning vs. Arch + light on recall, p = 0.1; Arch + light on learning vs. Arch + light on recall, p = 0.03; mC + light on recall vs. Arch + light on recall, p = 0.04. m. Normalized interaction time during the repetitive social presentation test in the same mice. The laser was on during trials 1-4 of the Arch-light and mC-light groups (4 mice and 3 mice respectively). Laser was not on for the Arch-no light group (4 mice). Two-way ANOVA: trial x group F 8,40 = 2.50, p = 0.03; trial F 4,40 = 19.06, p < 0.0001; group F 2,40 = 27.95, p < 0.0001. For the entire figure, bar graphs represent mean ± S.E.M. Grey dots are different mice.

Article Snippet: We injected AAV2/DJ hSyn.FLEX.mGFP.2A.Synatophysin-mRuby (Addgene #71760 prepared by the Stanford University vector core #1930), AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (Addgene #44362-AAV8), AAV2/8 hSyn.DIO.mCherry (Addgene #50459-AAV8), AAV2/5-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, #44361-AAV5), AAV2/5 hsyn.DIO.mCherry (Addgene #50459-AAV5), AAV2/1 syn.FLEX.GCaMP6f.WPRE.SV40 (Addgene #100833-AAV1), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA anti -Crh ), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled), AAV2/2 CAG.FLEX.ArchT-TdTomato (Addgene #28305 prepared by the University of North Carolina vector core) and AAV2/2 CAG.FLEX.TdTomato (Addgene #28306 prepared by the University of North Carolina vector core) into the ILA of CRH-Cre mice.

Techniques: Injection, shRNA, In Situ Hybridization, Expressing, Infection

Journal: bioRxiv

Article Title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

doi: 10.1101/2022.03.15.484224

Figure Lengend Snippet: a. Normalized interaction times during repetitive object presentation following expression of a shRNA against Crh or a scrambled shRNA in ILA CRH cells. b. Total interaction time during learning and recall of the social novelty preference test in the same mice. c. Total interaction time during learning and recall of the social novelty preference test following antalarmin infusion. d-e. Total interaction time during learning and recall of the social novelty preference test following Arch or mCherry expression in ILA CRH and silencing of their terminals in LS. For the entire figure, bar graphs represent mean ± S.E.M. Grey dots are different mice.

Article Snippet: We injected AAV2/DJ hSyn.FLEX.mGFP.2A.Synatophysin-mRuby (Addgene #71760 prepared by the Stanford University vector core #1930), AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (Addgene #44362-AAV8), AAV2/8 hSyn.DIO.mCherry (Addgene #50459-AAV8), AAV2/5-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, #44361-AAV5), AAV2/5 hsyn.DIO.mCherry (Addgene #50459-AAV5), AAV2/1 syn.FLEX.GCaMP6f.WPRE.SV40 (Addgene #100833-AAV1), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA anti -Crh ), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled), AAV2/2 CAG.FLEX.ArchT-TdTomato (Addgene #28305 prepared by the University of North Carolina vector core) and AAV2/2 CAG.FLEX.TdTomato (Addgene #28306 prepared by the University of North Carolina vector core) into the ILA of CRH-Cre mice.

Techniques: Expressing, shRNA

Journal: bioRxiv

Article Title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

doi: 10.1101/2022.03.15.484224

Figure Lengend Snippet: a. C57BL/6J wild-type mice injected in rdLS with AAV2/1 Syn.GCaMP6f and implanted with an optical ferrule above rdLS. b. Immunohistochemistry image showing GCaMP expression in rdLS. Scale bar: 500 µm. c. Decoding performance for familiarity versus novelty from individual recordings or pseudo-simultaneous data. Small black dots on the left are the results from each individual recording sessions using 20 cross-validation iterations, large red dot is the average. Red dot on the right is the result of pseudo-population analysis from 100 cross-validation iterations. Grey areas denote chance level computed using permutation tests (2.5 – 97.5 percentiles in distribution of shuffled decoding performances). In both cases, statistical significance is determined by the probability of drawing the observed decoding performance from the distribution of shuffled decoding performances (null-hypothesis). p < 0.001 (two-tailed permutation test, see Methods) d. Schematic of the repetitive social presentation test. e. Fiber-photometry recording during repetitive social presentation test (10 recording sessions in 5 mice). One-way ANOVA (trial 1-4) F 3,27 = 3.389, p = 0.03 followed by Dunnett’s multiple comparison tests compared to trial 1: trial 2 p = 0.5, trial 3 p = 0.01 and trial 4 p = 0.1. f. Interaction times (trial 1 to trial 4) vs. z-scores during the same experiment.

Article Snippet: We injected AAV2/DJ hSyn.FLEX.mGFP.2A.Synatophysin-mRuby (Addgene #71760 prepared by the Stanford University vector core #1930), AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (Addgene #44362-AAV8), AAV2/8 hSyn.DIO.mCherry (Addgene #50459-AAV8), AAV2/5-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, #44361-AAV5), AAV2/5 hsyn.DIO.mCherry (Addgene #50459-AAV5), AAV2/1 syn.FLEX.GCaMP6f.WPRE.SV40 (Addgene #100833-AAV1), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA anti -Crh ), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled), AAV2/2 CAG.FLEX.ArchT-TdTomato (Addgene #28305 prepared by the University of North Carolina vector core) and AAV2/2 CAG.FLEX.TdTomato (Addgene #28306 prepared by the University of North Carolina vector core) into the ILA of CRH-Cre mice.

Techniques: Injection, Immunohistochemistry, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

doi: 10.1101/2022.03.15.484224

Figure Lengend Snippet: a. Percentage of familiar choice during development, 19 mice. b. Discrimination index for familiar kin before and after postnatal day 16. Each point represents a mouse, 19 mice. Unpaired t test, p < 0.001. c. CRH-Cre;Ai9 mice. d. mPFC images of CRH-Cre;Ai9 mice at P7, P15 or P21. Scale bars: 500 µm. e. Number of CRH + cells in ILA, PLA and ACA during development. Each point represents one observation made on each side of 2 section, 3 mice per group. f. Fold-increase of CHR + cells between P7 and P21. P21 values compared to the average P7 value. One-way ANOVA: F 2,33 = 75.68, p < 0.0001. Dunnett’s multiple comparisons tests: ILA vs. PLA, p < 0.0001 and ILA vs. ACA, p < 0.0001. g. Number of CRH + cells per ILA layers during development. Each point represents one observation made on each side of 2 section, 3 mice per group. h. Percentage of familiar choice during development in CRH-Cre mice injected in ILA with AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(anti- Crh ) to downregulate Crh or control AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled). 12 pups per group. Chi-square test: p < 0.0001. i. Discrimination index for familiar kin before and after postnatal day 16. Each point represents a mouse, 12 pups per group. Unpaired t tests: p = 0.3 and p < 0.0001. For the entire figure, bar graphs represent mean ± S.E.M.

Article Snippet: We injected AAV2/DJ hSyn.FLEX.mGFP.2A.Synatophysin-mRuby (Addgene #71760 prepared by the Stanford University vector core #1930), AAV2/8 hSyn.DIO.hM4D(Gi)-mCherry (Addgene #44362-AAV8), AAV2/8 hSyn.DIO.mCherry (Addgene #50459-AAV8), AAV2/5-hSyn-DIO-hM3D(Gq)-mCherry (Addgene, #44361-AAV5), AAV2/5 hsyn.DIO.mCherry (Addgene #50459-AAV5), AAV2/1 syn.FLEX.GCaMP6f.WPRE.SV40 (Addgene #100833-AAV1), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA anti -Crh ), AAV2/9 CMV-DIO-(mCherry-U6)-shRNA(scrambled), AAV2/2 CAG.FLEX.ArchT-TdTomato (Addgene #28305 prepared by the University of North Carolina vector core) and AAV2/2 CAG.FLEX.TdTomato (Addgene #28306 prepared by the University of North Carolina vector core) into the ILA of CRH-Cre mice.

Techniques: Injection, shRNA

Journal: iScience

Article Title: TDP-43 deficiency in suprachiasmatic nucleus perturbs rhythmicity of neuroactivity in prefrontal cortex

doi: 10.1016/j.isci.2024.109522

Figure Lengend Snippet:

Article Snippet: Virus: pAAV-U6-shRNA(CON)-CMV-EGFP-WPRE (Serotype: AAV2/9) , Shanghai OBiO Technology , Cat# Y11982.

Techniques: Generated, Virus, Recombinant, Luciferase, SYBR Green Assay, Cloning, shRNA, Knockdown, Amplification, Plasmid Preparation, Software, Lysis

Journal: iScience

Article Title: TDP-43 deficiency in suprachiasmatic nucleus perturbs rhythmicity of neuroactivity in prefrontal cortex

doi: 10.1016/j.isci.2024.109522

Figure Lengend Snippet:

Article Snippet: Virus: pAAV-U6-shRNA( Tdp-43 )-CMV-EGFP-WPRE (Serotype: AAV2/9) , Shanghai OBiO Technology , Cat# Y25761.

Techniques: Generated, Virus, Recombinant, Luciferase, SYBR Green Assay, Cloning, shRNA, Knockdown, Amplification, Plasmid Preparation, Software, Lysis