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(A) Heatmap showing mRNA expression levels of muscarinic receptor subtypes ( <t>Chrm1</t> – Chrm5 ) in layer 1 cortical neurons derived from Allen Brain Atlas scRNA-seq database. (B) Confocal image of vRSPL1 showing Vgat + somata (red) co-labeled with RNAScope probes for Chrm1 (green) and Chrm3 (gray). (C) Transcript abundance quantified by counting puncta per cell using a z-projected image ( Chrm3 : 2.0 ± 2.1 puncta per cell. Chrm1 : 13.9 ± 5.7 puncta per cell. p = 4.13e-07 (signed-rank test). n = 65 cells, N = 3 mice). (D) Schematic of the surgery performed. (E) Schematic of the experiment performed. (F) Comparison of freezing between non-stressed mice with tdTomato-expression (black), stressed mice with tdTomato expression (red) and stressed mice with shRNA-expression (yellow) during TFC. Two-way ANOVA. For control vs stress-tdTom mice: main effect of phase, F (9,130) = 16.95, p = 3.14e-18, effect of group, F (1,130) = 2.86, p = 0.0932; interaction, F (9,130) = 1.15, p = 0.3358. For control vs stress-shRNA mice: main effect of phase, F (9,220) = 28.39, p = 2.26e-32, effect of group, F (1,220) = 1.24, p = 0.267; interaction, F (9,220) = 1.22, p = 0.283. For stress-tdTom vs stress-shRNA mice: main effect of phase, F (9,210) = 29.55, p = 7.14e-33, effect of group, F (1,210) = 0.92, p = 0.340; interaction, F (9,210) = 0.76, p = 0.658. (G) Freezing during context test between three groups (For control vs stress-tdTom mice, p = 0.03 (unpaired t-test). For control vs stress-shRNA mice, p = 1.1e-4 (unpaired t-test). For stress-tdTom vs stress-shRNA mice, p = 0.680 (unpaired t-test)). (H) Freezing during TFC test between three groups. Two-way ANOVA. For control vs stress-tdTom mice: main effect of phase, F (9,130) = 8.11, p = 1.72e-9, effect of group, F (1,130) = 12.24, p = 6.0e-4; interaction, F (9,130) = 0.850, p = 0.571. For control vs stress-shRNA mice: effect of phase, F (9,220) = 11.48, p = 1.01e-14, effect of group, F (1,220) = 0.98, p = 0.322; interaction, F (9,220) = 0.34, p = 0.959. For stress-tdTom vs stress-shRNA mice: effects of phase, F (9,210) = 9.6, p = 3.10e-12; effect of group, F (1,210) = 7.38, p = 0.0071, interaction, F (9,210) = 1.0, p = 0.444.
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(A) Heatmap showing mRNA expression levels of muscarinic receptor subtypes ( <t>Chrm1</t> – Chrm5 ) in layer 1 cortical neurons derived from Allen Brain Atlas scRNA-seq database. (B) Confocal image of vRSPL1 showing Vgat + somata (red) co-labeled with RNAScope probes for Chrm1 (green) and Chrm3 (gray). (C) Transcript abundance quantified by counting puncta per cell using a z-projected image ( Chrm3 : 2.0 ± 2.1 puncta per cell. Chrm1 : 13.9 ± 5.7 puncta per cell. p = 4.13e-07 (signed-rank test). n = 65 cells, N = 3 mice). (D) Schematic of the surgery performed. (E) Schematic of the experiment performed. (F) Comparison of freezing between non-stressed mice with tdTomato-expression (black), stressed mice with tdTomato expression (red) and stressed mice with shRNA-expression (yellow) during TFC. Two-way ANOVA. For control vs stress-tdTom mice: main effect of phase, F (9,130) = 16.95, p = 3.14e-18, effect of group, F (1,130) = 2.86, p = 0.0932; interaction, F (9,130) = 1.15, p = 0.3358. For control vs stress-shRNA mice: main effect of phase, F (9,220) = 28.39, p = 2.26e-32, effect of group, F (1,220) = 1.24, p = 0.267; interaction, F (9,220) = 1.22, p = 0.283. For stress-tdTom vs stress-shRNA mice: main effect of phase, F (9,210) = 29.55, p = 7.14e-33, effect of group, F (1,210) = 0.92, p = 0.340; interaction, F (9,210) = 0.76, p = 0.658. (G) Freezing during context test between three groups (For control vs stress-tdTom mice, p = 0.03 (unpaired t-test). For control vs stress-shRNA mice, p = 1.1e-4 (unpaired t-test). For stress-tdTom vs stress-shRNA mice, p = 0.680 (unpaired t-test)). (H) Freezing during TFC test between three groups. Two-way ANOVA. For control vs stress-tdTom mice: main effect of phase, F (9,130) = 8.11, p = 1.72e-9, effect of group, F (1,130) = 12.24, p = 6.0e-4; interaction, F (9,130) = 0.850, p = 0.571. For control vs stress-shRNA mice: effect of phase, F (9,220) = 11.48, p = 1.01e-14, effect of group, F (1,220) = 0.98, p = 0.322; interaction, F (9,220) = 0.34, p = 0.959. For stress-tdTom vs stress-shRNA mice: effects of phase, F (9,210) = 9.6, p = 3.10e-12; effect of group, F (1,210) = 7.38, p = 0.0071, interaction, F (9,210) = 1.0, p = 0.444.
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(A) Heatmap showing mRNA expression levels of muscarinic receptor subtypes ( Chrm1 – Chrm5 ) in layer 1 cortical neurons derived from Allen Brain Atlas scRNA-seq database. (B) Confocal image of vRSPL1 showing Vgat + somata (red) co-labeled with RNAScope probes for Chrm1 (green) and Chrm3 (gray). (C) Transcript abundance quantified by counting puncta per cell using a z-projected image ( Chrm3 : 2.0 ± 2.1 puncta per cell. Chrm1 : 13.9 ± 5.7 puncta per cell. p = 4.13e-07 (signed-rank test). n = 65 cells, N = 3 mice). (D) Schematic of the surgery performed. (E) Schematic of the experiment performed. (F) Comparison of freezing between non-stressed mice with tdTomato-expression (black), stressed mice with tdTomato expression (red) and stressed mice with shRNA-expression (yellow) during TFC. Two-way ANOVA. For control vs stress-tdTom mice: main effect of phase, F (9,130) = 16.95, p = 3.14e-18, effect of group, F (1,130) = 2.86, p = 0.0932; interaction, F (9,130) = 1.15, p = 0.3358. For control vs stress-shRNA mice: main effect of phase, F (9,220) = 28.39, p = 2.26e-32, effect of group, F (1,220) = 1.24, p = 0.267; interaction, F (9,220) = 1.22, p = 0.283. For stress-tdTom vs stress-shRNA mice: main effect of phase, F (9,210) = 29.55, p = 7.14e-33, effect of group, F (1,210) = 0.92, p = 0.340; interaction, F (9,210) = 0.76, p = 0.658. (G) Freezing during context test between three groups (For control vs stress-tdTom mice, p = 0.03 (unpaired t-test). For control vs stress-shRNA mice, p = 1.1e-4 (unpaired t-test). For stress-tdTom vs stress-shRNA mice, p = 0.680 (unpaired t-test)). (H) Freezing during TFC test between three groups. Two-way ANOVA. For control vs stress-tdTom mice: main effect of phase, F (9,130) = 8.11, p = 1.72e-9, effect of group, F (1,130) = 12.24, p = 6.0e-4; interaction, F (9,130) = 0.850, p = 0.571. For control vs stress-shRNA mice: effect of phase, F (9,220) = 11.48, p = 1.01e-14, effect of group, F (1,220) = 0.98, p = 0.322; interaction, F (9,220) = 0.34, p = 0.959. For stress-tdTom vs stress-shRNA mice: effects of phase, F (9,210) = 9.6, p = 3.10e-12; effect of group, F (1,210) = 7.38, p = 0.0071, interaction, F (9,210) = 1.0, p = 0.444.

Journal: bioRxiv

Article Title: Cholinergic disruption of state-dependent retrosplenial layer 1 activity causes temporal associative memory deficit under stress

doi: 10.1101/2025.05.27.656297

Figure Lengend Snippet: (A) Heatmap showing mRNA expression levels of muscarinic receptor subtypes ( Chrm1 – Chrm5 ) in layer 1 cortical neurons derived from Allen Brain Atlas scRNA-seq database. (B) Confocal image of vRSPL1 showing Vgat + somata (red) co-labeled with RNAScope probes for Chrm1 (green) and Chrm3 (gray). (C) Transcript abundance quantified by counting puncta per cell using a z-projected image ( Chrm3 : 2.0 ± 2.1 puncta per cell. Chrm1 : 13.9 ± 5.7 puncta per cell. p = 4.13e-07 (signed-rank test). n = 65 cells, N = 3 mice). (D) Schematic of the surgery performed. (E) Schematic of the experiment performed. (F) Comparison of freezing between non-stressed mice with tdTomato-expression (black), stressed mice with tdTomato expression (red) and stressed mice with shRNA-expression (yellow) during TFC. Two-way ANOVA. For control vs stress-tdTom mice: main effect of phase, F (9,130) = 16.95, p = 3.14e-18, effect of group, F (1,130) = 2.86, p = 0.0932; interaction, F (9,130) = 1.15, p = 0.3358. For control vs stress-shRNA mice: main effect of phase, F (9,220) = 28.39, p = 2.26e-32, effect of group, F (1,220) = 1.24, p = 0.267; interaction, F (9,220) = 1.22, p = 0.283. For stress-tdTom vs stress-shRNA mice: main effect of phase, F (9,210) = 29.55, p = 7.14e-33, effect of group, F (1,210) = 0.92, p = 0.340; interaction, F (9,210) = 0.76, p = 0.658. (G) Freezing during context test between three groups (For control vs stress-tdTom mice, p = 0.03 (unpaired t-test). For control vs stress-shRNA mice, p = 1.1e-4 (unpaired t-test). For stress-tdTom vs stress-shRNA mice, p = 0.680 (unpaired t-test)). (H) Freezing during TFC test between three groups. Two-way ANOVA. For control vs stress-tdTom mice: main effect of phase, F (9,130) = 8.11, p = 1.72e-9, effect of group, F (1,130) = 12.24, p = 6.0e-4; interaction, F (9,130) = 0.850, p = 0.571. For control vs stress-shRNA mice: effect of phase, F (9,220) = 11.48, p = 1.01e-14, effect of group, F (1,220) = 0.98, p = 0.322; interaction, F (9,220) = 0.34, p = 0.959. For stress-tdTom vs stress-shRNA mice: effects of phase, F (9,210) = 9.6, p = 3.10e-12; effect of group, F (1,210) = 7.38, p = 0.0071, interaction, F (9,210) = 1.0, p = 0.444.

Article Snippet: AAV5-hEF1a-dlox-EGFP(rev)-dlox (v217-5, VVF) AAV1-hSyn1-dlox-jGCaMP8s(rev) (v627-1, VVF) AAV8-hSyn-DIO-hM4Di (Gi) – mCherry (44362-AAV8, Addgene) AAV5-CAG-GFP (37525-AAV5, Addgene) AAV5-CAG.hChR2(H134R)-mCherry (10054-AAV5, Addgene) AAV5-EF1a-DIO-hChR2(E123T/T159C)-EYFP (35509-AAV5, Addgene) AAV1-hSyn1-dlox-TVA_2A_mCherry_2A_oG(rev)_dlox (v306-1, VVF) Rabies-GFP (NTNU viral core facility) AAV9-CAG-DIO-ChroME-ST-P2A-H2B-mRuby3 (108912-AAV9, Addgene) AAV9-FLEX-Syn1-mCherry-shRNA- Chrm1 (custom ordered , VectorBuilder)

Techniques: Expressing, Derivative Assay, Labeling, RNAscope, Comparison, shRNA, Control

(A) Confocal image of a vRSPL1 section from Vgat ::Ai14 mice, stained with RNAscope for Chrna7 (yellow) and Chrm1 (green). The pia and L1/2 border are indicated with black and tan arrows, respectively. White arrows mark the location of Vgat + neurons. A cell highlighted with a yellow arrow is expanded below to show individual RNA puncta. (B) The same as in (A) but from mice injected with Cre-dependent Chrm1 -shRNA into vRSPL1. (C) Transcript abundance quantified by counting puncta per cell using s single plane (non-z-projected) image. Control mice: 11.2 ± 6.4 puncta for Chrm1 and 16.5 ± 10.8 for Chrna7 (p = 1.68e-4 signed-rank test, n = 35 cells, N = 3). shRNA-injected mice: 2.0 ± 1.8 for Chrm1 and 13.0 ± 9.6 for Chrna7 (p = 1.51e-4, signed-rank test, n = 20 cells, N = 3). Comparison between Chrm1 expression in control and shRNA-injected mice: p = 5.10e-8 (rank-sum test). For Chrna7 : p = 0.241 (unpaired t-test).

Journal: bioRxiv

Article Title: Cholinergic disruption of state-dependent retrosplenial layer 1 activity causes temporal associative memory deficit under stress

doi: 10.1101/2025.05.27.656297

Figure Lengend Snippet: (A) Confocal image of a vRSPL1 section from Vgat ::Ai14 mice, stained with RNAscope for Chrna7 (yellow) and Chrm1 (green). The pia and L1/2 border are indicated with black and tan arrows, respectively. White arrows mark the location of Vgat + neurons. A cell highlighted with a yellow arrow is expanded below to show individual RNA puncta. (B) The same as in (A) but from mice injected with Cre-dependent Chrm1 -shRNA into vRSPL1. (C) Transcript abundance quantified by counting puncta per cell using s single plane (non-z-projected) image. Control mice: 11.2 ± 6.4 puncta for Chrm1 and 16.5 ± 10.8 for Chrna7 (p = 1.68e-4 signed-rank test, n = 35 cells, N = 3). shRNA-injected mice: 2.0 ± 1.8 for Chrm1 and 13.0 ± 9.6 for Chrna7 (p = 1.51e-4, signed-rank test, n = 20 cells, N = 3). Comparison between Chrm1 expression in control and shRNA-injected mice: p = 5.10e-8 (rank-sum test). For Chrna7 : p = 0.241 (unpaired t-test).

Article Snippet: AAV5-hEF1a-dlox-EGFP(rev)-dlox (v217-5, VVF) AAV1-hSyn1-dlox-jGCaMP8s(rev) (v627-1, VVF) AAV8-hSyn-DIO-hM4Di (Gi) – mCherry (44362-AAV8, Addgene) AAV5-CAG-GFP (37525-AAV5, Addgene) AAV5-CAG.hChR2(H134R)-mCherry (10054-AAV5, Addgene) AAV5-EF1a-DIO-hChR2(E123T/T159C)-EYFP (35509-AAV5, Addgene) AAV1-hSyn1-dlox-TVA_2A_mCherry_2A_oG(rev)_dlox (v306-1, VVF) Rabies-GFP (NTNU viral core facility) AAV9-CAG-DIO-ChroME-ST-P2A-H2B-mRuby3 (108912-AAV9, Addgene) AAV9-FLEX-Syn1-mCherry-shRNA- Chrm1 (custom ordered , VectorBuilder)

Techniques: Staining, RNAscope, Injection, shRNA, Control, Comparison, Expressing