The anterior cingulate cortex and its role in controlling contextual fear memory to predatory threats
- Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, Brazil
- Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Brazil
- Cellular and Molecular Neurobiology Laboratory (LaNeC) - Center for Mathematics, Computing and Cognition (CMCC), Federal University of ABC, Brazil
Figures
Figure 1
Pharmacogenetic inhibition of the ACA during acquisition and expression of contextual fear to predator threat.
(A) Fluorescence photomicrograph illustrating the bilateral injection in the ACA of a viral vector expressing inhibitory DREADD (hM4D Gi) fused with mCitrine. (B) Pharmacogenetic inhibition of the …
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Figure 1—source data 1
Spreadsheet of raw values for panels (B) and (C).
- https://cdn.elifesciences.org/articles/67007/elife-67007-fig1-data1-v1.docx
Figure 2
Pattern of activation of the different sources of inputs to the ACA during the exposure to the cat.
Animals received unilateral deposit of a retrograde tracer (Fluoro Gold) in the ACA (n=6, A).After 7 days, animals were exposed to the cat (PET, B) and perfused 90 min after. (C) Bar chart presents, …
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Figure 2—source data 1
Spreadsheet of raw values for panel (C).
- https://cdn.elifesciences.org/articles/67007/elife-67007-fig2-data1-v1.docx
Figure 3
Pattern of activation of the different sources of inputs to the ACA during the exposure to predatory context.
Animals received unilateral deposit of a retrograde tracer (Fluoro Gold) in the ACA (n=6, A).After 7 days, animals were exposed to the cat (PET) and 24 hr later to the Predatory Context (B) and …
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Figure 3—source data 1
Spreadsheet of raw values for panel (C).
- https://cdn.elifesciences.org/articles/67007/elife-67007-fig3-data1-v1.docx
Figure 4
Optogenetic inhibition of anteromedial thalamic nucleus>ACA pathway during cat exposure.
(A) Schematics showing the location of the bilateral AAV viral vector injection in the anteromedial thalamic nucleus (AM) and the position of bilateral optical fibers implanted in the ACA. (B) …
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Figure 4—source data 1
Spreadsheet of raw values for panel (E).
- https://cdn.elifesciences.org/articles/67007/elife-67007-fig4-data1-v1.docx
Figure 5
Optogenetic silencing and functional tracing of the ACA>BLA pathway during the acquisition and expression of contextual fear to predator threat.
(A) Fluorescence photomicrograph illustrating the bilateral injection in the ACA of a viral vector expressing halorhodopsin-3.0 (eNpHR3.0) fused with mCherry. (B) Schematic drawing (center) and …
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Figure 5—source data 1
Behavior data – spreadsheet of raw values for panels (D) and (F).
Cell counting – spreadsheet of raw values for panels (E) and (G).
- https://cdn.elifesciences.org/articles/67007/elife-67007-fig5-data1-v1.docx
Figure 6
Optogenetic silencing and functional tracing of the ACA>PERI pathway during the acquisition and expression of contextual fear to predator threat.
(A) Schematic drawing illustrating the bilateral injection in the ACA of a viral vector expressing halorhodopsin-3.0 (eNpHR3.0) fused with mCherry. (B) Schematic drawing (center) and fluorescence …
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Figure 6—source data 1
Behavior data – spreadsheet of raw values for panels (D) and (F).
Cell counting – spreadsheet of raw values for panels (E) and (G).
- https://cdn.elifesciences.org/articles/67007/elife-67007-fig6-data1-v1.docx
Figure 7
Optogenetic silencing and functional tracing of the ACA>POST pathway during the acquisition and expression of contextual fear to predator threat.
(A) Schematic drawing illustrating the bilateral injection in the ACA of a viral vector expressing halorhodopsin-3.0 (eNpHR3.0) fused with mCherry. (B) Schematic drawing (center) and fluorescence …
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Figure 7—source data 1
Behavior data – spreadsheet of raw values for panels (D) and (F).
Cell counting – spreadsheet of raw values for panels (E) and (G).
- https://cdn.elifesciences.org/articles/67007/elife-67007-fig7-data1-v1.docx
Figure 8
Optogenetic silencing and functional tracing of the ACA>PAGdl pathway during the acquisition and expression of contextual fear to predator threat.
(A) Schematic drawing illustrating the bilateral injection in the ACA of a viral vector expressing halorhodopsin-3.0 (eNpHR3.0) fused with mCherry. (B) Schematic drawing (left) and fluorescence …
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Figure 8—source data 1
Behavior data – spreadsheet of raw values for panels (D) and (F).
Cell counting – spreadsheet of raw values of panels (E) and (G).
- https://cdn.elifesciences.org/articles/67007/elife-67007-fig8-data1-v1.docx
Appendix 1—figure 1
Experimental apparatus used for exposure to the Predator and Predatory Context.
Appendix 1—figure 2
Timeline to illustrate the experimental procedure.
Appendix 1—figure 3
Comparison of ACA activity in response to cat and novel non-threat stimulus.
On the left, drawings to illustrate the experimental conditions PET (predator exposure test), Context (real cat related context), PCE (plush cat exposure), and PCC (plush cat context). On the right, …
Appendix 1—figure 4
Impact of CNO on resting membrane potential and neuronal excitability in hM4D+ transfected neurons.
(A) Representative traces of resting membrane potential from hM4D− neurons (control – upper panel) or hM4D+ neurons (lower panel). hM4D+ neurons hyperpolarized as they underwent extracellular CNO. …
Appendix 1—figure 5
Light-induced hyperpolarization in Halorhodopsin positive neurons (HR+).
(A) Representative trace of membrane potential from an HR+ neuron. The arrows and bars represent the light on and off, respectively. The percentage on the top of the panel indicates the intensity of …
Appendix 1—figure 6
Efficiency of pre-synaptic inhibition.
(A) Representative EPSC traces before and after illumination at 585 nm (25 μW) with the arrows marking the stimulation (stimulus artifact). The upper traces show that there was no rebound excitation …
Appendix 1—figure 7
ACA projections.
(A) Fluorescence photomicrograph illustrating the extent and location of unilateral viral injection in the ACA. (B–E) On the left, schematic drawings from the Allen Mouse Brain Atlas to indicate the …
Appendix 1—figure 8
Density of ACA terminal fields in BLA, POST, PERI, and PAGdl.
Upper panel: Corrected total field fluorescence (CTFF) values for the cases 70, 71, and 72 showing the CTFF values for the ACA terminal fields to the BAL, POST, PERI, and PAGdl in a selected case …
Appendix 1—figure 9
Triple retrograde tracing illustrating the distribution of ACA neurons projecting to the BLA, PERI, and PAGdl.
Upper panel: Fluorescence photomicrographs illustrating the extent and location of unilateral Fluoro Gold deposit in the BLA (A), AAV-Retro-Ef1a-mcherry-IRES-Cre deposit in the PAGdl (B), and …
Appendix 1—figure 10
Optic fiber location for optogenetic silencing of the ACA>POST pathway.
Fluorescence photomicrographs illustrating the optic fiber position in five experiments of the group HR+ that received optogenetic silencing of the ACA>POST pathway during cat exposure. Note that in …
Appendix 1—figure 11
Histological analysis on the surroundings of the optic fiber tips.
Frontal sections showing the optic fibers (of) tip surroundings at the dorsolateral PAG (PAGdl; A–C), postsubiculum (POST; D–F), perirhinal area (PERI; G–I), and basolateral amygdalar nucleus (BLA; J…
Videos
Appendix 1—video 1
Exposure to the predator and predatory context.
Video to illustrate the behavioral responses of a male mice (C57BL/6) exposed to the cat (PET condition) and the predator-associated environment (Context condition).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6 | Local breeding facilities | ||
| Strain, strain background (Felis catus) | Male cat | Domestic | ||
| Transfected construct(adeno-associated virus) | AAV5-hSyn-HA-hM4D(Gi)-IRES-mCitrine | Addgene plasmid #50464(RRID: Addegene_50464) | Dr. Bryan Roth; titer≥7×10¹² vg/ml | |
| Transfected construct (adeno-associated virus) | AAV5- hSyn-eGFP | Addgene viral prep #50465 | Dr. Bryan Roth; titer≥7×10¹² vg/ml | |
| Transfected construct(adeno-associated virus) | AAV5-hSyn-eNpHR3-mCherry | University of North Carolina, Vector Core | Titer ≥1×10¹³ vg/ml | |
| Transfected construct(adeno-associated virus) | AAV5-hSyn-mCherry | University of North Carolina, Vector Core | Titer ≥7×1012 vg/ml | |
| Transfected construct(adeno-associated virus) | AAV-Retro-Ef1a-mcherry- IRES-Cre | Addgene catalog #55632-AAVrg | Karl Deisseroth; Titer ≥7×10¹² vg/ml | |
| Antibody | (Polyclonal rabbit anti-c-Fos) | PC-38; Calbiochem-Millipore (RRID:AB_2106755) | (1:20,000) | |
| Antibody | (Polyclonal anti-rabbit Alexa 594 goat IgG) (H+L) | Invitrogen (RRID:AB_2534079) | (1:500) | |
| Antibody | (Polyclonal rabbit anti-FG antibody) | Chemicon International, CA | (1:5000) | |
| Antibody | (Polyclonal anti-rabbit Alexa 488 Goat IgG) (H+L) | Invitrogen (RRID:AB_143165) | (1:1000) | |
| Antibody | (Polyclonal anti-rabbit Alexa 405 goat IgG) (H+L) | Invitrogen (RRID:AB_221605) | (1:500) | |
| Antibody | (Polyclonal goat anti-CTb) | List Biological Laboratories, Campbell, CA | (1:20,000) | |
| Antibody | (Polyclonal anti-goat Alexa 488 donkey IgG) (H+L) | Invitrogen (RRID:AB_2534102) | (1:500) | |
| Chemical compound, drug | Fluoro Gold | Fluorochrome Inc, CO | FG | |
| Chemical compound, drug | Isoforine | Cristália Laboratories, SP, Brazil | ||
| Chemical compound, drug | Clozapine-N-oxide | Tocris Bioscience, UK | CNO | |
| Chemical compound, drug | Subunit B of the cholera toxin | List Biological Laboratories, Campbell, CA | CTb | |
| Software, algorithm | Fiji | https://imagej.net/software/fiji/ (RRID:SCR_002285) | ||
| Software, algorithm | BORIS software | DOI: 10.1111/2041–210X.12584 (RRID:SCR_021434) | Behavior Observation Research Interactive Software | |
| Software, algorithm | pClamp 10.7 | Molecular Devices, USA (RRID:SCR_011323) | ||
| Software, algorithm | Clampfit 10.7 | Molecular Devices, USA | ||
| Software, algorithm | CorelDraw 2018 | https://www.coreldraw.com/br/pages/coreldraw-2018/ (RRID:SCR_014235) | ||
| Software, algorithm | Statistica 7.0 | StatSoft (RRID:SCR_014213) | ||
| Other | DAPI | Sigma-Aldrich | 1:20,000 |
Appendix 1—table 1
Behavioral data from male (n=6) and female (n=6) mice during the PET and Context conditions.
| Predator exposure test (PET) | Context | |||
|---|---|---|---|---|
| Male | Female | Male | Female | |
| Freezing | 102.8±4.1 | 98.2±5.5 | 0±0 | 0±0 |
| Risk assessment | 107.6±3.5 | 110.9±6.2 | 161.6±8.2 | 162.8±6.9 |
| Exploration | 25.8±2.5 | 31.9±3.0 | 37.9±4.4 | 34.7±5.2 |
Appendix 1—table 2
Escape responses.
| ACA – Pharmaco inhibition | AM>ACA Opto inhibition | |||||||
|---|---|---|---|---|---|---|---|---|
| Phase | GROUP | PET_Esc | CONT_Esc | Phase | GROUP | PET_Esc | Cont_Esc | |
| Acquisition | hM4D+ | 1.57±0.3 | 0.43±0.2 | Acquisition | HR+ | 1.5±0.28 | 0.5±0.26 | |
| Acquisition | hM4D− | 1.67±0.21 | 0.33±0.33 | Acquisition | HR− | 1.43±0.3 | 0.43±0.2 | |
| Expression | hM4D+ | 1.57±0.3 | 0.57± 0.2 | |||||
| Expression | hM4D− | 1.6±0.4 | 0.4±0.24 | |||||
| ACA>BLA Opto inhibition | ACA>PERI Opto inhibition | |||||||
| Phase | GROUP | PET_Esc | CONT_Esc | Phase | GROUP | PET_Esc | CONT_Esc | |
| Acquisition | HR+ | 1.5±0.26 | 0.37±0.18 | Acquisition | HR+ | 1.66±0.33 | 0.16±0.16 | |
| Acquisition | HR− | 1.33±0.21 | 0.33±0.21 | Acquisition | HR− | 1.4± 0.24 | 0.4±0.24 | |
| Expression | HR+ | 1.28±0.18 | 0.5±0.27 | Expression | HR+ | 1.5±0.22 | 0.16±0.16 | |
| Expression | HR− | 1.5±0.34 | 0.33±0.21 | Expression | HR− | 1±0 | 0.2±0.19 | |
| ACA>POST Opto inhibition | ACA>PAG Opto inihibition | |||||||
| Phase | GROUP | PET_Esc | CONT_Esc | Phase | GROUP | PET_Esc | CONT_Esc | |
| Acquisition | HR+ | 1.33±0.21 | 0±0 | Acquisition | HR+ | 1.43±0.14 | 0.14±0.14 | |
| Acquisition | HR− | 1.2±0.37 | 0.4±0.24 | Acquisition | HR− | 1.4±0.24 | 0.2±0.22 | |
| Expression | HR+ | 1.16±0.18 | 0.33±0.18 | Expression | HR+ | 1.5±0.24 | 0.33±0.23 | |
| Expression | HR− | 1.2±0.19 | 0±0 | Expression | HR− | 1±0 | 0.2±0.19 | |
