A parabrachial to hypothalamic pathway mediates defensive behavior
- Department of Neurobiology and Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, China
- Liangzhu Laboratory, Zhejiang University Medical Center, MOE Frontier Science Center for Brain Science and Brain-machine Integration, State Key Labotatory of Brain-machine intelligence, Zhejiang University, China
- NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University, China
- Institute for Translational Brain Research, MOE Frontiers Center for Brain Science, Fudan University, China
- The Institute of Brain and Cognitive Sciences, Zhejiang University City College, China
- Chuanqi Research and Development Center of Zhejiang University, China
Figures
Figure 1 with 2 supplements
LPBCCK neurons project to the PVN.
(A) Scheme of viral strategy for retrograde tracing from the PVN in Cck-cre mice using AAV2-Retro virus. (B) Representative image showing the injection site as marked by CTB555. (C) Representative …
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Figure 1—source data 1
Quantification of the labeled cells, EPSCs and IPSCs.
- https://cdn.elifesciences.org/articles/85450/elife-85450-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Distribution and identification of CCK neurons in the LPB.
(A) Schematic of mice crossing to generate the Cck-ires-cre::Ai14 mice. (B) Representative coronal section images illustrating the expression of CCK-tdT in the LPB; scale bar, 100 μm. (C) …
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Figure 1—figure supplement 1—source data 1
Quantification of LPB CCK-tdT cells.
- https://cdn.elifesciences.org/articles/85450/elife-85450-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Anterograde mapping of LPBCCK neurons.
(A–I) Representative images showing the projections from LPBCCK neurons in the median preoptic nucleus (MnPO), lateral preoptic area (LPO), paraventricular thalamic nucleus (PVT), ventromedial …
Figure 2 with 2 supplements
Activation of LPBCCK neurons triggers aversion, defensive-like flight-to-nest behavior and autonomic responses.
(A) Left, schematic of light stimulation of and patch-clamp recording from ChR2-EYFP expressing CCK neurons in the LPB. Right, example of action potentials evoked by optogenetic stimulation LPBCCK …
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Figure 2—source data 1
Quantification of the flight-to-nest behavior and autonomic responses upon activation of LPB CCK neurons.
- https://cdn.elifesciences.org/articles/85450/elife-85450-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Photostimulation of LPBCCK neurons induces anxiety-like behaviors.
(A) Schematic of behavioral test protocol. OFT, open-field test; EPM, elevated plus maze test. (B) Experimental apparatus: Open field. (C) Example movement traces of a control (AAV2/9-EF1a-DIO-EYFP, …
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Figure 2—figure supplement 1—source data 1
Quantification of the anxiety-like behavior.
- https://cdn.elifesciences.org/articles/85450/elife-85450-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Images of ChR2-EYFP expression in the LPB and optical fiber implantation above the LPB (A-G), with circles (H-I) indicating the location of optical fibers.
Figure 3 with 3 supplements
Threat stimuli recruit LPBCCK neurons to elicit defensive behaviors.
(A) Schematic of the in vivo recording system for the Ca2+ signal. (B) Schematic showing the injections and recording of LPB neurons in Cck-cre mice. (C) A representative image (left panel) and a …
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Figure 3—source data 1
Plot depicting the difference of Ca2+ activity.
- https://cdn.elifesciences.org/articles/85450/elife-85450-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
LPBCCK neurons respond to threat stimuli.
(A) A schematic of the heat assay. (B) Representative trial-by-trial heatmap presentation of calcium transients evoked by heat exposure in five trials from one mouse. (C) Representative peri-event …
Figure 3—figure supplement 2
Images of AAV-DIO-synapse-jGCaMP7b virus infection at the LPB (A) and downstream PVN and PSTh (B-C).
Figure 3—figure supplement 3
Images of Gcamp7s expression in the LPB and optical fiber implantation in the LPB (A-E), with circles (F-G) indicating the location of optical fibers.
Figure 4 with 1 supplement
Optogenetic inhibition of LPBCCK neurons suppresses predator- and visual predatory cue-evoked innate flight responses.
(A) Left, schematic of light stimulation of and patch-clamp recording of GtACR1-expressing CCK neurons in the LPB. Right, example of action potentials evoked by optogenetic inhibition of LPBCCK …
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Figure 4—source data 1
Quantification of the defensive responses upon inhibition of LPB CCK neurons.
- https://cdn.elifesciences.org/articles/85450/elife-85450-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Images of GtACR1-EYFP expression in the LPB and optical fiber implantation above the LPB (A-I), with circles (J-L) indicating the location of optical fibers.
Figure 5 with 1 supplement
Photostimulation of the LPBCCK-PVN pathway induces defensive-like flight-to-nest behavior.
(A) Schematic diagram of optogenetic activation of LPBCCK-PVN pathway. (B–C) A representative image showing the ChR2-EYFP expression in the LPB (B) and the optical fiber tip locations in the PVN (C) …
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Figure 5—source data 1
Quantification of the flight-to-nest behavior and autonomic responses upon activation of LPB CCK-PVN pathway.
- https://cdn.elifesciences.org/articles/85450/elife-85450-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Images of ChR2-EYFP expression in the LPB and optical fiber implantation above the PVN (A-F).
The square boxes indicate the location of virus expression and circles represent optical fiber location (G-L).
Figure 6 with 3 supplements
PVN is involved in the defensive-like flight-to-nest behavior evoked by LPBCCK neurons.
(A) Schematic of experimental setup. (B) Left, representative image showing the ChR2-EYFP expression in the LPB of a Cck-cre mouse. Scale bar, 100 μm. Right, representative image showing the …
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Figure 6—source data 1
Quantification of the flight-to-nest behavior upon manipulation of PVN neurons.
- https://cdn.elifesciences.org/articles/85450/elife-85450-fig6-data1-v1.xlsx
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Figure 6—source data 2
Maps for virus expression and optical fiber location.
- https://cdn.elifesciences.org/articles/85450/elife-85450-fig6-data2-v1.docx
Figure 6—figure supplement 1
Stimulation of LPBCCK-PVN pathway activates PVNCRH neurons, but activation of PVNCRH neurons does not trigger flight-to-nest behavior.
(A–B) Representative histological images showing Cckar and Cckbr in the PVN. (C) C-fos expression and colocalization (arrows) with CRH in the PVN with blue light activation of LPB-originating …
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Figure 6—figure supplement 1—source data 1
Quantification of the CRH neurons and flight-to-nest behavior.
- https://cdn.elifesciences.org/articles/85450/elife-85450-fig6-figsupp1-data1-v1.xlsx
Figure 6—figure supplement 2
Images of ISH data from the Allen Brain Atlas.
ISH data from the Allen Brain Atlas showed that Vglut2 neurons are located in the PVN (A), whereas GABAergic neurons are almost excluded from the PVN region by surrounding the PVN (B).
Figure 6—figure supplement 3
Images of ChR2-EYFP expression in the PVN and optical fiber implantation above the PVN (A-F).
The circles represent optical fiber location (G-I).
Author response image 1
ISH data from the Allen Brain Atlas.
Author response image 2
Representative figures of AAV-DIO-synapse-jGCaMP7b infection at the LPB and downstream PVN and PSTh.
Author response image 3
Representative images of fibers implantation above the LPB after injection of AAV-DIO-GtACR1-EYFP virus.
Author response image 4
Analysis of flight latency and speed after optic inhibition of LPBCCK neurons using GtACR1 virus.
(For latency, p = 0.1866, t = 1.394, df = 13; unpaired t test; for speed, p = 0.6926, t = 0.4043, df = 13; unpaired t test).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Cck-ires-cre(Ccktm1.1(cre)Zjh/J) | The Jackson Laboratory | JAX012706 | |
| Strain, strain background (Mus musculus) | Crh-ires-cre (B6(Cg))-Crhtm1(cre)Zjh/J | The Jackson Laboratory | JAX012704 | |
| Strain, strain background (Mus musculus) | Vglut2-ires-Cre (Slc17a6tm2(cre)Lowl/J) | The Jackson Laboratory | JAX 016963 | |
| Strain, strain background (Mus musculus) | Ai14 (B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) | The Jackson Laboratory | JAX007908 | |
| Strain, strain background (Rattus norvegicus) | SD | Shanghai SLAC Laboratory Animal Co. Ltd | http://www.slaccas.com/ | |
| Strain, strain background (Mus musculus) | C57BL/6 J | Shanghai SLAC Laboratory Animal Co. Ltd | http://www.slaccas.com/ | |
| Antibody | Anti-Dsred, rabbit polyclonal | Takara | Cat# 632496 RRID: AB_10013483 | (1:800) |
| Antibody | Anti-CGRP, mouse monoclonal | Abcam | Cat# 81887 RRID: AB_1658411 | (1:800) |
| Antibody | Anti-GFP, goat polyclonal | Abcam | Cat# 5450 RRID: AB_304896 | (1:500) |
| Antibody | Anti-CRH, rabbit polyclonal | Phoenix Biotech | Cat# H-019–06 | (1:500) |
| Antibody | Anti-c-fos, Guinea pig polyclonal | Synaptic Systems | Cat# 226004 RRID: AB_2619946 | (1:10,000) |
| Antibody | Alexa Fluor 488 donkey anti-guinea pig IgG (H+L) polyclonal | Jackson | Cat#112-486-068 RRID: AB_2617153 | (1:1000) |
| Antibody | Alexa Fluor 555 donkey anti-rabbit IgG (H+L) polyclonal | Invitrogen | Cat#A31572 RRID: AB_162543 | (1:1000) |
| Antibody | Alexa Fluor 488 donkey anti-mouse IgG (H+L) polyclonal | Invitrogen | Cat# R37114 RRID: AB_2556542 | (1:1000) |
| Antibody | Alexa Fluor 488 donkey anti-goat IgG (H+L) polyclonal | Invitrogen | Cat# A11055 RRID: AB_2534102 | (1:1000) |
| Antibody | Alexa Fluor 488 donkey anti-rabbit IgG (H+L) polyclonal | Jackson | Cat# R37118 RRID: AB_2556546 | (1:1000) |
| Commercial assay or kit | RNAscope Multiplex Fluorescent Reagent Kit v2 | Advanced Cell Diagnostics | Cat# 323100 | |
| Sequence-based reagent | RNAscope probe Slc17a6 | Advanced Cell Diagnostics | accession number NM_080853.3 | probe region 1986–2998 |
| Sequence-based reagent | RNAscope probe Slc32a1 | Advanced Cell Diagnostics | Accession number NM_009508.2 | probe region 894–2037 |
| Sequence-based reagent | RNAscope probe Cckar | Advanced Cell Diagnostics | accession number NM_009827.2 | probe region 328–1434 |
| Sequence-based reagent | RNAscope probe Cckbr | Advanced Cell Diagnostics | accession number NM_007627.4 | probe region 136–1164 |
| Chemical compound, drug | CNQX disodium salt hydrate | Sigma-Aldrich | Cat#1045 | |
| Chemical compound, drug | Devazepide | Sigma-Aldrich | Cat#2304 | |
| Chemical compound, drug | Clozapine N-oxide | Sigma-Aldrich | Cat#C0832 | |
| Chemical compound, drug | L-365,260 | Sigma-Aldrich | Cat#143626 | |
| Chemical compound, drug | DAPI | Sigma-Aldrich | N/A | |
| Chemical compound, drug | Tween-20 | Sigma-Aldrich | N/A | |
| Strain, strain background (AAV2/9) | AAV2/9-hEF1a-DIO-hChR2(H134R)-EYFP-WPRE-pA | Shanghai Taitool Bioscience Co. | Cat# S0199-9 | Viral titers: 2.95x1013 particles/ml |
| Strain, strain background (AAV2/9) | AAV2/9-hEF1a-DIO-EYFP-WPRE-pA | Shanghai Taitool Bioscience Co. | Cat#S0196-9 | Viral titers:1.0x1012 particles/ml |
| Strain, strain background (AAV2/9) | AAV2/9-CAG-DIO-hGtACR1-P2A-EGFP-WPRE-pA | Shanghai Taitool Bioscience Co. | Cat# S0311-9 | Viral titers:5×1013 particles/ml |
| Strain, strain background (AAV2/9) | rAAV2/9-hSyn-DIO-mGFP-2A-Synaptophysin-mRuby | Shanghai Taitool Bioscience Co. | Cat# S0250-9 | Viral titers:1.55x1013 particles/ml |
| Strain, strain background (AAV2/2) | rAAV2/2-Retro-hEF1a-DIO-EYFP-WPRE-pA | Shanghai Taitool Bioscience Co. | Cat# S0196-2R | Viral titers: 2.52x1013 particles/ml |
| Strain, strain background (AAV2/9) | AAV2/9-hSyn-mCherry-WPRE-pA | Shanghai Taitool Bioscience Co. | Cat# S0238-9 | Viral titers:≥1.0 × 1013 particles/ml |
| Strain, strain background (AAV2/9) | AAV2/9-hSyn-hM4D(Gi)-mCherry-WPRE-pA | Shanghai Taitool Bioscience Co. | Cat# S0279-9 | Viral titers:≥1.0 × 1013 particles/ml |
| Strain, strain background (AAV2/9) | AAV2/9-hsyn-DIO-jGCaMP7s-WPRE-pA | Shanghai Taitool Bioscience Co. | Cat# S0590-9 | Viral titers:≥1.0 × 1013 particles/ml |
| Software, algorithm | ANY-Maze software 5.3 | Global Biotech Inc | http://www.anymaze.co.uk/ | |
| Software, algorithm | Image J | NIH | https://imagej.nih.gov/ij/index.html;%20 RRID:SCR_003070 | |
| Software, algorithm | GraphPad Prism 6 | GraphPad Software | https://www.graphpad.com/scientificsoftware/prism/; RRID: SCR_002798 | |
| Software, algorithm | MatLab R2016a | MathWorks | https://www.mathworks.com/products.html; RRID:SCR_001622 |
Author response table 1
Numbers of retrogradely traced cells in CCK-Cre mice.
| Animal 1 | ||||||
|---|---|---|---|---|---|---|
| Region | Slice 1 | Slice 2 | Slice 3 | total | percentage | average |
| CC | 94 | 103 | 95 | 292 | 25.75% | 97 |
| DI | 11 | 34 | 21 | 66 | 5.82% | 22 |
| LPB | 71 | 103 | 69 | 243 | 21.43% | 81 |
| PAG | 26 | 34 | 19 | 79 | 6.97% | 26 |
| MO | 137 | 175 | 142 | 454 | 10.04% | 151 |
| Animal 2 | ||||||
| Region | Slice 1 | Slice 2 | Slice 3 | total | percentage | average |
| CC | 82 | 93 | 75 | 250 | 21.82% | 83 |
| DI | 18 | 23 | 17 | 58 | 5.06% | 19 |
| LPB | 94 | 102 | 91 | 287 | 25.04% | 96 |
| PAG | 11 | 23 | 15 | 49 | 4.28% | 16 |
| MO | 166 | 172 | 164 | 502 | 43.80% | 167 |
| Animal 3 | ||||||
| Region | Slice 1 | Slice 2 | Slice 3 | Total | percentage | average |
| CC | 80 | 95 | 76 | 251 | 24.25% | 84 |
| DI | 27 | 32 | 28 | 87 | 8.41% | 29 |
| LPB | 67 | 82 | 63 | 212 | 20.48% | 71 |
| PAG | 12 | 19 | 16 | 47 | 4.54% | 16 |
| MO | 139 | 157 | 142 | 438 | 42.32% | 146 |
| Region | Total average | |||||
| CC | 88 | |||||
| DI | 23 | |||||
| LPB | 83 | |||||
| PAG | 19 | |||||
| MO | 155 |
Author response table 2
Data of behavioral tests after inhibition of LPBCCK neurons.
| Animal | Stage | Hot zone : time | Safe zone : latency to first entry | Hot zone time/total time | Optical fiber location | |
|---|---|---|---|---|---|---|
| GtACR1 | 1 | Habitat stage | 96.5 | Optical fibers in only one side | ||
| Test stage | 36.7 | 13.5 | 12.23% | |||
| 2 | Habitat stage | 60.8 | Optical fibers in only one side | |||
| Test stage | 70 | 67.0 | 23.33% | |||
| 3 | Habitat stage | 13.4 | ||||
| Test stage | 265.7 | 265.7 | 88.57% | |||
| 4 | Habitat stage | 151.2 | ||||
| Test stage | 300 | 300.0 | 100.00% | |||
| 5 | Habitat stage | 176.7 | ||||
| Test stage | 300 | 300.0 | 100.00% | |||
| 6 | Habitat stage | 70.6 | Optical fibers in only one side | |||
| Test stage | 93 | 91.7 | 31.00% | |||
| 7 | Habitat stage | 33.7 | ||||
| Test stage | 137.9 | 106.7 | 45.97% | |||
| EYFP | 8 | Habitat stage | 84.8 | |||
| Test stage | 72.9 | 2.3 | 24.30% | |||
| 9 | Habitat stage | 41.9 | ||||
| Test stage | 18.4 | 5.6 | 6.13% | |||
| 10 | Habitat stage | 14.9 | ||||
| Test stage | 6.7 | 2.4 | 2.23% | |||
| 11 | Habitat stage | 77.6 | ||||
| Test stage | 44.9 | 9.0 | 14.97% | |||
| 12 | Habitat stage | 137.3 | ||||
| Test stage | 52 | 7.5 | 17.33% | |||
| 13 | Habitat stage | 75.8 | ||||
| Test stage | 41.3 | 38.0 | 13.77% | |||
| 14 | Habitat stage | 149.4 | ||||
| Test stage | 108.9 | 108.9 | 36.30% | |||
| 15 | Habitat stage | 30.8 | ||||
| Test stage | 6.8 | 4.1 | 2.27% |
Author response table 3
Number of labeled cells.
| Number of labeled cells | |||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| animal 1 | animal 2 | animal 3 | total average | ||||||||||||||||||
| region | slice 1 | slice 2 | slice 3 | total | percentage | average | region | slice 1 | slice 2 | slice 3 | total | percentage | average | region | slice 1 | slice 2 | slice 3 | total | percentage | average | |
| CC | 94 | 103 | 95 | 292 | 25.75% | 97 | CC | 82 | 93 | 75 | 250 | 21.82% | 83 | CC | 80 | 95 | 76 | 251 | 24.25% | 84 | 88 |
| DI | 11 | 34 | 21 | 66 | 5.82% | 22 | DI | 18 | 23 | 17 | 58 | 5.06% | 19 | DI | 27 | 32 | 28 | 87 | 8.41% | 29 | 23 |
| LPB | 71 | 103 | 69 | 243 | 21.43% | 81 | LPB | 94 | 102 | 91 | 287 | 25.04% | 96 | LPB | 67 | 82 | 63 | 212 | 20.48% | 71 | 83 |
| PAG | 26 | 34 | 19 | 79 | 6.97% | 26 | PAG | 11 | 23 | 15 | 49 | 4.28% | 16 | PAG | 12 | 19 | 16 | 47 | 4.54% | 16 | 19 |
| MO | 137 | 175 | 142 | 454 | 40.04% | 151 | MO | 166 | 172 | 164 | 502 | 43.80% | 167 | MO | 139 | 157 | 142 | 438 | 42.32% | 146 | 155 |
