Dissociable control of unconditioned responses and associative fear learning by parabrachial CGRP neurons
- Department of Biochemistry, University of Washington, United States
- Howard Hughes Medical Institute, University of Washington, United States
- Graduate Program in Neuroscience, University of Washington, United States
- Center for Integrative Brain Research, Seattle Children’s Research Institute, United States
Figures
Figure 1 with 2 supplements
CGRPPBN neurons potentiate fear behavior, drive associative learning and robustly activate forebrain targets.
(A) Bilateral injections of AAV1-DIO-ChR2:YFP or AAV1-DIO-YFP and fiberoptic cannula implants above the PBN of CalcaCre/+ mice. (B) Photostimulation (30 Hz) of CGRPPBN neurons generated robust …
Figure 1—figure supplement 1
Fiber placement, autonomic measurements, and contralateral projection strength.
(A) Position of fiberoptic cannula tips for CGRPPBN neuron stimulation. (B) Example of freezing behavior in response to repeated 30 Hz photostimulation of CGRPPBN neurons. (C) Latency to freeze …
Figure 1—video 1
Freezing behavior generated by activating CGRPPBNneurons.
Supplement to Figure 1.
Figure 2 with 1 supplement
CGRPPBN neurons broadly collateralize to forebrain targets.
(A) Injections of rAAV2-retro-Flp into projection targets and INTRSECT viruses into the PBN of CalcaCre/+ mice to isolate target-projecting (Target +, Cre-on Flp-on) or non-projecting (Target -, …
Figure 2—figure supplement 1
Collateralization to forebrain targets by CGRPPBN neurons.
(A) Fluorescent images of CGRPPBN neurons and their projection targets from mice expressing YFP in either all CGRPPBN neurons or those projecting to the VPMpc, PSTN, CeA, or ovBNST. Scale bar: 100 …
Figure 3 with 3 supplements
Photostimulation of CGRPPBN neuron terminals in individual downstream targets exerts diverse effects on physiology and behavior.
(A) Activating terminals in the VPMpc (n = 8,5) (ChR2, YFP) elicited freezing behavior but had no effect on (G) heart rate (M) respiration or (S) vasoconstriction. (B) Photostimulating terminals in …
Figure 3—figure supplement 1
Verification of terminal stimulation of CGRPPBN neuron projections.
(A–B) Postsynaptic neurons are reliably activated by 15- (top) and 30 Hz (bottom) photostimulation of CGRPPBN neuron terminals (five cells from two mice, two cells represented). (C) Position of …
Figure 3—figure supplement 2
Freezing behavior elicited by photostimulation of CGRPPBN neuron terminals.
(A) Freezing behavior from each animal collapsed across stimulation epochs. Photostimulation of CGRPPBN neurons resulted in robust freezing behavior not replicated by activation of any individual …
Figure 3—figure supplement 3
Physiological responses to photostimulation of CGRPPBN neuron terminals.
(A) Autonomic responses to 15 Hz photostimulation of CGRPPBN neuron terminals in the VPMpc (n = 8, paired t-test, heart rate t(7) = 1.47, p=0.19; respiration t(7) = 0.03, p>0.05). (B) Autonomic …
Figure 4 with 1 supplement
Stimulating CGRPPBN neuron terminals in ovBNST is anxiogenic while stimulating most other projections is aversive.
(A) Experimental timeline and example responses to stimulation of CGRPPBN neuron terminals or somata during measurements of anxiety-like behavior. (B) Activation of CGRPPBN neurons reduced time …
Figure 4—figure supplement 1
Activation of CGRPPBN terminals in the rCeA potentiates nocifensive responses.
(A) Response latency on 52°C hot plate increased by stimulating CGRPPBN neurons or projections to the cCeA, rCeA, or ovBNST prior to exposure, consistent with stress-induced analgesia (significance …
Figure 5
Photostimulating terminals in the VPMpc or SI can promote associative fear learning.
(A) Illustration of experimental paradigm for cue-dependent optogenetic conditioning. (B) Conditioned-freezing responses to CS paired with CGRPPBN terminal stimulation in the VPMpc during training …
Figure 6 with 3 supplements
Combined activation of CGRPPBN neuron terminals in the VPMpc and cCeA scales freezing responses and produces bradycardia.
(A) Schematic showing configuration for implantation of 3 fiberoptic cannulae into one hemisphere allowing simultaneous photostimulation of multiple CGRPPBN-neuron terminal fields. (B) Freezing …
Figure 6—figure supplement 1
Coincident activation of CGRPPBN neuron projections using ChrimsonR causes profound freezing responses.
(A) Survival curve comparing latencies of freezing and bradycardia responses to simultaneous 30 Hz photostimulation of CGRPPBN neuron terminals in the VPMpc and cCeA (n = 4 freezing, n = 5 heart …
Figure 6—video 1
Freezing behavior generated by activating the SI and caudal CeA simultaneously supplement to Figure 6.
Figure 6—video 2
Freezing behavior generated by activating the caudal CeA and VPMpc simultaneously supplement to Figure 6.
Figure 7 with 1 supplement
Foot shock-induced activation of the VPMpc and SI by CGRPPBN neurons contributes to associative fear learning.
(A) Bilateral injections of AAV1-DIO-JAWS:GFP or AAV1-DIO-YFP and fiber-optic cannula implants above the PBN of CalcaCre/+ mice for photoinhibition of CGRPPBN neurons. (B) Photoinhibition of CGRPPBN …
Figure 7—figure supplement 1
Photoinhibition of CGRPPBN neurons or projections does not affect nocifensive responses or alter place preference.
(A) Representative recording of action potentials from a CGRPPBN neuron. Red-light photostimulation (3 s on and 1 s ramp-down) effectively suppressed firing rate of CGRPPBN neurons with minimal …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Calca-Cre, C57BL6/J | Carter et al., 2013 | RRID:IMSR_JAX:033168 | |
| Strain, strain background (AAV1) | pAAV1-Ef1alpha-DIO hChR2(H134R) eYFP | Carter et al., 2013 | Addgene Plasmid #20298 RRID:Addgene_20298 | |
| Strain, strain background (AAV1) | pAAV1-Ef1alpha-DIO YFP | Carter et al., 2013 | Addgene Plasmid #27056 RRID:Addgene_27056 | |
| Strain, strain background (AAV1) | pAAV1-Ef1alpha-DIO JAWS GFP | Jo et al., 2018 | RRID:Addgene_78174 | |
| Strain, strain background (AAV1) | pAAV1-nEF-Con/Fon-ChR2-mCherry | Fenno et al., 2014 | Addgene Plasmid #137142 RRID:Addgene_137142 | |
| Strain, strain background (AAV1) | pAAV1-nEF-Con/Foff 2.0-ChR2-mCherry | Fenno et al., 2014 | Addgene Plasmid #137143 RRID:Addgene_137143 | |
| Strain, strain background (rAAV2-retro) | AAV2-retro-CBA-Flippase-dsRed | This paper | N/A | palmiter@uw.edu |
| Strain, strain background (AAV9) | AAV9-Syn-ChrimsonR-tdTomato | UNC Vector Core | Cat# AV6556B A RRID:Addgene_62723 | |
| Antibody | Anti-c-Fos (Rabbit polyclonal) | Abcam | Cat#: ab190289 RRID:AB_2737414 | (1:1000) |
| Antibody | Anti-GFP (Chicken polyclonal) | Abcam | Cat#: ab13970 RRID:AB_300798 | (1:10,000) |
| Antibody | Anti-dsRed (Rabbit monoclonal) | Takara | Cat#: 632496 RRID:AB_10013483 | (1:1000) |
| Antibody | Alexa Fluor 488 anti-Chicken (Donkey monoclonal) | Jackson ImmunoResearch | Cat#: 703-545-155 RRID:AB_2340375 | (1:500) |
| Antibody | Alexa Fluor 594 anti-Rabbit (Donkey monoclonal) | Jackson ImmunoResearch | Cat#: 711-585-152 RRID:AB_2340621 | (1:500) |
| Antibody | Cy5 anti-rabbit (Donkey monoclonal) | Jackson ImmunoResearch | Cat#: 711-175-152 RRID:AB_2340607 | (1:500) |
| Other | Normal donkey serum | Jackson ImmunoResearch | Cat#:017-000-121 RRID:AB_2337258 | |
| Other | Pulse oximeter | STARR Life Sciences | Part#: 015000 | |
| Other | Pulse oximeter collar sensor | STARR Life Sciences | Part#: 015021 | |
| Software, algorithm | MouseOxPlus conscious applications module | STARR Life Sciences | Part#: 015002 | |
| Software, algorithm | Ethovision XT 10 | Noldus Technology | www.noldus.com RRID:SCR_000441 |
Additional files
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Supplementary file 1
Full statistical information for all data figures.
- https://cdn.elifesciences.org/articles/59799/elife-59799-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/59799/elife-59799-transrepform-v2.pdf
