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Encoding of danger by parabrachial CGRP neurons

Abstract

Animals must respond to various threats to survive. Neurons that express calcitonin gene-related peptide in the parabrachial nucleus (CGRPPBN neurons) relay sensory signals that contribute to satiation and pain-induced fear behaviour, but it is unclear how they encode these distinct processes. Here, by recording calcium transients in vivo from individual neurons in mice, we show that most CGRPPBN neurons are activated by noxious cutaneous (shock, heat, itch) and visceral stimuli (lipopolysaccharide). The same neurons are inhibited during feeding, but become activated during satiation, consistent with evidence that CGRPPBN neurons prevent overeating. CGRPPBN neurons are also activated during consumption of novel foods or by an auditory cue that has previously been paired with electrical footshocks. Correspondingly, silencing of CGRPPBN neurons attenuates the expression of food neophobia and conditioned fear responses. Therefore, in addition to transducing primary sensory danger signals, CGRPPBN neurons promote affective-behavioural states that limit harm in response to potential threats.

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Figure 1: CGRPPBN neurons are activated by painful stimuli.
Figure 2: CGRPPBN neurons are activated by non-painful noxious stimuli.
Figure 3: Rapid control of CGRPPBN neurons before and during feeding.
Figure 4: CGRPPBN neurons are activated in response to a novel and palatable high-fat diet.
Figure 5: CGRPPBN neurons are activated during recall of a pain memory.
Figure 6: Silencing of CGRPPBN neurons attenuates fear response during recall of pain memory.

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Acknowledgements

Research was supported by a fellowship grant from Hope Funds for Cancer Research (C.A.C.), a National Institutes of Health (NIH) training grant (C.W.R., T32DK007247), and an NIH grant (R.D.P., R01-DA24908). Inscopix provided the calcium imaging equipment via their DECODE program. We thank C. de Solages and L. Cardy (Inscopix) for advice regarding calcium imaging, Y. S. Jo for help with conditioning equipment, M. Chiang for maintaining the mouse colony, and S. Han for making the GCaMP6m virus.

Author information

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Authors

Contributions

C.A.C. designed and conducted the calcium imaging studies. A.J.B. performed the stereotaxic surgeries for loss-of-function studies. C.A.C. and A.J.B. designed and conducted the fear studies. C.A.C. and C.W.R. designed and conducted the itch studies. R.D.P. provided guidance and resources. C.A.C. wrote the manuscript with input from the other authors.

Corresponding authors

Correspondence to Carlos A. Campos or Richard D. Palmiter.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks C. Alexander and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 CGRPPBN neurons are activated by facial pain and electrical shock.

a, b, Percentage of cells activated by tail pinch and placing a warm metal rod on the lip of anaesthetized mice (n = 6 mice, 397 neurons). c, Representative calcium traces of the same individual CGRP neurons during baseline and 2 h after LPS injection in awake mice. d, Average calcium fluorescence activity 1 s before (Base) and during a footshock (0.5 mA, 0.5 s duration; n = 5 mice, 317 neurons); bar graph is mean ± s.e.m.. e, Calcium activity time course of CGRP neurons (n = 3 mice, 158 neurons) and oxytocin receptor neurons (n = 3 mice, 65 neurons) in response to electrical tail shock (2 mA, 2 s duration) in anaesthetized mice; baseline was 2-s period before shock. AAV-DIO-GCaMP6m was injected into the lateral PBN of CalcaCre/+ and OxtrCre/+ mice. For imaging oxytocin receptor neurons, the lens was placed over the dorsal lateral PBN, in contrast to external lateral PBN targeting for CGRP neurons. Line graph is mean ± s.e.m. (shaded region). ***P < 0.001, paired Student’s t-test (two-tailed). Related to Fig. 1. For statistical analysis, see Supplementary Information.

Source data

Extended Data Figure 2 CGRPPBN neurons encode stimulus intensity.

a, Plot shows calcium fluorescence activity of neurons in an anaesthetized mouse following placement of the tail in a water bath ranging from 40 °C to 52 °C (n = 56 neurons). b, Area under the curve of calcium activity following placement of tail in 40–52 °C water bath (n = 5 mice, 286 neurons). c, Percentage of cells activated by placement of tail in 56 or 60 °C water in anaesthetized mice; data points are individual mice (n = 4 mice) and line represents mean. d, Time course and peak neuronal responses to tail being placed in 56 or 60 °C water (n = 4 mice, 111 neurons). Baseline was 10-s period before placing tail in water. e, Area under the curve of calcium activity following placement of tail in water bath. f, Time course and peak neuronal responses to tail receiving graded intensities of electrical current (2–6 mA, 2 s duration; n = 3 mice, 105 neurons) in anaesthetized mice. Baseline was 2-s period before shock. g, Time course and peak calcium activity of neurons in an awake mouse (46 neurons) during exposure to a hotplate (40–56 °C, 20-s duration). Baseline was 2-min period in home cage before the hotplate test. h, Area under the curve of calcium activity following placement of awake mice on the hotplate (n = 3 mice, 126 neurons). Bar graphs are mean ± s.e.m.; line graphs are mean ± s.e.m. (shaded region). ***P < 0.001; dissimilar letters above columns indicate statistical differences between treatments. b, h, One-way repeated measures ANOVA, Tukey’s post-hoc; e, paired Student’s t-test (two-tailed). Related to Fig. 1. For statistical analysis, see Supplementary Information.

Source data

Extended Data Figure 3 Additional analysis of calcium imaging from fast–refeed study.

a, Average calcium fluorescence activity 5 s before and 5 s after presentation of standard chow pellet (n = 5 mice, 282 neurons); mean ± s.e.m.. b, Calcium activity relative to taking a bite (n = 5 mice, 286 neurons). c, d, Plot showing calcium activity (normalized ΔF/F) relative to placement of chow in cage (c) and relative to bite (d). Plots show neurons (rows) from all experimental mice (n = 5 mice, 282 neurons). e, f, Representative calcium traces extracted using CNMF analysis, relative to chow availability (e) and taking a bite (f). Βaseline was 60-s period before chow availability for all data shown. ***P < 0.001; dissimilar letters above columns indicate statistical differences between time points; a, paired Student’s t-test (two-tailed); c, one-way repeated measures ANOVA, Tukey’s post-hoc. Related to Fig. 3. For statistical analysis, see Supplementary Information.

Source data

Extended Data Figure 4 Additional analysis of calcium imaging from food neophobia studies.

ac, Change in calcium fluorescence activity in response to a novel, high-fat diet (HFD) pellet. Test trials were conducted on consecutive days. Line graphs are means ± s.e.m. (shaded region) for raw ΔF/F values and plots show normalized ΔF/F values for neurons (rows) of all experimental mice. df, Average calcium activity 5 s before and 5 s after presentation of HFD pellet during three test trials. gi, Average calcium activity 5 s before and 5 s after taking a bite from the HFD pellet. Bar graphs are means ± s.e.m. Test trial 1, n = 5 mice, 247 neurons; trial 2, n = 5 mice, 244 neurons; trial 3, n = 5 mice, 254 neurons. Βaseline was 60-s period before HFD pellet was presented. ***P < 0.001; di, paired Student’s t-test (two-tailed). Related to Fig. 4. For statistical analysis, see Supplementary Information.

Source data

Extended Data Figure 5 CGRPPBN neurons are activated during exposure to a novel marble.

ac, Change in calcium activity in response to a marble being placed in home cage. Line graphs are means ± s.e.m. (shaded region) for raw ΔF/F values and plots show normalized ΔF/F values for neurons (rows) of all experimental mice. df, Average calcium activity 5 s before and 10 s after placing a marble in the cage. Graphs are means ± s.e.m. Test trial 1, n = 4 mice, 220 neurons; trial 2, n = 4 mice, 218 neurons; trial 3, n = 4 mice, 222 neurons. Βaseline was 60-s period before marble was presented. ***P < 0.001; df, paired Student’s t-test (two-tailed). Related to Fig. 4. See also Supplementary Video 4. For statistical analysis, see Supplementary Information.

Source data

Extended Data Figure 6 Additional calcium imaging analysis from fear recall studies.

a, b, Average calcium fluorescence activity during 10-s tone presentations in fear-conditioned (a) and sham-conditioned (b) mice during tone trials 1, 15, and 30. Relative fluorescence was calculated from a 5-s baseline period before tone presentation. Graphs are mean ± s.e.m.; fear-conditioned mice, n = 4 mice, 214 neurons; sham-conditioned mice, n = 4 mice, 169 neurons. c, Contextual fear conditioning paradigm in which mice were placed in a shock chamber before (Pre 1 and Pre 2) and after (Post 1 and Post 2) footshock conditioning. d, Calcium activity of individual neurons (horizontal lines are means) during a 5-min exposure to the shock chamber before and after fear conditioning (Pre 1, n = 3 mice, 120 neurons; Pre 2, n = 3 mice, 125 neurons; Post 1, n = 3 mice, 124 neurons; Post 2, n = 3 mice, 121 neurons). Relative fluorescence was normalized to a 2-min baseline recording period. e, Calcium activity of individual neurons (horizontal lines are means) during freezing and non-freezing behaviour after conditioning. f, Percentage of time spent freezing during exposure to the shock chamber after conditioning. Data points represent individual mice (n = 3). g, Representative individual neuron traces showing calcium activity while in home cage versus shock chamber after contextual fear conditioning. The dotted line denotes when the shock chamber recording began. The shaded regions annotate when the mouse was freezing. Dissimilar letters above columns indicate statistical differences between trials. ***P < 0.001; a, d, e, One-way repeated measures ANOVA, Tukey’s post-hoc. Related to Fig. 5. For statistical analysis, see Supplementary Information.

Source data

Extended Data Figure 7 Lens placement over CGRPPBN neurons.

Representative images of lens placement for imaging CGRP neurons (green). Dashed lines are approximations of lens placement. Scale bar is 100 μm.

Supplementary information

Life Sciences Reporting Summary (PDF 72 kb)

Supplementary Information

This file contains the detailed statistical analysis. (PDF 130 kb)

CGRPPBN neurons in anesthetized mice are activated by tail pinch

This is an unprocessed video file from calcium imaging. (MP4 5527 kb)

CGRPPBN neurons are inhibited before food consumption

Calcium recording was processed using CNMF, and the fluorescence of each neuron was normalized to its own maximum fluorescence intensity in the video. (MP4 7901 kb)

CGRPPBN neurons are active during exploration of novel, palatable food

Calcium recording was processed using CNMF, and the fluorescence of each neuron was normalized to its own maximum fluorescence intensity in the video. Video is 2x speed. (MP4 11980 kb)

CGRPPBN neurons are active during exploration of novel marble

Calcium recording was processed using CNMF, and the fluorescence of each neuron was normalized to its own maximum fluorescence intensity in the video. Video is 2x speed. (MP4 25197 kb)

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Campos, C., Bowen, A., Roman, C. et al. Encoding of danger by parabrachial CGRP neurons. Nature 555, 617–622 (2018). https://doi.org/10.1038/nature25511

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