The peptidergic control circuit for sighing

Journal name:
Nature
Volume:
530,
Pages:
293–297
Date published:
DOI:
doi:10.1038/nature16964
Received
Accepted
Published online

Abstract

Sighs are long, deep breaths expressing sadness, relief or exhaustion. Sighs also occur spontaneously every few minutes to reinflate alveoli, and sighing increases under hypoxia, stress, and certain psychiatric conditions. Here we use molecular, genetic, and pharmacologic approaches to identify a peptidergic sigh control circuit in murine brain. Small neural subpopulations in a key breathing control centre, the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), express bombesin-like neuropeptide genes neuromedin B (Nmb) or gastrin-releasing peptide (Grp). These project to the preBötzinger Complex (preBötC), the respiratory rhythm generator, which expresses NMB and GRP receptors in overlapping subsets of ~200 neurons. Introducing either neuropeptide into preBötC or onto preBötC slices, induced sighing or in vitro sigh activity, whereas elimination or inhibition of either receptor reduced basal sighing, and inhibition of both abolished it. Ablating receptor-expressing neurons eliminated basal and hypoxia-induced sighing, but left breathing otherwise intact initially. We propose that these overlapping peptidergic pathways comprise the core of a sigh control circuit that integrates physiological and perhaps emotional input to transform normal breaths into sighs.

At a glance

Figures

  1. NMB neuropeptide pathway neurons in breathing centre.
    Figure 1: NMB neuropeptide pathway neurons in breathing centre.

    a, P0 mouse brain section probed for Nmb mRNA (green) with DAPI counterstain (nuclei, blue). Scale bar, 1 mm. b, Boxed region (a) showing specific expression in RTN/pFRG. Scale bar, 100 μm. c, Whole mount P0 brainstem (ventral view) showing Nmb-GFP transgene expression (GFP, green) bilaterally in RTN/pFRG. Scale bar, 0.5 mm. d, e, Three-dimensional reconstruction (sagittal (d), coronal (e) projections) of CLARITY-cleared P14 Nmb-GFP brainstem. Note, RTN/pFRG expression ventral, dorsal, and lateral to facial nucleus. Numbers, representative neurons. A, anterior; V, ventral; M, medial. Scale bar, 100 μm. f, P0 Nmb-GFP-expressing neurons (green) in RTN/pFRG (dashed) co-express RTN marker PHOX2B (red). Scale bar, 50 μm. g, P7 Nmb-GFP-expressing neurons (green) project to preBötC (dashed). SST (somatostatin), preBötC marker (white). Asterisk indicates isolated GFP-labelled neuron in facial nucleus. Scale bar, 100 μm. h, Boxed region (g) with NMB co-stain (z-stack projection; optical sections, Extended Data Fig. 2). Arrowhead, NMB puncta (red) in Nmb-GFP-expressing projection (green) abutting preBötC neuron (SST, white). Scale bars, 10 μm (1 μm, inset). i, P7 ventral medulla section probed for Nmbr mRNA (purple) showing preBötC expression. Scale bar, 100 μm. j, Tiled image (left) and tracing (right) of Nmb-GFP neuron as in g projecting to preBötC. Scale bar, 30 μm.

  2. NMB effect on breathing.
    Figure 2: NMB effect on breathing.

    ac, Breathing activity of anaesthetized rat following bilateral NMB injection (100 nl, 3 μM) into preBötC. Note increased sighing (spikes in tidal volume (VT), integrated diaphragm activity (∫Dia)), but little change in respiratory rate (frequency, f). Bar, 1 min. b, c, Similar, stereotyped waveforms of spontaneous (b) and NMB-induced (c) sighs (from a; also Extended Data Fig. 3a, c–f). Bar, 2 s. d, Quantification of (a). Top: raster plots of sighs (tics) in five rats following NMB injection (grey); numbers, highest instantaneous sigh rate (red tics). Bottom: instantaneous sigh rate of bottom raster plot; numbers, average instantaneous sigh rate before and maximum (and fold increase) after injection. e, Integrated hypoglossal nerve (∫XII; black) and preBötC neural activity (∫preBötC; grey) in preBötC slices containing indicated NMB concentrations. NMB increases doublets (*), a sigh signature in slices. Bar, 10 s. f, Quantification of (e) (data as mean ± s.d. n = 7; *P < 0.05 by paired t-test). g, Basal sigh rate in C57BL/6 wild-type (WT) and Nmbr−/− mice. n = 4; data as mean ± s.d.; *P < 0.001 by unpaired t-test. h, Effect on sighing in anaesthetized rats of bilateral preBötC injection (grey) of NMBR antagonist BIM23042 (100 nl, 6 μM). Top: raster plots; numbers, longest inter-sigh intervals (s, seconds) following injection. Bottom: sliding average sigh rate (bin 4 min; slide 30 s); numbers, average rate before (left) and minimum binned rate after injection (right).

  3. GRP neuropeptide pathway expression and function in breathing.
    Figure 3: GRP neuropeptide pathway expression and function in breathing.

    a, b, Sagittal ventral medulla sections of P7 mice probed for Grp (a) or Grpr (b) mRNA (purple). Scale bar, 200 μm. c, Effect on sighing of bilateral preBötC injection of GRP (100 nl, 3 μM), as in Fig. 2d. d, Effect of GRP on doublets (sighs) in preBötC slices, as in Fig. 2f. Data as mean ± s.d. n = 9; *P < 0.05 by paired t-test. e, Basal sigh rate in C57BL/6 wild-type (WT) and Grpr−/− mice. n = 4; data as mean ± s.d.; *P < 0.001 by Mann–Whitney U-test. f, Effect on sighing of bilateral preBötC injection of GRPR antagonist RC3095 (100 nl, 6 μM), as in Fig. 2h.

  4. Interactions between NMB and GRP pathways in sighing.
    Figure 4: Interactions between NMB and GRP pathways in sighing.

    ad, RTN/pFRG section of P7 Nmb-GFP mouse immunostained for GFP (green, arrowheads) and probed for Grp mRNA (red, arrows). Note no expression overlap. Scale bar, 30 μm. eh, preBötC section of P28 mouse probed for Nmbr mRNA (green, arrowheads) and Grpr mRNA (red, arrows). Note partial expression overlap. Scale bar, 30 μm. i, Effect on sighing of bilateral preBötC injection of both NMB (100 nl, 3 μM) and GRP (100 nl, 3 μM) as in Fig. 2d. j, Effect on sighing of bilateral preBötC injection (100 nl, 6 μM) of both NMBR and GRPR antagonists (BIM23042, RC3095) as in Fig. 2h.

  5. Effect on sighing of ablating preBötC NMBR-expressing and GRPR-expressing neurons.
    Figure 5: Effect on sighing of ablating preBötC NMBR-expressing and GRPR-expressing neurons.

    a, b, Basal (a) and hypoxia-induced (b) sigh rates before (control) and 3 or 5 days after preBötC injections of bombesin–saporin (200 nl, 6.2 ng; BBN–SAP ablation) to ablate NMBR- and GRPR-expressing neurons, or 5 days after saporin alone (200 nl, 6.2 ng; Blank–SAP). Data as mean ± s.d.; P < 0.05 (BBN–SAP ablation vs control rats at day 3 or 5 for both room air (a) and hypoxia (b)). Sample size n = 6 (control), 3 (Blank–SAP), 7 (day 3), 6 (day 5). c, Model of peptidergic sigh control circuit. NMB- and GRP-expressing neurons in RTN/pFRG (and perhaps GRP-expressing neurons in NTS and PBN) receive physiological and perhaps emotional input from other brain regions, stimulating neuropeptide secretion. This activates receptor-expressing preBötC neurons expressing their receptors, which transform the normal preBötC rhythm to sighs. (Because neuropeptides induce sighs separated by normal breaths (Fig. 2a), there must be some refractory mechanism in or downstream of receptor-expressing neurons that temporarily prevents a second sigh.)

  6. Expression of Nmb in rodent brain.
    Extended Data Fig. 1: Expression of Nmb in rodent brain.

    a, b, Sagittal sections of P7 mouse (a) and P7 rat (b) brain showing RTN/pFRG region probed for Nmb mRNA expression (purple) by in situ hybridization as in Fig. 1. Scale bars, 100 μm. c, d, Nmb expression as in a showing regions outside ventrolateral medulla. Nmb is expressed in mouse olfactory bulb (c) and hippocampus (d). Scale bars, 200 μm (c) and 100 μm (d). e–h, Section through RTN/pFRG brain region of P0 transgenic Nmb-GFP mouse immunostained for GFP (green) and probed for Nmb mRNA (red) by in situ hybridization. Blue, DAPI nuclear stain. Nmb-GFP and Nmb mRNA are mainly co-expressed in the same cells. Scale bar, 100 μm.

  7. Serial confocal preBötC sections showing Nmb-GFP projections contain puncta of NMB.
    Extended Data Fig. 2: Serial confocal preBötC sections showing Nmb-GFP projections contain puncta of NMB.

    a–d, Serial confocal optical sections (0.6 μm apart) through preBötC brain region of Nmb-GFP mouse immunostained for GFP (green), NMB (red), preBötC marker SST (white), and DAPI (blue) as in Fig. 2h. Note the GFP-positive projection with a puncta of NMB (yellow, open arrows in b, c) directly abutting an SST positive neuron (asterisk). Most NMB puncta (open arrowheads) were detected within GFP-positive projections as expected, and only a small fraction of NMB puncta (closed arrowhead) were detected outside them; NMB outside Nmb-GFP projections could be secreted protein or the rare Nmb-expressing cells that do not co-express the Nmb-GFP transgene (see Extended Data Fig. 1e–h). Scale bar, 20 μm.

  8. Sighing after surgery and bilateral injection of saline into preBötC.
    Extended Data Fig. 3: Sighing after surgery and bilateral injection of saline into preBötC.

    a, Example of a sigh in a breathing activity trace of a urethane anaesthetized rat after surgery as in Fig. 2a–c. VT, tidal volume; ∫Dia, integrated diaphragm activity; Dia, raw diaphragm activity trace. b, Sigh rate before (control) and after (saline) bilateral saline injection into preBötC. There is no effect of saline injection (data are mean ± s.d., n = 5, P = 0.83 by paired t-test). cf, Breathing activity trace as in a (but also showing airflow). Note stereotyped waveform of sighs (df). Bars, 1 min (c), 1 s (df).

  9. Effects on sighing in individual rats following bilateral injection into preBötC of NMB, GRP and both NMB/GRP.
    Extended Data Fig. 4: Effects on sighing in individual rats following bilateral injection into preBötC of NMB, GRP and both NMB/GRP.

    ae, Raster plot of sighs (upper) and instantaneous sigh rates (lower) before and after NMB injection for the five experiments (ae) shown in Fig. 2d. fj, Raster plot of sighs (upper) and instantaneous sigh rates (lower) before and after GRP injection for the five experiments (fj) shown in Fig. 3c. ko, Raster plot of sighs (upper) and instantaneous sigh rates (lower) before and after NMB/GRP injection for the five experiments (ko) shown in Fig. 4i. Grey, injection period; arrowhead in raster plots, maximum instantaneous sigh rate; numbers, basal (left) and maximal instantaneous sigh rate (right) and fold induction (in parentheses) after neuropeptide injection.

  10. Effect of NMB and GRP on rhythmic activity of preBötC slice.
    Extended Data Fig. 5: Effect of NMB and GRP on rhythmic activity of preBötC slice.

    a, Neuronal activity trace(∫XII, black; ∫preBötC population activity, grey) of preBötC slice containing 30 nM NMB, as in Fig. 2e. Note the extreme effect of NMB in which every burst (‘breath’) in the trace is a doublet (‘sigh’, asterisk). Bar, 5 s. b, c, NMB increases the doublet rate by increasing the fraction of total events that are doublets (b) and decreasing the interval following a doublet (c). Data are mean ± s.d., *P < 0.05 by paired t-test, n = 7. d, e, GRP also increases the doublet rate by increasing the fraction of total events that are doublets (d) and decreasing the interval following a doublet (e). Data are mean ± s.d., *P < 0.05 by paired t-test, n = 9. Note that post-doublet intervals are significantly longer than post-burst intervals under all conditions, consistent with longer post-sigh apneas in vivo.

  11. Effects on sighing in individual rats following bilateral injection of BIM23042, RC3095 and BIM23042/RC3095 into preBötC.
    Extended Data Fig. 6: Effects on sighing in individual rats following bilateral injection of BIM23042, RC3095 and BIM23042/RC3095 into preBötC.

    ad, Raster plot of sighs (upper) and binned sigh rates (lower; bin size 4 min; slide 30 s) before and after injection of the NMBR antagonist BIM23042 for the four experiments shown in Fig. 2h. eh, Raster plot of sighs (upper) and binned sigh rates (lower; bin size 4 min; slide 30 s) before and after injection of the GRPR antagonist RC3095 for the four experiments shown in Fig. 3f. in, Raster plot of sighs (upper) and binned sigh rates (lower; bin size 4 min; slide 30 s) before and after BIM23042 and RC3095 injection for the six experiments shown in Fig. 4j. Grey, injection period; numbers, longest inter-sigh intervals (s, seconds) following injection.

  12. Specificity of antagonists BIM23042 and RC3095 in preBötC slice.
    Extended Data Fig. 7: Specificity of antagonists BIM23042 and RC3095 in preBötC slice.

    a, BIM 23042 (100 nM) blocks the effect of NMB (10 nM), but not GRP (3 nM) in preBötC slices. Data are mean ± s.d., *P < 0.05 by paired t-test, n = 7. b, RC3095 (100 nM) shows the opposite specificity, blocking the effect of GRP (3 nM), but not NMB (10 nM). Data are mean ± s.d., *P < 0.05 by paired t-test, n = 9.

  13. Expression of Grp in rodent brain.
    Extended Data Fig. 8: Expression of Grp in rodent brain.

    a, b, In situ hybridization of mouse brain slices as in Fig. 3a showing expression of Grp (purple) in parabrachial nucleus (PBN) (a) and nucleus tractus solitarius (NTS) (b). Scale bar, 200 μm. ce, In situ hybridization of rat brain slices showing expression of Grp in PBN (c), NTS (d), RTN/pFRG (e). Scale bar, 200 μm. f, Tiled image showing GRP-positive projection (red) from RTN/pFRG region to preBötC region containing SST-positive neuron (green). Scale bar, 20 μm. gi, Serial confocal optical sections (0.8 μm apart) through mouse preBötC stained for GRP (red) and SST (green) focusing on short segment of GRP-positive projection where a GRP puncta (red) directly abuts (arrowhead) an SST-positive neuron. Scale bar, 10 μm.

  14. Effect of bombesin injection on sighing following BBN–SAP-induced ablation of NMBR-expressing and GRPR-expressing preBötC neurons.
    Extended Data Fig. 9: Effect of bombesin injection on sighing following BBN–SAP-induced ablation of NMBR-expressing and GRPR-expressing preBötC neurons.

    a, b, 10 min plethysmography traces of a control rat (a) and a day 5 BBN–SAP injected rat (b) during eupneic breathing (left). Indicated parts (10 s) of traces are expanded at right. Note presence of sighs with stereotyped waveform in control rat, and no sighs detectable in BBN–SAP injected rat. c, Sigh rate before (control) and after 10 μg bombesin injection (BBN) into the cisterna magna of rats before BBN–SAP injection (WT) and at day 4 and day 6 after BBN–SAP injection (BBN–SAP) into the preBötC to ablate NMBR-expressing and GRPR-expressing neurons as in Fig. 5a, b. Values shown are mean ± s.d. (WT, n = 10; BBN–SAP, n = 7 for day 4 and n = 5 for day 6), *P < 0.05 by paired t-test; n.s., not significant.

Videos

  1. Video 1: 3-D view of Nmb-GFP neurons in RTN/pFRG region of CLARITY-processed P14 brainstem
    Video 1: Video 1: 3-D view of Nmb-GFP neurons in RTN/pFRG region of CLARITY-processed P14 brainstem
    Nmb-GFP neurons surround the lateral half of the facial nucleus, as shown in Figure 1d, e.

References

  1. Haldane, J. S., Meakins, J. C. & Priestley, J. G. The effects of shallow breathing. J. Physiol. (Lond.) 52, 433453 (1919)
  2. McCutcheon, F. H. Atmospheric respiration and the complex cycles in mammalian breathing mechanisms. J. Cell. Physiol. 41, 291303 (1953)
  3. Knowlton, G. C. & Larrabee, M. G. A unitary analysis of pulmonary volume receptors. Am. J. Physiol. 147, 100114 (1946)
  4. Reynolds, L. B. Characteristics of an inspiration-augmenting reflex in anesthetized cats. J. Appl. Physiol. 17, 683688 (1962)
  5. Bartlett, D. Origin and regulation of spontaneous deep breaths. Respir. Physiol. 12, 230238 (1971)
  6. Maytum, C. K. Sighing dyspnea: A clinical syndrome. J. Allergy 10, 5055 (1938)
  7. Smith, J. C., Ellenberger, H. H., Ballanyi, K., Richter, D. W. & Feldman, J. L. Pre-Bötzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254, 726729 (1991)
  8. Gray, P. A., Janczewski, W. A., Mellen, N., McCrimmon, D. R. & Feldman, J. L. Normal breathing requires preBötzinger complex neurokinin-1 receptor-expressing neurons. Nature Neurosci. 4, 927930 (2001)
  9. Tan, W. et al. Silencing preBötzinger complex somatostatin-expressing neurons induces persistent apnea in awake rat. Nature Neurosci. 11, 538540 (2008)
  10. Feldman, J. L., Del Negro, C. A. & Gray, P. A. Understanding the rhythm of breathing: so near, yet so far. Annu. Rev. Physiol. 75, 423452 (2013)
  11. Kam, K., Worrell, J. W., Janczewski, W. A., Cui, Y. & Feldman, J. L. Distinct inspiratory rhythm and pattern generating mechanisms in the preBötzinger complex. J. Neurosci. 33, 92359245 (2013)
  12. Lieske, S. P., Thoby-Brisson, M., Telgkamp, P. & Ramirez, J. M. Reconfiguration of the neural network controlling multiple breathing patterns: eupnea, sighs and gasps. Nature Neurosci. 3, 600607 (2000)
  13. Ruangkittisakul, A. et al. Generation of eupnea and sighs by a spatiochemically organized inspiratory network. J. Neurosci. 28, 24472458 (2008)
  14. Caughey, J. L. Jr. Analysis of breathing patterns. Am. Rev. Tuberc. 48, 382 (1943)
  15. Niewoehner, D. E., Levine, A. S. & Morley, J. E. Central effects of neuropeptides on ventilation in the rat. Peptides 4, 277281 (1983)
  16. Ramirez, J. M. The integrative role of the sigh in psychology, physiology, pathology, and neurobiology. Prog. Brain Res. 209, 91129 (2014)
  17. Janczewski, W. A., Pagliardini, S., Cui, Y. & Feldman, J. L. Sighing after vagotomy. Proc. Society of Neuroscience Conference (New Orleans, 2012)
  18. Smith, J. C., Morrison, D. E., Ellenberger, H. H., Otto, M. R. & Feldman, J. L. Brainstem projections to the major respiratory neuron populations in the medulla of the cat. J. Comp. Neurol. 281, 6996 (1989)
  19. Onimaru, H. & Homma, I. A novel functional neuron group for respiratory rhythm generation in the ventral medulla. J. Neurosci. 23, 14781486 (2003)
  20. Diez-Roux, G. et al. A high resolution atlas of the transcriptome in the mouse embryo. PLoS Biol. 9, e1000582 (2011)
  21. Tomer, R. et al. Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nature Protocols 9, 16821697 (2014)
  22. Mulkey, D. K. et al. Respiratory control by ventral surface chemoreceptor neurons in rats. Nature Neurosci. 7, 13601369 (2004)
  23. Stornetta, R. L. et al. Expression of Phox2b by brainstem neurons involved in chemosensory integration in the adult rat. J. Neurosci. 26, 1030510314 (2006)
  24. Guyenet, P. G. & Bayliss, D. A. Neural control of breathing and CO2 homeostasis. Neuron 87, 946961 (2015)
  25. Lazarenko, R. M. et al. Acid sensitivity and ultrastructure of the retrotrapezoid nucleus in Phox2b–EGFP transgenic mice. J. Comp. Neurol. 517, 6986 (2009)
  26. Bendixen, H. H., Smith, G. M. & Mead, J. Pattern of ventilation in young adults. J. Appl. Phyiol . 19, 195198 (1964)
  27. Jensen, R. T. et al., International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states. Pharmacol. Rev. 60, 142 (2008)
  28. Guyenet, P. G., Stornetta, R. L. & Bayliss, D. A. Central respiratory chemoreception. J. Comp. Neurol. 518, 38833906 (2010)
  29. Lee, H., Naughton, N. N., Woods, J. H. & Ko, M. C. Characterization of scratching responses in rats following centrally administered morphine or bombesin. Behav. Pharmacol. 14, 501508 (2003)
  30. Pagliardini, S. et al. Active expiration induced by excitation of ventral medulla in adult anesthetized rats. J. Neurosci. 31, 28952905 (2011)
  31. Ohki-Hamazaki, H. et al. Functional properties of two bombesin-like peptide receptors revealed by the analysis of mice lacking neuromedin B receptor. J. Neurosci. 19, 948954 (1999)
  32. Wada, E. et al. Generation and characterization of mice lacking gastrin-releasing peptide receptor. Biochem. Biophys. Res. Commun. 239, 2833 (1997)
  33. Orbuch, M. et al. Discovery of a novel class of neuromedin B receptor antagonists, substituted somatostatin analogues. Mol. Pharmacol. 44, 841850 (1993)
  34. Qin, Y. et al. Inhibitory effect of bombesin receptor antagonist RC-3095 on the growth of human pancreatic cancer cells in vivo and in vitro. Cancer Res. 54, 10351041 (1994)

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Author information

  1. These authors contributed equally to this work.

    • Peng Li,
    • Wiktor A. Janczewski &
    • Kevin Yackle
  2. Present addresses: Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064, USA (K.K.); Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2E1, Canada (S.P.).

    • Kaiwen Kam &
    • Silvia Pagliardini
  3. These authors jointly supervised this project.

    • Mark A. Krasnow &
    • Jack L. Feldman

Affiliations

  1. Department of Biochemistry and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305, USA

    • Peng Li,
    • Kevin Yackle &
    • Mark A. Krasnow
  2. Systems Neurobiology Laboratory, Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90095, USA

    • Wiktor A. Janczewski,
    • Kaiwen Kam,
    • Silvia Pagliardini &
    • Jack L. Feldman

Contributions

W.A.J. and S.P. performed experiments showing the effects on sighing of bombesin injection into the preBötC and ablation of receptor-expressing neurons with bombesin–saporin. K.Y. performed the screen that discovered Nmb expression in the respiratory centres. P.L. performed experiments identifying and characterizing expression of Nmb, Grp and their receptors. W.A.J., P.L., and K.Y. performed genetic and pharmacology experiments on Nmb and Grp pathways. K.K. performed in vitro slice experiments. W.A.J., K.K., P.L., S.P., and K.Y. analysed data. J.L.F., W.A.J., K.K., M.A.K., P.L., and K.Y. conceived experiments, interpreted data and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Expression of Nmb in rodent brain. (1,141 KB)

    a, b, Sagittal sections of P7 mouse (a) and P7 rat (b) brain showing RTN/pFRG region probed for Nmb mRNA expression (purple) by in situ hybridization as in Fig. 1. Scale bars, 100 μm. c, d, Nmb expression as in a showing regions outside ventrolateral medulla. Nmb is expressed in mouse olfactory bulb (c) and hippocampus (d). Scale bars, 200 μm (c) and 100 μm (d). e–h, Section through RTN/pFRG brain region of P0 transgenic Nmb-GFP mouse immunostained for GFP (green) and probed for Nmb mRNA (red) by in situ hybridization. Blue, DAPI nuclear stain. Nmb-GFP and Nmb mRNA are mainly co-expressed in the same cells. Scale bar, 100 μm.

  2. Extended Data Figure 2: Serial confocal preBötC sections showing Nmb-GFP projections contain puncta of NMB. (1,100 KB)

    a–d, Serial confocal optical sections (0.6 μm apart) through preBötC brain region of Nmb-GFP mouse immunostained for GFP (green), NMB (red), preBötC marker SST (white), and DAPI (blue) as in Fig. 2h. Note the GFP-positive projection with a puncta of NMB (yellow, open arrows in b, c) directly abutting an SST positive neuron (asterisk). Most NMB puncta (open arrowheads) were detected within GFP-positive projections as expected, and only a small fraction of NMB puncta (closed arrowhead) were detected outside them; NMB outside Nmb-GFP projections could be secreted protein or the rare Nmb-expressing cells that do not co-express the Nmb-GFP transgene (see Extended Data Fig. 1e–h). Scale bar, 20 μm.

  3. Extended Data Figure 3: Sighing after surgery and bilateral injection of saline into preBötC. (98 KB)

    a, Example of a sigh in a breathing activity trace of a urethane anaesthetized rat after surgery as in Fig. 2a–c. VT, tidal volume; ∫Dia, integrated diaphragm activity; Dia, raw diaphragm activity trace. b, Sigh rate before (control) and after (saline) bilateral saline injection into preBötC. There is no effect of saline injection (data are mean ± s.d., n = 5, P = 0.83 by paired t-test). cf, Breathing activity trace as in a (but also showing airflow). Note stereotyped waveform of sighs (df). Bars, 1 min (c), 1 s (df).

  4. Extended Data Figure 4: Effects on sighing in individual rats following bilateral injection into preBötC of NMB, GRP and both NMB/GRP. (162 KB)

    ae, Raster plot of sighs (upper) and instantaneous sigh rates (lower) before and after NMB injection for the five experiments (ae) shown in Fig. 2d. fj, Raster plot of sighs (upper) and instantaneous sigh rates (lower) before and after GRP injection for the five experiments (fj) shown in Fig. 3c. ko, Raster plot of sighs (upper) and instantaneous sigh rates (lower) before and after NMB/GRP injection for the five experiments (ko) shown in Fig. 4i. Grey, injection period; arrowhead in raster plots, maximum instantaneous sigh rate; numbers, basal (left) and maximal instantaneous sigh rate (right) and fold induction (in parentheses) after neuropeptide injection.

  5. Extended Data Figure 5: Effect of NMB and GRP on rhythmic activity of preBötC slice. (199 KB)

    a, Neuronal activity trace(∫XII, black; ∫preBötC population activity, grey) of preBötC slice containing 30 nM NMB, as in Fig. 2e. Note the extreme effect of NMB in which every burst (‘breath’) in the trace is a doublet (‘sigh’, asterisk). Bar, 5 s. b, c, NMB increases the doublet rate by increasing the fraction of total events that are doublets (b) and decreasing the interval following a doublet (c). Data are mean ± s.d., *P < 0.05 by paired t-test, n = 7. d, e, GRP also increases the doublet rate by increasing the fraction of total events that are doublets (d) and decreasing the interval following a doublet (e). Data are mean ± s.d., *P < 0.05 by paired t-test, n = 9. Note that post-doublet intervals are significantly longer than post-burst intervals under all conditions, consistent with longer post-sigh apneas in vivo.

  6. Extended Data Figure 6: Effects on sighing in individual rats following bilateral injection of BIM23042, RC3095 and BIM23042/RC3095 into preBötC. (223 KB)

    ad, Raster plot of sighs (upper) and binned sigh rates (lower; bin size 4 min; slide 30 s) before and after injection of the NMBR antagonist BIM23042 for the four experiments shown in Fig. 2h. eh, Raster plot of sighs (upper) and binned sigh rates (lower; bin size 4 min; slide 30 s) before and after injection of the GRPR antagonist RC3095 for the four experiments shown in Fig. 3f. in, Raster plot of sighs (upper) and binned sigh rates (lower; bin size 4 min; slide 30 s) before and after BIM23042 and RC3095 injection for the six experiments shown in Fig. 4j. Grey, injection period; numbers, longest inter-sigh intervals (s, seconds) following injection.

  7. Extended Data Figure 7: Specificity of antagonists BIM23042 and RC3095 in preBötC slice. (163 KB)

    a, BIM 23042 (100 nM) blocks the effect of NMB (10 nM), but not GRP (3 nM) in preBötC slices. Data are mean ± s.d., *P < 0.05 by paired t-test, n = 7. b, RC3095 (100 nM) shows the opposite specificity, blocking the effect of GRP (3 nM), but not NMB (10 nM). Data are mean ± s.d., *P < 0.05 by paired t-test, n = 9.

  8. Extended Data Figure 8: Expression of Grp in rodent brain. (942 KB)

    a, b, In situ hybridization of mouse brain slices as in Fig. 3a showing expression of Grp (purple) in parabrachial nucleus (PBN) (a) and nucleus tractus solitarius (NTS) (b). Scale bar, 200 μm. ce, In situ hybridization of rat brain slices showing expression of Grp in PBN (c), NTS (d), RTN/pFRG (e). Scale bar, 200 μm. f, Tiled image showing GRP-positive projection (red) from RTN/pFRG region to preBötC region containing SST-positive neuron (green). Scale bar, 20 μm. gi, Serial confocal optical sections (0.8 μm apart) through mouse preBötC stained for GRP (red) and SST (green) focusing on short segment of GRP-positive projection where a GRP puncta (red) directly abuts (arrowhead) an SST-positive neuron. Scale bar, 10 μm.

  9. Extended Data Figure 9: Effect of bombesin injection on sighing following BBN–SAP-induced ablation of NMBR-expressing and GRPR-expressing preBötC neurons. (79 KB)

    a, b, 10 min plethysmography traces of a control rat (a) and a day 5 BBN–SAP injected rat (b) during eupneic breathing (left). Indicated parts (10 s) of traces are expanded at right. Note presence of sighs with stereotyped waveform in control rat, and no sighs detectable in BBN–SAP injected rat. c, Sigh rate before (control) and after 10 μg bombesin injection (BBN) into the cisterna magna of rats before BBN–SAP injection (WT) and at day 4 and day 6 after BBN–SAP injection (BBN–SAP) into the preBötC to ablate NMBR-expressing and GRPR-expressing neurons as in Fig. 5a, b. Values shown are mean ± s.d. (WT, n = 10; BBN–SAP, n = 7 for day 4 and n = 5 for day 6), *P < 0.05 by paired t-test; n.s., not significant.

Supplementary information

Video

  1. Video 1: Video 1: 3-D view of Nmb-GFP neurons in RTN/pFRG region of CLARITY-processed P14 brainstem (2.62 MB, Download)
    Nmb-GFP neurons surround the lateral half of the facial nucleus, as shown in Figure 1d, e.

Additional data