The peptidergic control circuit for sighing

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.

Extended Data Figure 1. Expression of neuromedin b (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 Figure 1. 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). 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 largely co-expressed in same cells. Bar, 100 μm.

Extended Data Figure 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 Figure 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 Figure 1e-h). Bar, 20 μm.

Extended Data Figure 3. Sighing after surgery and bilateral injection of saline into preBötC

a, Example of a sigh in a breathing activity trace of a urethane-anesthetized rat after surgery as in Fig. 2a-c. V_T, 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 (n=5, p=0.83). c-f, Breathing activity trace as in a (but also showing airflow). Note stereotyped waveform of sighs (d-f). Bars, 1.min (c), 1 second (d-f).

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

a-e, Raster plot of sighs (upper) and instantaneous sigh rates (lower) before and after NMB injection for the five experiments (a-e) shown in Figure 2d. f-j, Raster plot of sighs (upper) and instantaneous sigh rates (lower) before and after GRP injection for the five experiments (f-j) shown in Figure 3c. k-o, Raster plot of sighs (upper) and instantaneous sigh rates (lower) before and after NMB/GRP injection for the five experiments (k-o) shown in Figure 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.

Extended Data Figure 5. Effect of NMB 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 Figure 2e. Note the extreme effect of NMB in which every burst (“breath”) in the trace is a doublet (“sigh”, *). 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). *, p<0.05, 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). *, p<0.05, 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.

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

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

Extended Data Figure 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. *, p<0.05, n=7. b, RC3095 (100 nM) shows the opposite specificity, blocking the effect of GRP (3 nM), but not NMB (10 nM) . *, p<0.05, n=9.

Extended Data Figure 8. Expression of Grp in rodent brain

a,b, In situ hybridization of mouse brain slices as in Figure 3a showing expression of Grp (purple) in parabrachial nucleus (PBN) (a) and nucleus tractus solitarius (NTS) (b). Bar, 200 μm. c-e, In situ hybridization of rat brain slices showing expression of Grp in PBN (c), NTS (d), RTN/pFRG (e). Bar, 200 μm. f, Tiled image showing GRP-positive projection (red) from RTN/pFRG region to preBötC region containing SST-positive neuron (green). Bar, 20 μm. g-i, 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. Bar, 10 μm.

Extended Data Figure 9. Effect of bombesin injection on sighing following bombesin-saporin (BBN-SAP)-induced ablation of NMBR 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 secs) 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 preBötC of rats prior to BBN-SAP injection (WT) and at day 4 and day 6 after BBN-SAP injection (BBN-SAP) into the preBötC to ablate NMBR and GRPR-expressing neurons as in Figure 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; n.s., not significant.

Figure 1. NMB neuropeptide pathway neurons in breathing center

a, P0 mouse brain section probed for Nmb mRNA (green) with DAPI counterstain (nuclei, blue). Bar, 1mm. b, Boxed region (a) showing specific expression in RTN/pFRG. Bar, 100μm. c, Whole mount P0 brainstem (ventral view) showing Nmb-GFP transgene expression (GFP, green) bilaterally in RTN/pFRG. Bar, 0.5mm. d,e, 3D 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. Bar, 100μm. f, P0 Nmb-GFP-expressing neurons (green) in RTN/pFRG (dashed) co-express RTN marker PHOX2B (red). Bar, 50μm. g, P7 Nmb-GFP-expressing neurons (green) project to preBötC (dashed). SST (somatostatin), preBötC marker (white). *, isolated GFP-labeled neuron in facial nucleus. Bar, 100μm. h, Boxed region (g) with NMB co-stain (z-stack projection; optical sections, ED Fig. 2). Arrowhead, NMB puncta (red) in Nmb-GFP-expressing projection (green) abutting preBötC neuron (SST, white). Bars, 10μm (1μm, inset). i, P7 ventral medulla section probed for Nmbr mRNA (purple) showing preBötC expression. Bar, 100μm. j, Tiled image (left) and tracing (right) of Nmb-GFP neuron as in (g) projecting to preBötC. Bar, 30μm.

Figure 2. NMB effect on breathing

a-c, Breathing activity of anesthetized rat following bilateral NMB injection (100nl, 3μM) into preBötC. Note increased sighing (spikes in tidal volume (V_T), 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 ED Fig. 3a,c-f). Bar, 2s. 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, 10s. f, Quantification of (e) (n=7; *, p<0.05). g, Basal sigh rate in C57BL/6 wild-type (WT) and Nmbr^−/− mice. n=4; bars, standard deviation of mean; *, p<0.001. h, Effect on sighing in anesthetized rats of bilateral preBötC injection (grey) of NMBR antagonist BIM23042 (100nl, 6μM). Top: raster plots; numbers, longest intersigh intervals (s, seconds) following injection. Bottom: sliding average sigh rate (bin 4 min; slide 30s); numbers, average rate before (left) and minimum binned rate after injection (right).

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). Bar, 200μm. c, Effect on sighing of bilateral preBötC injection of GRP (100nl, 3μM), as in Fig. 2d. d, Effect of GRP on doublets (sighs) in preBötC slices, as in Fig. 2f. n=9; *, p<0.05. e, Basal sigh rate in C57BL/6 wild-type (WT) and Grpr^−/− mice, as in Fig. 2g. f, Effect on sighing of bilateral preBötC injection of GRPR antagonist RC3095 (100nl, 6μM), as in Fig. 2h.

Figure 4. Interactions between NMB and GRP pathways in sighing

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

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 (200nl, 6.2ng; BBN-SAP ablation) to ablate NMBR and GRPR expressing neurons, or 5 days after saporin alone (200nl, 6.2ng; Blank-SAP). 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 (Figure 2A), there must be some refractory mechanism in or downstream of receptor-expressing neurons that temporarily prevents a second sigh.)