Abstract
Breathing must be tightly coordinated with other behaviours such as vocalization, swallowing, and coughing. These behaviours occur after inspiration, during a respiratory phase termed postinspiration1. Failure to coordinate postinspiration with inspiration can result in aspiration pneumonia, the leading cause of death in Alzheimer’s disease, Parkinson’s disease, dementia, and other neurodegenerative diseases2. Here we describe an excitatory network that generates the neuronal correlate of postinspiratory activity in mice. Glutamatergic–cholinergic neurons form the basis of this network, and GABA (γ-aminobutyric acid)-mediated inhibition establishes the timing and coordination relative to inspiration. We refer to this network as the postinspiratory complex (PiCo). The PiCo has autonomous rhythm-generating properties and is necessary and sufficient for postinspiratory activity in vivo. The PiCo also shows distinct responses to neuromodulators when compared to other excitatory brainstem networks. On the basis of the discovery of the PiCo, we propose that each of the three phases of breathing is generated by a distinct excitatory network: the preBötzinger complex, which has been linked to inspiration3,4; the PiCo, as described here for the neuronal control of postinspiration; and the lateral parafacial region (pFL), which has been associated with active expiration, a respiratory phase that is recruited during high metabolic demand4,5.
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Acknowledgements
Supported by grants from the National Institute of Health NS087828-01 (to T.M.A.), HL090554 (to J.-M.R.), and HL126523-01 (to J.-M.R.). We thank P. Gray for insights provided throughout the preparation of this manuscript, T. Dashevskiy for creating the heat map of postinspiratory activity, F. Bedogni for obtaining in situ hybridization reagents, and K. Cuthill for cryosectioning. J.-M.R. also thanks D. W. Richter and S. W. Schwarzacher for the inspiration to study postinspiration.
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Nature thanks J. Bouvier, O. Kiehn and H. Zoghbi for their contribution to the peer review of this work.
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T.M.A, A.J.G., and J.-M.R. designed all experiments. T.M.A., A.J.G., N.A.B, J.P., J.C.B., A.D.W., and K.G.R. performed the experiments. T.M.A., A.J.G., N.A.B, and J.P. analysed the data. T.M.A., A.J.G., N.A.B, J.P., and J.-M.R. contributed to manuscript preparation. T.M.A. and J.-M.R wrote the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Schematic of the horizontal slice from a sagittal view that retains the medullary ventral respiratory column in the brainstem.
Dotted lines represent approximate boundaries of the horizontal slice. Slice retains part of the superior olive (SO), and the entire retrotrapezoidal nucleus/para-facial respiratory group (RTN/pFRG), facial nucleus (VII N), Bötzinger complex (BötC), postinspiratory complex (PiCo), nucleus ambiguus (NA), preBötzinger complex (preBötC), lateral reticular nucleus (LRT), and the rostral and caudal ventral respiratory groups (rVRG and cVRG, respectively). The slice also retains a portion of the spinal cord and includes part of the phrenic motor nucleus (approximately cervical segments 3 and 4). The slice does not contain the dorsal portion of the medulla including the dorsal respiratory group (DRG). Dorsal (D), ventral (V), rostral (R), caudal (C).
Extended Data Figure 2 Norepinephrine dose response of preBötC and PiCo rhythms in horizontal and transverse slices.
The frequency of the PiCo rhythm (black) is highly sensitive to the application of low concentrations of norepinephrine while the preBötC rhythm (purple) stays relatively constant in both types of slice preparations. a, In horizontal slices the PiCo rhythm is slow under spontaneous conditions (n = 10), the two rhythms have similar burst frequencies in the presence of 2 μM norepinephrine (n = 6), and the PiCo rhythm significantly outpaces the preBötC rhythm under higher concentrations of norepinephrine (n = 4, 3–4 μM norepinephrine). b, Similarly, when isolated in transverse slices, the PiCo rhythm has a slow frequency under spontaneous conditions (n = 10), and the preBötC and PiCo have similar frequencies at 2 μM norepinephrine (n = 7; mean ± s.e.m.). Two-way ANOVA followed by a Bonferroni post-hoc test. ****P < 0.0001 comparing PiCo to preBötC, ϕP < 0.05 compared to baseline (Spon.), #P < 0.05 compared to 2 μM norepinephrine.
Extended Data Figure 3 Nucleus ambiguus neurons lack Vglut2–cre expression.
High magnification view at the level of PiCo from a Vglut2-cre;Ai6 (ZsGreen1; green) mouse immunolabelled with ChAT antibody (magenta) and Cre antibody (white). Note lack of green Vglut2–cre expression in the nucleus ambiguus. Scale bar, 100 μm.
Extended Data Figure 4 Progressive synaptic blockade in horizontal and transverse slices.
Left, graphs comparing frequency and normalized burst area between horizontal (n = 5) and paired transverse slices (n = 5) after the application of strychnine and gabazine. In both horizontal and paired transverse slices, PiCo and preBötC rhythms have nearly identical burst frequencies in the presence of gabazine (top). The burst area of both rhythms also significantly increases with the application of gabazine in both slice preparations (bottom). Two-way ANOVA followed by a Bonferroni post-hoc test. ϕP < 0.05 compared to baseline (2 μM norepinephrine). Right, synaptic blockers were progressively perfused over paired transverse slices at 10-min intervals. Both PiCo and preBötC rhythms persist in the presence of 1 μM strychnine, 10 μM gabazine, and 10 μM CPP. Population rhythms ceased in the presence of 20 μM CNQX, indicating that both rhythms are excitatory (n = 5). The asterisk denotes a characteristic sigh in the preBötC trace.
Extended Data Figure 5 Peri-event interval between preBötC and PiCo bursts during inhibitory block in the horizontal slice.
a, Peri-event interval, time between peak of preBötC and PiCo bursts, is constant in strychnine; however, gabazine initiates progressive synchronization between rhythms (shown here in a representative experiment). b, Average peri-event intervals at baseline and after sequential application of strychnine and gabazine (n = 6, mean ± s.e.m.). Repeated measures Friedman test followed by Dunn’s multiple comparisons post-hoc test. *P < 0.05.
Extended Data Figure 6 Blocking muscarinic and nicotinic acetylcholine receptors does not abolish the PiCo rhythm.
a, Raw population bursts from PiCo and contralateral preBötC with the progressive addition of 1 μM mecamylamine (nicotinic receptor antagonist), 10 μM atropine (muscarinic receptor antagonist), and 4 μM norepinephrine. b, The left two graphs show n = 5 experiments in which atropine was applied first, and the right graphs illustrate n = 3 experiments in which mecamylamine was applied first. Blockade of muscarinic receptors results in a larger decrease in PiCo burst frequency than blocking nicotinic receptors, while preBötC frequency does not change significantly (top graphs). Blockade of muscarinic receptors increases the amplitude of PiCo bursts (bottom graphs). The PiCo rhythm persists after concurrent blockade of both types of acetylcholine receptors, and PiCo burst frequency rebounds to near baseline levels when an additional 2 μM norepinephrine is applied (total 4 μM norepinephrine; top graphs; mean ± s.e.m.). Two-way ANOVA followed by a Bonferroni post-hoc test. **P < 0.01, *P < 0.05 comparing preBötC to PiCo, ϕP < 0.05 compared to baseline (2 μM norepinephrine).
Extended Data Figure 7 Synaptically isolated PiCo neurons decrease firing frequency in the presence of DAMGO.
a, Top traces show intracellular recordings from PiCo cells with concurrent extracellular preBötC population activity from a horizontal slice under 1 μM norepinephrine baseline conditions. Bottom traces show the same recordings after blocking fast synaptic transmission (1 μM strychnine, 10 μM gabazine, 10 μM CPP, 20 μM CNQX) to synaptically isolate PiCo neurons. Application of 10 nM DAMGO decreases the cell’s intrinsic firing frequency. b, Quantified data show that DAMGO significantly decreases action potential (AP) firing frequency of synaptically isolated PiCo neurons in both horizontal slices (black dots) and transverse PiCo slices (grey dots) (two-tailed paired t-test, *P < 0.05; n = 5).
Extended Data Figure 8 Differential PiCo and preBötC population responses to DAMGO and SST in horizontal and transverse slices.
a, After the application of 25 nM DAMGO, preBötC burst frequency decreases only slightly (n = 5), whereas PiCo bursting is nearly eliminated. b, Similar to results observed in horizontal slices, the PiCo rhythm is eliminated by 25 nM DAMGO in transverse slices that isolate PiCo and preBötC in the presence of 2 μM norepinephrine (n = 5). Periodic large amplitude bursts in the bottom preBötC trace are fictive sighs. c, DAMGO dose response of normalized preBötC and PiCo burst frequency in transverse slices, illustrating the differential sensitivity of the PiCo and preBötC rhythms to DAMGO; burst frequency values are normalized to baseline frequency in 2 μM norepinephrine (mean ± s.e.m., n = 8 with minimum replicates of 4 for each location and concentration). d, The PiCo rhythm is selectively and transiently inhibited by the application of 500 nM SST whereas the preBötC rhythm persists in horizontal slices. Graph shows normalized average burst frequencies of both rhythms at baseline, 1.5–3.5 min after SST application, and 8–10 min after SST application (n = 6). e, Similar to the horizontal slice, SST application results in a robust inhibition of PiCo bursting in paired transverse slices. f, Similar to d, complied normalized burst frequencies for n = 6 transverse slices before and after SST application. (mean ± s.e.m.) Two-way ANOVA followed by a Bonferroni post-hoc test. ****P < 0.0001 comparing PiCo to preBötC, ϕP < 0.05 compared to baseline.
Extended Data Figure 9 Light stimulation of cholinergic cells evokes postinspiratory activity in horizontal slices and in vivo.
a, Two population electrodes were placed at the level of PiCo (black dot and trace) and contralateral preBötC (purple dot and trace) in a horizontal slice from a Chat-cre;Ai27 mouse. Under spontaneous conditions (no norepinephrine), cholinergic neurons expressing channelrhodopsin-2 were light activated with an optical fibre (labelled ‘light’) placed over the PiCo ipsilateral to the preBötC electrode. PiCo population bursts were triggered upon the onset of a 1.5-s light pulse whereas no bursts were light evoked in the preBötC (n = 6). Figure shows 10 traces overlaid for each electrode with averaged traces below from a representative experiment. b, Photo-stimulating PiCo in adult anaesthetized Chat-cre;Ai27 mice reliably triggers cVN bursts. Figure shows 10 traces overlaid with averages below of cVN and XII activity during a 200-ms light stimulation of PiCo. c, Postinspiratory bursts can be photo-evoked both in vivo (n = 6) and in vitro (n = 6) at any phase except for during inspiration and just before inspiration (bottom left) owing to the inspiratory phase delay that occurs when PiCo is stimulated (mean ± s.e.m., bottom right).
Extended Data Figure 10 Elimination of phase delay by DAMGO and diversity of postinspiratory waveforms in vivo.
a, Injection of 5 μM DAMGO into the PiCo eliminates the phase delay elicited by photostimulation of PiCo in Chat-cre;Ai27 mice. A representative experiment showing cVN and XII recordings during a 200-ms light pulse before and after injection of PiCo with DAMGO (left; grey bars, expected phase; purple bars, inspiratory phase delay) and the average inspiratory phase delay (right) (mean ± s.e.m., two-tailed paired t-test, **P < 0.01; n = 6). b, Diverse postinspiratory vagal waveforms were recorded in vivo. Five examples of cVN (black) and XII (purple) recordings (overlaid) show that postinspiratory activity can vary from large decrementing patterns to small short bursts, potentially representing the neural basis of a variety of postinspiratory behaviours.
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Anderson, T., Garcia, A., Baertsch, N. et al. A novel excitatory network for the control of breathing. Nature 536, 76–80 (2016). https://doi.org/10.1038/nature18944
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DOI: https://doi.org/10.1038/nature18944
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