Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Optogenetic perturbation of preBötzinger complex inhibitory neurons modulates respiratory pattern

Nature Neuroscience volume 18, pages 408–414 (2015)Cite this article

Subjects

Abstract

Inhibitory neurons make up a substantial fraction of the neurons in the preBötzinger complex (preBötC), a site that is critical for mammalian eupneic breathing. We investigated the role of glycinergic preBötC neurons in respiratory rhythmogenesis in mice using optogenetically targeted excitation and inhibition. Channelrhodopsin-2 (ChR2) or Archaerhodopsin (Arch) were expressed in glycinergic preBötC neurons of glycine transporter 2 (Glyt2, also known as Slc6a5)-Cre mice. In ChR2-transfected mice, brief inspiratory-phase bilateral photostimulation targeting the preBötC prematurely terminated inspiration, whereas expiratory-phase photostimulation delayed the onset of the next inspiration. Prolonged photostimulation produced apneas lasting as long as the light pulse. Inspiratory-phase photoinhibition in Arch-transfected mice during inspiration increased tidal volume without altering inspiratory duration, whereas expiratory-phase photoinhibition shortened the latency until the next inspiration. During persistent apneas, prolonged photoinhibition restored rhythmic breathing. We conclude that glycinergic preBötC neurons modulate inspiratory pattern and are important for reflex apneas, but that the rhythm can persist after substantial dampening of their activity.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Cre-dependent ChR2 or Arch expression targeted to preBötC Glyt2 neurons.
Figure 2: Photostimulation of preBötC Glyt2 neurons depresses breathing.
Figure 3: Prolonged photostimulation of preBötC Glyt2 neurons results in apnea.
Figure 4: Photoinhibition of preBötC Glyt2 neurons augments breathing.
Figure 5: Photoinhibition of preBötC Glyt2 neurons during a reflex apnea rescues breathing.
Figure 6: Unit recording with concurrent ChR2 or Arch activation.

Similar content being viewed by others

References

  1. Tan, W. et al. Silencing preBotzinger complex somatostatin-expressing neurons induces persistent apnea in awake rat. Nat. Neurosci. 11, 538–540 (2008).

    Article  CAS  Google Scholar 

  2. Smith, J.C., Ellenberger, H.H., Ballanyi, K., Richter, D.W. & Feldman, J.L. Pre-Botzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254, 726–729 (1991).

    Article  CAS  Google Scholar 

  3. Winter, S.M. et al. Glycinergic interneurons are functionally integrated into the inspiratory network of mouse medullary slices. Pflugers Arch. 458, 459–469 (2009).

    Article  CAS  Google Scholar 

  4. Stornetta, R.L. et al. A group of glutamatergic interneurons expressing high levels of both neurokinin-1 receptors and somatostatin identifies the region of the pre-Botzinger complex. J. Comp. Neurol. 455, 499–512 (2003).

    Article  CAS  Google Scholar 

  5. Bouvier, J. et al. Hindbrain interneurons and axon guidance signaling critical for breathing. Nat. Neurosci. 13, 1066–1074 (2010).

    Article  CAS  Google Scholar 

  6. Gray, P.A. et al. Developmental origin of preBotzinger complex respiratory neurons. J. Neurosci. 30, 14883–14895 (2010).

    Article  CAS  Google Scholar 

  7. Koizumi, H. et al. Structural-functional properties of identified excitatory and inhibitory interneurons within pre-Botzinger complex respiratory microcircuits. J. Neurosci. 33, 2994–3009 (2013).

    Article  CAS  Google Scholar 

  8. Richter, D.W. & Smith, J.C. Respiratory rhythm generation in vivo. Physiology (Bethesda) 29, 58–71 (2014).

    CAS  PubMed Central  Google Scholar 

  9. Ballantyne, D. & Richter, D.W. Post-synaptic inhibition of bulbar inspiratory neurones in the cat. J. Physiol. (Lond.) 348, 67–87 (1984).

    Article  CAS  Google Scholar 

  10. Janczewski, W.A., Tashima, A., Hsu, P., Cui, Y. & Feldman, J.L. Role of inhibition in respiratory pattern generation. J. Neurosci. 33, 5454–5465 (2013).

    Article  CAS  Google Scholar 

  11. Morgado-Valle, C., Baca, S.M. & Feldman, J.L. Glycinergic pacemaker neurons in preBotzinger complex of neonatal mouse. J. Neurosci. 30, 3634–3639 (2010).

    Article  CAS  Google Scholar 

  12. Deisseroth, K. et al. Next-generation optical technologies for illuminating genetically targeted brain circuits. J. Neurosci. 26, 10380–10386 (2006).

    Article  CAS  Google Scholar 

  13. Chow, B.Y. et al. High-performance genetically targetable optical neural silencing by light-driven proton pumps. Nature 463, 98–102 (2010).

    Article  CAS  Google Scholar 

  14. Lin, J.Y., Lin, M.Z., Steinbach, P. & Tsien, R.Y. Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys. J. 96, 1803–1814 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Chalphin, A.V. & Saha, M.S. The specification of glycinergic neurons and the role of glycinergic transmission in development. Front. Mol. Neurosci. 3, 11 (2010).

    PubMed  PubMed Central  Google Scholar 

  16. Cardin, J.A. et al. Driving fast-spiking cells induces gamma rhythm and controls sensory responses. Nature 459, 663–667 (2009).

    Article  CAS  Google Scholar 

  17. Smith, J.C., Abdala, A.P., Koizumi, H., Rybak, I.A. & Paton, J.F. Spatial and functional architecture of the mammalian brain stem respiratory network: a hierarchy of three oscillatory mechanisms. J. Neurophysiol. 98, 3370–3387 (2007).

    Article  CAS  Google Scholar 

  18. Shao, X.M. & Feldman, J.L. Respiratory rhythm generation and synaptic inhibition of expiratory neurons in pre-Botzinger complex: differential roles of glycinergic and GABAergic neural transmission. J. Neurophysiol. 77, 1853–1860 (1997).

    Article  CAS  Google Scholar 

  19. Schreihofer, A.M., Stornetta, R.L. & Guyenet, P.G. Evidence for glycinergic respiratory neurons: Botzinger neurons express mRNA for glycinergic transporter 2. J. Comp. Neurol. 407, 583–597 (1999).

    Article  CAS  Google Scholar 

  20. Jiang, C. & Lipski, J. Extensive monosynaptic inhibition of ventral respiratory group neurons by augmenting neurons in the Botzinger complex in the cat. Exp. Brain Res. 81, 639–648 (1990).

    Article  CAS  Google Scholar 

  21. Ezure, K., Tanaka, I. & Kondo, M. Glycine is used as a transmitter by decrementing expiratory neurons of the ventrolateral medulla in the rat. J. Neurosci. 23, 8941–8948 (2003).

    Article  CAS  Google Scholar 

  22. Alheid, G.F. & McCrimmon, D.R. The chemical neuroanatomy of breathing. Respir. Physiol. Neurobiol. 164, 3–11 (2008).

    Article  CAS  Google Scholar 

  23. Dobbins, E.G. & Feldman, J.L. Brainstem network controlling descending drive to phrenic motoneurons in rat. J. Comp. Neurol. 347, 64–86 (1994).

    Article  CAS  Google Scholar 

  24. Dobbins, E.G. & Feldman, J.L. Differential innervation of protruder and retractor muscles of the tongue in rat. J. Comp. Neurol. 357, 376–394 (1995).

    Article  CAS  Google Scholar 

  25. Kam, K., Worrell, J.W., Ventalon, C., Emiliani, V. & Feldman, J.L. Emergence of population bursts from simultaneous activation of small subsets of preBötzinger complex inspiratory neurons. J. Neurosci. 33, 3332–3338 (2013).

    Article  CAS  Google Scholar 

  26. Burns, B.D. The central control of respiratory movements. Br. Med. Bull. 19, 7–9 (1963).

    Article  CAS  Google Scholar 

  27. Bradley, G.W., von Euler, C., Marttila, I. & Roos, B. A model of the central and reflex inhibition of inspiration in the cat. Biol. Cybern. 19, 105–116 (1975).

    Article  CAS  Google Scholar 

  28. von Euler, C. On the central pattern generator for the basic breathing rhythmicity. J. Appl. Physiol. 55, 1647–1659 (1983).

    Article  CAS  Google Scholar 

  29. Feldman, J.L. & Cowan, J.D. Large-scale activity in neural nets II: a model for the brainstem respiratory oscillator. Biol. Cybern. 17, 39–51 (1975).

    Article  CAS  Google Scholar 

  30. Richter, D.W. Generation and maintenance of the respiratory rhythm. J. Exp. Biol. 100, 93–107 (1982).

    CAS  PubMed  Google Scholar 

  31. Schmid, K., Foutz, A.S. & Denavit-Saubie, M. Inhibitions mediated by glycine and GABAA receptors shape the discharge pattern of bulbar respiratory neurons. Brain Res. 710, 150–160 (1996).

    Article  CAS  Google Scholar 

  32. Xia, L., Damon, T., Niblock, M.M., Bartlett, D. & Leiter, J.C. Unilateral microdialysis of gabazine in the dorsal medulla reverses thermal prolongation of the laryngeal chemoreflex in decerebrate piglets. J. Appl. Physiol. 103, 1864–1872 (2007).

    Article  CAS  Google Scholar 

  33. Curran, A.K., Xia, L., Leiter, J.C. & Bartlett, D. Jr. Elevated body temperature enhances the laryngeal chemoreflex in decerebrate piglets. J. Appl. Physiol. 98, 780–786 (2005).

    Article  CAS  Google Scholar 

  34. Heman-Ackah, Y.D., Pernell, K.J. & Goding, G.S. The laryngeal chemoreflex: an evaluation of the normoxic response. Laryngoscope 119, 370–379 (2009).

    Article  Google Scholar 

  35. Adamantidis, A.R., Zhang, F., Aravanis, A.M., Deisseroth, K. & de Lecea, L. Neural substrates of awakening probed with optogenetic control of hypocretin neurons. Nature 450, 420–424 (2007).

    Article  CAS  Google Scholar 

  36. Gradinaru, V. et al. Targeting and readout strategies for fast optical neural control in vitro and in vivo. J. Neurosci. 27, 14231–14238 (2007).

    Article  CAS  Google Scholar 

  37. Paxinos, G., Franklin, K.B.J. & Franklin, K.B.J. The Mouse Brain in Stereotaxic Coordinates (Academic Press, 2001).

  38. Pagliardini, S. et al. Active expiration induced by excitation of ventral medulla in adult anesthetized rats. J. Neurosci. 31, 2895–2905 (2011).

    Article  CAS  Google Scholar 

  39. Poyatos, I., Ponce, J., Aragon, C., Gimenez, C. & Zafra, F. The glycine transporter GLYT2 is a reliable marker for glycine-immunoreactive neurons. Brain Res. Mol. Brain Res. 49, 63–70 (1997).

    Article  CAS  Google Scholar 

  40. Zeilhofer, H.U. et al. Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J. Comp. Neurol. 482, 123–141 (2005).

    Article  CAS  Google Scholar 

  41. McKay, L.C., Janczewski, W.A. & Feldman, J.L. Sleep-disordered breathing after targeted ablation of preBotzinger complex neurons. Nat. Neurosci. 8, 1142–1144 (2005).

    Article  CAS  Google Scholar 

  42. Lewis, J., Bachoo, M., Polosa, C. & Glass, L. The effects of superior laryngeal nerve stimulation on the respiratory rhythm: phase-resetting and aftereffects. Brain Res. 517, 44–50 (1990).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank G. Li for excellent technical work and N. Brecha, T. Otis and K. Kam for thoughtful discussion. This work was supported by US National Institutes of Health grants NS72211 and NS58280.

Author information

Authors and Affiliations

  1. Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, California, USA

    David Sherman, Jason W Worrell, Yan Cui & Jack L Feldman

  2. Department of Physiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, Sichuan, People's Republic of China.,

    Yan Cui

Authors
  1. David Sherman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jason W Worrell
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yan Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jack L Feldman
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.S. and J.L.F. conceived of the study and designed the experiments. D.S. performed the experiments with help from J.W.W. and Y.C., and D.S. and J.W.W. analyzed the data. D.S. and J.L.F. made the figures and wrote the manuscript with help from J.W.W.

Corresponding author

Correspondence to Jack L Feldman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sherman, D., Worrell, J., Cui, Y. et al. Optogenetic perturbation of preBötzinger complex inhibitory neurons modulates respiratory pattern. Nat Neurosci 18, 408–414 (2015). https://doi.org/10.1038/nn.3938

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.3938

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing