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Neuronal pacemaker for breathing visualized in vitro

Abstract

Breathing movements in mammals arise from a rhythmic pattern of neural activity, thought to originate in the pre-Bötzinger complex1 in the lower brainstem. The mechanisms generating the neural rhythm in this region are unknown2,3,4,5. The central question is whether the rhythm is generated by a network of bursting pacemaker neurons coupled by excitatory synapses that synchronize pacemaker activity. Here we visualized the activity of inspiratory pacemaker neurons at single-cell and population levels with calcium-sensitive dye. We developed methods to label these neurons retrogradely with the dye in neonatal rodent brainstem slices that retain the rhythmically active respiratory network. We simultaneously used infrared structural imaging to allow patch-clamp recording from the identified neurons. After we pharmacologically blocked glutamatergic synaptic transmission, a subpopulation of inspiratory neurons continued to burst rhythmically but asynchronously. The intrinsic bursting frequency of these pacemaker neurons depended on the baseline membrane potential, providing a cellular mechanism for respiratory frequency control. These results provide evidence that the neuronal kernel for rhythm generation consists of a network of synaptically-coupled pacemaker neurons.

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Figure 1: CaG injection site and localized Ca2+ activity in pre-Bötzinger complex.
Figure 2: Functional and structural imaging of inspiratory neuron with intrinsic bursting properties.
Figure 3: Intrinsic Ca2+ activity of inspiratory neuron after blockade of non-NMDA glutamatergic synaptic transmission.
Figure 4: Ca2+ activity and voltage-dependent pacemaker properties of inspiratory neuron.
Figure 5: Desynchronization of Ca2+ activities of pacemaker neurons after blockade of glutamatergic synaptic transmission.

References

  1. 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, 726–729 (1991).

    ADS  CAS  Article  Google Scholar 

  2. Richter, D. W., Ballanyi, K. & Schwarzacher, S. Mechanisms of respiratory rhythm generation. Curr. Opin. Neurobiol. 2, 788–793 (1992).

    CAS  Article  Google Scholar 

  3. Ramirez, J. M. & Richter, D. W. The neuronal mechanisms of respiratory rhythm generation. Curr. Opin. Neurobiol. 6, 817–825 (1996).

    CAS  Article  Google Scholar 

  4. Smith, J. C. in Neurons, Networks, and Motor Behavior (eds Stein, P., Grillner, S., Selverston, A. & Stuart, D.) 97–104 (MIT Press, Cambridge, Massachusetts, 1997).

    Google Scholar 

  5. Rekling, J. C. & Feldman, J. L. PreBötzinger complex and pacemaker neurons: hypothesized site and kernel for respiratory rhythm generation. Annu. Rev. Physiol. 60, 385–405 (1998).

    CAS  Article  Google Scholar 

  6. Smith, J. C., Funk, G. D., Johnson, S. M. & Feldman, J. L. in Ventral Brainstem Mechanisms and Control of Respiration and Blood Pressure (eds Trouth, C. O., Millis, R., Kiwull-Schone, H. & Schlafke, M.) 463–496 (Dekker, New York, 1995).

    Google Scholar 

  7. Koshiya, N. & Guyenet, P. G. Tonic sympathetic chemoreflex after blockade of respiratory rhythmogenesis in the rat. J. Physiol. (Lond.) 491, 859–869 (1996).

    CAS  Article  Google Scholar 

  8. Ramirez, J. M., Schwarzacher, S. W., Pierrefiche, O., Olivera, B. M. & Richter, D. W. Selective lesioning of the cat pre-Bötzinger complex in vivo eliminates breathing but not gasping. J. Physiol. (Lond.) 507, 895–907 (1998).

    CAS  Article  Google Scholar 

  9. Funk, G. D., Smith, J. C. & Feldman, J. L. Generation and transmission of respiratory oscillations in medullary slices: role of excitatory amino acids. J. Neurophysiol. 70, 1497–1515 (1993).

    CAS  Article  Google Scholar 

  10. Ramirez, J. M., Quellmalz, U. J. A. & Richter, D. W. Postnatal changes in the mammalian respiratory network as revealed by the transverse brainstem slice of mice. J. Physiol. (Lond.) 491, 799–812 (1996).

    CAS  Article  Google Scholar 

  11. Johnson, S. M., Smith, J. C., Funk, G. D. & Feldman, J. L. Pacemaker behavior of respiratory neurons in medullary slices from neonatal rat. J. Neurophysiol. 72, 2598–2608 (1994).

    CAS  Article  Google Scholar 

  12. Koshiya, N. & Smith, J. C. Real-time functional and structural imaging of rhythmically active respiratory neurons with calcium-sensitive dyes and IR-DIC in medullary slices in vitro. Soc. Neurosci. Abstr. 23, 1252 (1997).

    Google Scholar 

  13. Smith, J. C., Greer, J. J., Liu, G. & Feldman, J. L. Neural mechanisms generating respiratory pattern in mammalian brain stem-spinal cord in vitro. I. Spatiotemporal patterns of motor and medullary neuron activity. J. Neurophysiol. 64, 1149–1169 (1990).

    CAS  Article  Google Scholar 

  14. Schwarzacher, S. W., Smith, J. C. & Richter, D. W. Pre-Bötzinger complex in the cat. J. Neurophysiol. 73, 1452–1461 (1995).

    CAS  Article  Google Scholar 

  15. Friel, D. D. & Tsien, R. W. Phase-dependent contributions from Ca2+ entry and Ca2+ release to caffeine-induced [Ca2+]ioscillations in bullfrog sympathetic neurons. Neuron 8, 1109–1125 (1992).

    CAS  Article  Google Scholar 

  16. Li, Y. X., Keizer, J., Stojilkovic, S. S. & Rinzel, J. Ca2+ excitability of the ER membrane: an explanation for IP3-induced Ca2+ oscillations. Am. J. Physiol. 269, C1079–1092 (1995).

    CAS  Article  Google Scholar 

  17. Wilson, C. G., Koshiya, N. & Smith, J. C. Real-time functional imaging of hypoglossal pre-motor and motor neurons in thin medullary slices in vitro. Soc. Neurosci. Abstr. 23, 875 (1998).

    Google Scholar 

  18. Butera, R. J., Rinzel, J. & Smith, J. C. Models of respiratory rhythm generation in the pre-Bötzinger complex: I. Bursting pacemaker neurons. J. Neurophysiol. 82, 382–397 (1999).

    Article  Google Scholar 

  19. Butera, R. J., Rinzel, J. & Smith, J. C. Models of respiratory rhythm generation in the pre-Bötzinger complex: II. Populations of coupled pacemaker neurons. J. Neurophysiol. 82, 398–415 (1999).

    Article  Google Scholar 

  20. Bianchi, A. L., Denavit-Saubie, M. & Champagnat, J. Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters. Physiol. Rev. 75, 1–45 (1995).

    CAS  Article  Google Scholar 

  21. O'Donovan, M., Ho, S. & Yee, W. Calcium imaging of rhythmic network activity in the developing spinal cord of the chick embryo. J. Neurosci. 11, 6354–6369 (1994).

    Article  Google Scholar 

  22. McClellan, A. D., McPherson, D. & O'Donovan, M. J. Combined retrograde labeling and calcium imaging in spinal cord and brainstem neurons of the lamprey. Brain Res. 663, 61–68 (1994).

    CAS  Article  Google Scholar 

  23. Fetcho, J. R. & O'Malley, D. M. Visualization of active neural circuitry in the spinal cord of intact zebrafish. J. Neurophysiol. 73, 399–406 (1995).

    CAS  Article  Google Scholar 

  24. Regehr, W. G. & Atluri, P. P. Calcium transients in cerebellar granule cell presynaptic terminals. Biophys. J. 68, 2156–2170 (1995).

    ADS  CAS  Article  Google Scholar 

  25. Sabatini, B. L. & Regehr, W. G. Timing of neurotransmission at fast synapses in the mammalian brain. Nature 384, 170–172 (1996).

    ADS  CAS  Article  Google Scholar 

  26. Inoué, S. & Spring, K. R. Video Microscopy: the Fundamentals 2nd edn (Plenum New York, 1997).

    Book  Google Scholar 

  27. Torrence, C. & Compo, G. P. Apractical guide to wavelet analysis. Bull. Amer. Meteor. Soc. 79, 61–78 (1998).

    Article  Google Scholar 

  28. Dodt, H. U. & Zieglgänsberger, W. Infrared videomicroscopy: a new look at neuronal structure and function. Trends Neurosci. 17, 453–458 (1994).

    CAS  Article  Google Scholar 

  29. Foskett, J. K. Simultaneous Nomarski and fluorescence imaging during video microscopy of cells. Am. J. Physiol. (suppl.) C566–C571 (1988).

  30. Tsubokawa, H. & Ross, W. N. IPSPs modulate spike backpropagation and associated [Ca2+]ichanges in the dendrites of hippocampal CA1 pyramidal neurons. J. Neurophysiol. 76, 2896–2906 (1996).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank M. J. O'Donovan and K. R. Spring for advice on optical recording; W.S.Rasband for NIH Image programs; G. P. Compo for the CWT denoising algorithm; and R. E. Burke and A. Lev-Tov for critical comments on the manuscript.

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Correspondence to Naohiro Koshiya.

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Koshiya, N., Smith, J. Neuronal pacemaker for breathing visualized in vitro. Nature 400, 360–363 (1999). https://doi.org/10.1038/22540

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