Skip to main content
SpringerLink
Log in

The Sagittally Sectioned Rat Hindbrain Preparation: Improved Access to the Brainstem Respiratory Network

  • Protocol
  • First Online:
Multidisciplinary Tools for Investigating Synaptic Plasticity

Part of the book series: Neuromethods ((NM,volume 81))

Abstract

Neurons that control breathing are distributed along the ventral respiratory column (VRC) in the ventrolateral brain stem, extending from the pons to the spinomedullary junction. These networks can be isolated in the en bloc brainstem-spinal cord preparation, which remains spontaneously active for hours under physiological conditions, but which only permits blind single-unit recordings from respiratory network constituents. The transverse slice preparation isolates the minimal neural substrate sufficient for respiratory rhythmogenesis, but does not permit the study of neuronal signaling along the column. Here, the sagittally sectioned rat hindbrain preparation is described. This preparation exposes the VRC transected along its long axis, at its surface, permitting optical recording of network activity using membrane-permeant Ca2+ indicators, as well as single-unit recording under visual control using differential interference contrast microscopy to patch onto cells identified using Ca2+ indicators. Because the VRC does not run parallel with the midline or the dorsal brainstem surface, exposing respiratory networks at the preparation’s surface requires that the sagittal surface be cut at the appropriate ventrodorsal (18.6°, relative to the dorsal surface) and rostrocaudal tilt (8.5°, relative to the midline), and mediolateral level. These empirically derived parameters conform with anatomical features of the brainstem: the rostrocaudal tilt aligns with the nucleus ambiguus; the ventrodorsal tilt lies parallel with reticular nuclei striations; the mediolateral level of section abuts a branch of the basilar artery. Detailed methods for isolating the SSRH, stabilizing it in the recording chamber, and recording respiratory networks using a membrane-permeant Ca2+ indicator are provided here.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Flourens M-J-P (1858) Nouveaux details sur le noeud vital. C R Seances Acad Sci III 47:803–807

    Google Scholar 

  2. Lindsey BG et al (1994) Distributed actions and dynamic associations in respiratory-related neuronal assemblies of the ventrolateral medulla and brain stem midline: evidence from spike train analysis. J Neurophysiol 72(4):1830–1851

    PubMed  CAS  Google Scholar 

  3. Solomon IC, Edelman NH, Neubauer JA (1999) Patterns of phrenic motor output evoked by chemical stimulation of neurons located in the pre-Botzinger complex in vivo. J Neurophysiol 81(3):1150–1161

    PubMed  CAS  Google Scholar 

  4. Nuding SC et al (2009) Pontine-ventral respiratory column interactions through raphe circuits detected using multi-array spike train recordings. J Neurophysiol 101(6):2943–2960

    Article  PubMed  Google Scholar 

  5. Smith JC, Feldman JL (1987) In vitro brainstem-spinal cord preparations for study of motor systems for mammalian respiration and locomotion. J Neurosci Methods 21(2–4):321–333

    Article  PubMed  CAS  Google Scholar 

  6. Suzue T (1984) Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat. J Physiol 354:173–183

    PubMed  CAS  Google Scholar 

  7. Smith JC et al (1991) Pre-Botzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254(5032):726–729

    Article  PubMed  CAS  Google Scholar 

  8. Del Negro CA et al (2001) Models of respiratory rhythm generation in the pre-Botzinger complex. III. Experimental tests of model predictions. J Neurophysiol 86(1):59–74

    PubMed  Google Scholar 

  9. Thoby-Brisson M, Ramirez JM (2001) Identification of two types of inspiratory pacemaker neurons in the isolated respiratory neural network of mice. J Neurophysiol 86(1):104–112

    PubMed  CAS  Google Scholar 

  10. Del Negro CA, Morgado-Valle C, Feldman JL (2002) Respiratory rhythm: an emergent network property? Neuron 34(5):821–830

    Article  PubMed  Google Scholar 

  11. Mellen NM et al (2003) Opioid-induced quantal slowing reveals dual networks for respiratory rhythm generation. Neuron 37(5):821–826

    Article  PubMed  CAS  Google Scholar 

  12. Onimaru H, Homma I (2003) A novel functional neuron group for respiratory rhythm generation in the ventral medulla. J Neurosci 23(4):1478–1486

    PubMed  CAS  Google Scholar 

  13. Thoby-Brisson M et al (2009) Genetic identification of an embryonic parafacial oscillator coupling to the preBotzinger complex. Nat Neurosci 12(8):1028–1035

    Article  PubMed  CAS  Google Scholar 

  14. Pagliardini S et al (2011) Active expiration induced by excitation of ventral medulla in adult anesthetized rats. J Neurosci 31(8):2895–2905

    Article  PubMed  CAS  Google Scholar 

  15. Janczewski WA, Feldman JL (2006) Distinct rhythm generators for inspiration and expiration in the juvenile rat. J Physiol 570(Pt 2):407–420

    PubMed  CAS  Google Scholar 

  16. Guyenet PG et al (2005) Retrotrapezoid nucleus: a litmus test for the identification of central chemoreceptors. Exp Physiol 90(3):247–253, discussion 253–7

    Article  PubMed  CAS  Google Scholar 

  17. Guyenet PG, Mulkey DK (2010) Retrotrapezoid nucleus and parafacial respiratory group. Respir Physiol Neurobiol 173(3):244–255

    Article  PubMed  CAS  Google Scholar 

  18. Bouvier J et al (2010) Hindbrain interneurons and axon guidance signaling critical for breathing. Nat Neurosci 13(9):1066–1074

    Article  PubMed  CAS  Google Scholar 

  19. Chatonnet F et al (2003) From hindbrain segmentation to breathing after birth: developmental patterning in rhombomeres 3 and 4. Mol Neurobiol 28(3):277–294

    Article  PubMed  CAS  Google Scholar 

  20. Thoby-Brisson M et al (2005) Emergence of the pre-Botzinger respiratory rhythm generator in the mouse embryo. J Neurosci 25(17):4307–4318

    Article  PubMed  CAS  Google Scholar 

  21. Schwarzacher S et al (1991) The medullary respiratory network in the rat. J Physiol 435:631–644

    PubMed  CAS  Google Scholar 

  22. McCrimmon D et al (2003) Functional compartmentalization of ventrolateral medullary respiratory neurons in vivo. In: IXth Oxford conference on modeling and control of breathing, Paris

    Google Scholar 

  23. Lindsey BG et al (2000) Respiratory neuronal assemblies. Respir Physiol 122(2–3):183–196

    Article  PubMed  CAS  Google Scholar 

  24. Onimaru H, Homma I (1992) Whole cell recordings from respiratory neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats. Pflugers Arch 420:399–406

    Article  PubMed  CAS  Google Scholar 

  25. Smith J, Ballanyi K, Richter D (1992) Whole-cell patch-clamp recordings from respiratory neurons in neonatal rat brainstem in vitro. Neurosci Lett 134(2):153–156

    Article  PubMed  CAS  Google Scholar 

  26. Mellen NM, Feldman JL (2001) Phasic vagal sensory feedback transforms respiratory neuron activity in vitro. J Neurosci 21:7363–7371

    PubMed  CAS  Google Scholar 

  27. Paton JF, Ramirez JM, Richter DW (1994) Functionally intact in vitro preparation generating respiratory activity in neonatal and mature mammals. Pflugers Arch 428(3–4):250–260

    Article  PubMed  CAS  Google Scholar 

  28. Viemari JC et al (2003) Perinatal maturation of the mouse respiratory rhythm-generator: in vivo and in vitro studies. Eur J Neurosci 17(6):1233–1244

    Article  PubMed  Google Scholar 

  29. Abu-Shaweesh JM et al (1999) Changes in respiratory timing induced by hypercapnia in maturing rats. J Appl Physiol 87(2):484–490

    PubMed  CAS  Google Scholar 

  30. Thoby-Brisson M, Ramirez J (2000) Role of inspiratory pacemaker neurons in mediating the hypoxic response of the respiratory network in vitro. J Neurosci 20(15):5858–5866

    PubMed  CAS  Google Scholar 

  31. Ramirez JM et al (1998) The hypoxic response of neurones within the in vitro mammalian respiratory network. J Physiol 507(Pt 2):571–582

    Article  PubMed  CAS  Google Scholar 

  32. Gourine AV et al (2005) Release of ATP in the ventral medulla during hypoxia in rats: role in hypoxic ventilatory response. J Neurosci 25(5):1211–1218

    Article  PubMed  CAS  Google Scholar 

  33. Mellen NM (2008) A vibrating microtome attachment for cutting brain slice preparations at reproducible compound angles relative to the midline. J Neurosci Methods 168(1):113–118

    Article  PubMed  Google Scholar 

  34. Ruangkittisakul A et al (2006) High sensitivity to neuromodulator-activated signaling pathways at physiological [K+] of confocally imaged respiratory center neurons in on-line-calibrated newborn rat brainstem slices. J Neurosci 26(46):11870–11880

    Article  PubMed  CAS  Google Scholar 

  35. Barnes BJ, Tuong CM, Mellen NM (2007) Functional imaging reveals respiratory network activity during hypoxic and opioid challenge in the neonate rat tilted sagittal slab preparation. J Neurophysiol 97(3):2283–2292

    Article  PubMed  Google Scholar 

  36. Kitagawa K et al (1998) Cerebral ischemia after bilateral carotid artery occlusion and intraluminal suture occlusion in mice: evaluation of the patency of the posterior communicating artery. J Cereb Blood Flow Metab 18(5):570–579

    Article  PubMed  CAS  Google Scholar 

  37. Grapengiesser E (1993) Cell photodamage, a potential hazard when measuring cytoplasmic Ca2+ with fura-2. Cell Struct Funct 18(1):13–17

    Article  PubMed  CAS  Google Scholar 

  38. Ranganathan GN, Koester HJ (2010) Optical recording of neuronal spiking activity from unbiased populations of neurons with high spike detection efficiency and high temporal precision. J Neurophysiol 104(3):1812–1824

    Article  PubMed  Google Scholar 

  39. Tsien RY, Ernst LA, Waggoner AS (2006) Fluorophores for confocal microscopy: photophysics and photochemistry. In: Pawley J (ed) Handbook of confocal microscopy. Plenum Press, New York, pp 351–365

    Google Scholar 

  40. Mellen NM, Mishra D (2010) Functional anatomical evidence for respiratory rhythmogenic function of endogenous bursters in rat medulla. J Neurosci 30(25):8383–8392

    Article  PubMed  CAS  Google Scholar 

  41. Escobar AL et al (1997) Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis. Pflugers Arch 434(5):615–631

    Article  PubMed  CAS  Google Scholar 

  42. Yaksi E, Friedrich RW (2006) Reconstruction of firing rate changes across neuronal populations by temporally deconvolved Ca2+ imaging. Nat Methods 3(5):377–383

    Article  PubMed  CAS  Google Scholar 

  43. Mellen NM, Tuong CM (2009) Neuroimage 47(4):1331–1340

    Article  PubMed  CAS  Google Scholar 

  44. Darnall RA (2010) The role of CO(2) and central chemoreception in the control of breathing in the fetus and the neonate. Respir Physiol Neurobiol 173(3):201–212

    Article  PubMed  Google Scholar 

  45. Funke F, Dutschmann M, Muller M (2007) Imaging of respiratory-related population activity with single-cell resolution. Am J Physiol Cell Physiol 292(1):C508–C516

    Article  PubMed  CAS  Google Scholar 

  46. Ruangkittisakul A, Panaitescu B, Ballanyi K (2011) K(+) and Ca(2)(+) dependence of inspiratory-related rhythm in novel “calibrated” mouse brainstem slices. Respir Physiol Neurobiol 175(1):37–48

    Article  PubMed  CAS  Google Scholar 

  47. Garaschuk O, Milos RI, Konnerth A (2006) Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo. Nat Protoc 1(1):380–386

    Article  PubMed  CAS  Google Scholar 

  48. Nagayama S et al (2007) In vivo simultaneous tracing and Ca(2+) imaging of local neuronal circuits. Neuron 53(6):789–803

    Article  PubMed  CAS  Google Scholar 

  49. Prinz AA, Bucher D, Marder E (2004) Similar network activity from disparate circuit parameters. Nat Neurosci 7(12):1345–1352

    Article  PubMed  CAS  Google Scholar 

  50. Marder E, Goaillard JM (2006) Variability, compensation and homeostasis in neuron and network function. Nat Rev Neurosci 7(7):563–574

    Article  PubMed  CAS  Google Scholar 

  51. Marder E, Taylor AL (2011) Multiple models to capture the variability in biological neurons and networks. Nat Neurosci 14(2):133–138

    Article  PubMed  CAS  Google Scholar 

  52. Marder E (2011) Quantification of Behavior Sackler Colloquium: Variability, compensation, and modulation in neurons and circuits. Proc Natl Acad Sci U S A 108(Suppl 3):15542–15548

    Article  PubMed  CAS  Google Scholar 

  53. Grashow R, Brookings T, Marder E (2009) Reliable neuromodulation from circuits with variable underlying structure. Proc Natl Acad Sci U S A 106(28):11742–11746

    Article  PubMed  CAS  Google Scholar 

  54. Goaillard JM et al (2009) Functional consequences of animal-to-animal variation in circuit parameters. Nat Neurosci 12(11):1424–1430

    Article  PubMed  CAS  Google Scholar 

  55. MacLean JN et al (2003) Activity-independent homeostasis in rhythmically active neurons. Neuron 37(1):109–120

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Alberta Heritage Foundation for Medical Research (AHFMR), Canadian Institute for Health Research (CIHR), Canadian Foundation for Innovation (CFI), Alberta Science and Research Authority (ASRA). GDF is an AHFMR Scientist.

Author information

Authors and Affiliations

  1. Kosair Children’s Hospital Research Institute, University of Louisville, Louisville, KY, USA

    Nicholas M. Mellen

  2. Departments of Physiology and Pediatrics, Centre for Neuroscience, Women and Children’s Health Research Institute (WCHRI), University of Alberta, Edmonton, AB, Canada

    Gregory D. Funk

Authors
  1. Nicholas M. Mellen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gregory D. Funk
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nicholas M. Mellen .

Editor information

Editors and Affiliations

  1. School of Medicine, Department of Physiology, University of Alberta, Medical Sciences Building 7-14, Edmonton, T6G 2H7, Alberta, Canada

    Peter Nguyen

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Mellen, N.M., Funk, G.D. (2013). The Sagittally Sectioned Rat Hindbrain Preparation: Improved Access to the Brainstem Respiratory Network. In: Nguyen, P. (eds) Multidisciplinary Tools for Investigating Synaptic Plasticity. Neuromethods, vol 81. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-517-0_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-517-0_12

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-516-3

  • Online ISBN: 978-1-62703-517-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation