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Optogenetic analysis of respiratory neuronal networks in the ventral medulla of neonatal rats producing channelrhodopsin in Phox2b-positive cells

Abstract

Paired-like homeobox gene Phox2b is predominantly expressed in pre-inspiratory neurons in the parafacial respiratory group (pFRG) in newborn rat rostral ventrolateral medulla. To analyse detailed local networks of the respiratory centre using optogenetics, the effects of selective activation of Phox2b-positive neurons in the ventral medulla on respiratory rhythm generation were examined in brainstem–spinal cord preparations isolated from transgenic newborn rats with Phox2b-positive cells expressing channelrhodopsin variant ChRFR(C167A). Photostimulation up to 43 s increased the respiratory rate > 200% of control, whereas short photostimulation (1.5 s) of the rostral pFRG reset the respiratory rhythm. At the cellular level, photostimulation depolarised Phox2b-positive pre-inspiratory, inspiratory and respiratory-modulated tonic neurons and Phox2b-negative pre-inspiratory neurons. In contrast, changes in membrane potential of Phox2b-negative inspiratory and expiratory neurons varied depending on characteristics of ongoing synaptic connections in local respiratory networks in the rostral medulla. In the presence of tetrodotoxin, photostimulation depolarised Phox2b-positive cells, but caused no significant changes in membrane potential of Phox2b-negative cells. We concluded that depolarisation of Phox2b-positive neurons was due to cell-autonomous photo-activation and summation of excitatory postsynaptic potentials, whereas membrane potential changes of Phox2b-negative neurons depended on the network configuration. Our findings shed further light on local networks among respiratory-related neurons in the rostral ventrolateral medulla and emphasise the important role of pre-inspiratory neurons in respiratory rhythm generation in the neonatal rat en bloc preparation.

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References

  1. 1.

    Abbott SB, Stornetta RL, Coates MB, Guyenet PG (2011) Phox2b-expressing neurons of the parafacial region regulate breathing rate, inspiration, and expiration in conscious rats. J Neurosci 31:16410–16422

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Abbott SB, Stornetta RL, Fortuna MG, Depuy SD, West GH, Harris TE, Guyenet PG (2009) Photostimulation of retrotrapezoid nucleus phox2b-expressing neurons in vivo produces long-lasting activation of breathing in rats. J Neurosci 29:5806–5819

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Abdala AP, Rybak IA, Smith JC, Paton JF (2009) Abdominal expiratory activity in the rat brainstem-spinal cord in situ: patterns, origins and implications for respiratory rhythm generation. J Physiol 587:3539–3559

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Amiel J, Laudier B, Attié-Bitach T, Trang H, de Pontual L, Gener B, Trochet D, Etchevers H, Ray P, Simonneau M, Vekemans M, Munnich A, Gaultier C, Lyonnet S (2003) Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome. Nat Genet 33:459–461

    CAS  PubMed  Google Scholar 

  5. 5.

    Arata A, Onimaru H, Homma I (1998) Possible synaptic connections of expiratory neurons in the medulla of newborn rat in vitro. Neuroreport 9:743–746

    CAS  PubMed  Google Scholar 

  6. 6.

    Ballanyi K, Onimaru H, Homma I (1999) Respiratory network function in the isolated brainstem-spinal cord of newborn rats. Prog Neurobiol 59:583–634

    CAS  PubMed  Google Scholar 

  7. 7.

    Ballanyi K, Ruangkittisakul A, Onimaru H (2009) Opioids prolong and anoxia shortens delay between onset of preinspiratory (pFRG) and inspiratory (preBötC) network bursting in newborn rat brainstems. Pflugers Arch 458:571–587

    CAS  PubMed  Google Scholar 

  8. 8.

    Basting TM, Burke PG, Kanbar R, Viar KE, Stornetta DS, Stornetta RL, Guyenet PG (2015) Hypoxia silences retrotrapezoid nucleus respiratory chemoreceptors via alkalosis. J Neurosci 35:527–543

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Berndt A, Yizhar O, Gunaydin LA, Hegemann P, Deisseroth K (2009) Bi-stable neural state switches. Nat Neurosci 12:229–234

    CAS  PubMed  Google Scholar 

  10. 10.

    Bodineau L, Cayetanot F, Frugiere A (2000) Possible role of retrotrapezoid nucleus and parapyramidal area in the respiratory response to anoxia: an in vitro study in neonatal rat. Neurosci Lett 295:67–69

    CAS  PubMed  Google Scholar 

  11. 11.

    Brunet J-F, Goridis C (2008) Phox2b and the homeostatic brain. In: Gaultier C (ed) Genetic basis for respiratory control disorders. Springer, New York, pp 25–44

    Google Scholar 

  12. 12.

    Burke PG, Abbott SB, Coates MB, Viar KE, Stornetta RL, Guyenet PG (2014) Optogenetic stimulation of adrenergic C1 neurons causes sleep state-dependent cardiorespiratory stimulation and arousal with sighs in rats. Am J Respir Crit Care Med 190:1301–1310

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Czeisler CM, Silva TM, Fair SR, Liu J, Tupal S, Kaya B, Cowgill A, Mahajan S, Silva PE, Wang Y, Blissett AR, Göksel M, Borniger JC, Zhang N, Fernandes-Junior SA, Catacutan F, Alves MJ, Nelson RJ, Sundaresean V, Rekling J, Takakura AC, Moreira TS, Otero JJ (2019) The role of PHOX2B-derived astrocytes in chemosensory control of breathing and sleep homeostasis. J Physiol 597:2225–2251

    CAS  PubMed  Google Scholar 

  14. 14.

    Dubreuil V, Ramanantsoa N, Trochet D, Vaubourg V, Amiel J, Gallego J, Brunet JF, Goridis C (2008) A human mutation in Phox2b causes lack of CO2 chemosensitivity, fatal central apnea, and specific loss of parafacial neurons. Proc Natl Acad Sci U S A 105:1067–1072

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Durand E, Dauger S, Pattyn A, Gaultier C, Goridis C, Gallego J (2005) Sleep-disordered breathing in newborn mice heterozygous for the transcription factor Phox2b. Am J Respir Crit Care Med 172:238–243

    PubMed  Google Scholar 

  16. 16.

    Ellenberger HH, Feldman JL (1990) Brainstem connections of the rostral ventral respiratory group of the rat. Brain Res 513:35–42

    CAS  PubMed  Google Scholar 

  17. 17.

    Feldman JL, Del Negro CA (2006) Looking for inspiration: new perspectives on respiratory rhythm. Nat Rev Neurosci 7:232–242

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Fortuna MG, West GH, Stornetta RL, Guyenet PG (2008) Botzinger expiratory-augmenting neurons and the parafacial respiratory group. J Neurosci 28:2506–2515

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Guyenet PG, Bayliss DA, Stornetta RL, Fortuna MG, Abbott SB, DePuy SD (2009) Retrotrapezoid nucleus, respiratory chemosensitivity and breathing automaticity. Respir Physiol Neurobiol 168:59–68

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Guyenet PG, Mulkey DK, Stornetta RL, Bayliss DA (2005) Regulation of ventral surface chemoreceptors by the central respiratory pattern generator. J Neurosci 25:8938–8947

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Guyenet PG, Stornetta RL, Bayliss DA (2008) Retrotrapezoid nucleus and central chemoreception. J Physiol 586:2043–2048

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Hososhima S, Sakai S, Ishizuka T, Yawo H (2015) Kinetic evaluation of photosensitivity in bi-stable variants of chimeric channelrhodopsins. PLoS One 10:e0119558

    PubMed  PubMed Central  Google Scholar 

  23. 23.

    Huckstepp RT, Cardoza KP, Henderson LE, Feldman JL (2015) Role of parafacial nuclei in control of breathing in adult rats. J Neurosci 35:1052–1067

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Igarashi H, Ikeda K, Onimaru H, Kaneko R, Koizumi K, Beppu K, Nishizawa K, Takahashi Y, Kato F, Matsui K, Kobayashi K, Yanagawa Y, Muramatsu SI, Ishizuka T, Yawo H (2018) Targeted expression of step-function opsins in transgenic rats for optogenetic studies. Sci Rep 8:5435

    PubMed  PubMed Central  Google Scholar 

  25. 25.

    Igarashi H, Koizumi K, Kaneko R, Ikeda K, Egawa R, Yanagawa Y, Muramatsu S, Onimaru H, Ishizuka T, Yawo H (2016) A novel reporter rat strain that conditionally expresses the bright red fluorescent protein tdTomato. PLoS One 11:e0155687

    PubMed  PubMed Central  Google Scholar 

  26. 26.

    Ikeda K, Kaneko R, Yanagawa Y, Ogawa M, Kobayashi K, Arata S, Kawakami K, Onimaru H (2019) Analysis of the neuronal network of the medullary respiratory center in transgenic rats expressing archaerhodopsin-3 in Phox2b-expressing cells. Brain Res Bull 144:39–45

    CAS  PubMed  Google Scholar 

  27. 27.

    Ikeda K, Kawakami K, Onimaru H, Okada Y, Yokota S, Koshiya N, Oku Y, Iizuka M, Koizumi H (2017) The respiratory control mechanisms in the brainstem and spinal cord: integrative views of the neuroanatomy and neurophysiology. J Physiol Sci 67:45–62

    PubMed  Google Scholar 

  28. 28.

    Ikeda K, Takahashi M, Sato S, Igarashi H, Ishizuka T, Yawo H, Arata S, Southard-Smith EM, Kawakami K, Onimaru H (2015) A Phox2b BAC transgenic rat line useful for understanding respiratory rhythm generator neural circuitry. PLoS One 10:e0132475

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Janczewski WA, Onimaru H, Homma I, Feldman JL (2002) Opioid-resistant respiratory pathway from the preinspiratory neurones to abdominal muscles: in vivo and in vitro study in the newborn rat. J Physiol 545:1017–1026

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Kanbar R, Stornetta RL, Cash DR, Lewis SJ, Guyenet PG (2010) Photostimulation of Phox2b medullary neurons activates cardiorespiratory function in conscious rats. Am J Respir Crit Care Med 182:1184–1194

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Kang BJ, Chang DA, Mackay DD, West GH, Moreira TS, Takakura AC, Gwilt JM, Guyenet PG, Stornetta RL (2007) Central nervous system distribution of the transcription factor Phox2b in the adult rat. J Comp Neurol 503:627–641

    CAS  PubMed  Google Scholar 

  32. 32.

    Li C, Guan Z, Chan Y, Zheng Y (2004) Projections from facial nucleus interneurons to the respiratory groups of brainstem in the rat. Neurosci Lett 368:25–28

    CAS  PubMed  Google Scholar 

  33. 33.

    Lin ST, Onimaru H (2015) Effects of riluzole on respiratory rhythm generation in the brainstem-spinal cord preparation from newborn rat. Neurosci Res 94:28–36

    CAS  PubMed  Google Scholar 

  34. 34.

    Malheiros-Lima MR, Totola LT, Lana MVG, Strauss BE, Takakura AC, Moreira TS (2018) Breathing responses produced by optogenetic stimulation of adrenergic C1 neurons are dependent on the connection with preBötzinger complex in rats. Pflugers Arch 470:1659–1672

    CAS  PubMed  Google Scholar 

  35. 35.

    Marina N, Abdala AP, Trapp S, Li A, Nattie EE, Hewinson J, Smith JC, Paton JF, Gourine AV (2010) Essential role of Phox2b-expressing ventrolateral brainstem neurons in the chemosensory control of inspiration and expiration. J Neurosci 30:12466–12473

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Melchior JR, Ferris MJ, Stuber GD, Riddle DR, Jones SR (2015) Optogenetic versus electrical stimulation of dopamine terminals in the nucleus accumbens reveals local modulation of presynaptic release. J Neurochem 134:833–844

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Mellen NM, Janczewski WA, Bocchiaro CM, Feldman JL (2003) Opioid-induced quantal slowing reveals dual networks for respiratory rhythm generation. Neuron 37:821–826

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Moraes DJ, Dias MB, Cavalcanti-Kwiatkoski R, Machado BH, Zoccal DB (2012) Contribution of the retrotrapezoid nucleus/parafacial respiratory region to the expiratory-sympathetic coupling in response to peripheral chemoreflex in rats. J Neurophysiol 108:882–890

    PubMed  Google Scholar 

  39. 39.

    Nattie EE (2001) Central chemosensitivity, sleep, and wakefulness. Respir Physiol 129:257–268

    CAS  PubMed  Google Scholar 

  40. 40.

    Nattie EE, Li A (1994) Retrotrapezoid nucleus lesions decrease phrenic activity and CO2 sensitivity in rats. Respir Physiol 97:63–77

    CAS  PubMed  Google Scholar 

  41. 41.

    Oku Y, Masumiya H, Okada Y (2007) Postnatal developmental changes in activation profiles of the respiratory neuronal network in the rat ventral medulla. J Physiol 585:175–186

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Onimaru H, Arata A, Homma I (1988) Primary respiratory rhythm generator in the medulla of brainstem-spinal cord preparation from newborn rat. Brain Res 445:314–324

    CAS  PubMed  Google Scholar 

  43. 43.

    Onimaru H, Arata A, Homma I (1997) Neuronal mechanisms of respiratory rhythm generation: an approach using in vitro preparation. Jpn J Physiol 47:385–403

    CAS  PubMed  Google Scholar 

  44. 44.

    Onimaru H, Homma I (1987) Respiratory rhythm generator neurons in medulla of brainstem-spinal cord preparation from newborn rat. Brain Res 403:380–384

    CAS  PubMed  Google Scholar 

  45. 45.

    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

    CAS  PubMed  Google Scholar 

  46. 46.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Onimaru H, Homma I, Iwatsuki K (1992) Excitation of inspiratory neurons by preinspiratory neurons in rat medulla in vitro. Brain Res Bull 29:879–882

    CAS  PubMed  Google Scholar 

  48. 48.

    Onimaru H, Ikeda K, Kawakami K (2007) Defective interaction between dual oscillators for respiratory rhythm generation in Na+,K+-ATPase {alpha}2 subunit-deficient mice. J Physiol 584:271–284

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Onimaru H, Ikeda K, Kawakami K (2008) CO2-sensitive preinspiratory neurons of the parafacial respiratory group express Phox2b in the neonatal rat. J Neurosci 28:12845–12850

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Onimaru H, Ikeda K, Kawakami K (2009) Phox2b, RTN/pFRG neurons and respiratory rhythmogenesis. Respir Physiol Neurobiol 168:13–18

    CAS  PubMed  Google Scholar 

  51. 51.

    Onimaru H, Ikeda K, Kawakami K (2012) Postsynaptic mechanisms of CO(2) responses in parafacial respiratory neurons of newborn rats. J Physiol 590:1615–1624

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Onimaru H, Ikeda K, Mariho T, Kawakami K (2014) Cytoarchitecture and CO(2) sensitivity of Phox2b-positive Parafacial neurons in the newborn rat medulla. Prog Brain Res 209:57–71

    PubMed  Google Scholar 

  53. 53.

    Onimaru H, Nakamura S, Ikeda K, Kawakami K, Inoue T (2018) Confocal calcium imaging analysis of respiratory-related burst activity in the parafacial region. Brain Res Bull 139:16–20

    CAS  PubMed  Google Scholar 

  54. 54.

    Pagliardini S, Janczewski WA, Tan W, Dickson CT, Deisseroth K, Feldman JL (2011) Active expiration induced by excitation of ventral medulla in adult anesthetized rats. J Neurosci 31:2895–2905

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF (1997) Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis. Development 124:4065–4075

    CAS  PubMed  Google Scholar 

  56. 56.

    Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF (1999) The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives. Nature 399:366–370

    CAS  PubMed  Google Scholar 

  57. 57.

    Ruangkittisakul A, Schwarzacher SW, Secchia L, Poon BY, Ma Y, Funk GD, Ballanyi K (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 26:11870–11880

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Smith JC, Ellenberger HH, Ballanyi K, Richter DW, Feldman JL (1991) Pre-Bötzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254:726–729

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Smith JC, Morrison DE, Ellenberger HH, Otto MR, Feldman JL (1989) Brainstem projections to the major respiratory neuron populations in the medulla of the cat. J Comp Neurol 281:69–96

    CAS  PubMed  Google Scholar 

  60. 60.

    Song JH, Lucaci D, Calangiu I, Brown MTC, Park JS, Kim J, Brickley SG, Chadderton P (2018) Combining mGRASP and optogenetics enables high-resolution functional mapping of descending cortical projections. Cell Rep 24:1071–1080

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Stornetta RL, Moreira TS, Takakura AC, Kang BJ, Chang DA, West GH, Brunet JF, Mulkey DK, Bayliss DA, Guyenet PG (2006) Expression of Phox2b by brainstem neurons involved in chemosensory integration in the adult rat. J Neurosci 26:10305–10314

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Takakura AC, Moreira TS, Colombari E, West GH, Stornetta RL, Guyenet PG (2006) Peripheral chemoreceptor inputs to retrotrapezoid nucleus (RTN) CO2-sensitive neurons in rats. J Physiol 572:503–523

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Thoby-Brisson M, Karlen M, Wu N, Charnay P, Champagnat J, Fortin G (2009) Genetic identification of an embryonic parafacial oscillator coupling to the preBötzinger complex. Nat Neurosci 12:1028–1035

    CAS  PubMed  Google Scholar 

  65. 65.

    Tremoureux L, Raux M, Hudson AL, Ranohavimparany A, Straus C, Similowski T (2014) Does the supplementary motor area keep patients with Ondine's curse syndrome breathing while awake? PLoS One 9:e84534

    PubMed  PubMed Central  Google Scholar 

  66. 66.

    Trochet D, O'Brien LM, Gozal D, Trang H, Nordenskjöld A, Laudier B, Svensson PJ, Uhrig S, Cole T, Niemann S, Munnich A, Gaultier C, Lyonnet S, Amiel J (2005) PHOX2B genotype allows for prediction of tumor risk in congenital central hypoventilation syndrome. Am J Hum Genet 76:421–426

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Wenker IC, Abe C, Viar KE, Stornetta DS, Stornetta RL, Guyenet PG (2017) Blood pressure regulation by the rostral ventrolateral medulla in conscious rats: effects of hypoxia, hypercapnia, baroreceptor denervation, and anesthesia. J Neurosci 37:4565–4583

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Zhang C, Yan H, Li C, Zheng Y (2004) Possible involvement of the facial nucleus in regulation of respiration in rats. Neurosci Lett 367:283–288

    CAS  PubMed  Google Scholar 

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Funding

This study was supported by the Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and JSPS KAKENHI (16K07003, 25430012) and by the Program for the Strategic Research Foundation at Private Universities 2016–2017 (Showa University School of Medicine and Jichi Medical University).

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Correspondence to Hiroshi Onimaru.

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Experiments were approved by the Animal Ethics Committee of Showa University (approval nos. 57006, 58020) in accordance with Japanese Government Law No. 105 for the care and use of laboratory animals.

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Ikeda, K., Igarashi, H., Yawo, H. et al. Optogenetic analysis of respiratory neuronal networks in the ventral medulla of neonatal rats producing channelrhodopsin in Phox2b-positive cells. Pflugers Arch - Eur J Physiol 471, 1419–1439 (2019). https://doi.org/10.1007/s00424-019-02317-9

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Keywords

  • Phox2b
  • Parafacial respiratory group
  • Channelrhodopsin
  • Optogenetics
  • Transgenic rat