Skip to main content

Autonomic Nervous System In Vitro: Studying Tonically Active Neurons Controlling Vagal Outflow in Rodent Brainstem Slices

  • Protocol
  • First Online:
Book cover Isolated Central Nervous System Circuits

Part of the book series: Neuromethods ((NM,volume 73))

Abstract

Central mammalian neurons often show spontaneous discharge of single action potentials at regular rate. Several types of such ‘tonic’ neurons are involved in sensing or control of the intake and processing of food. Brain slices containing tonically active hypothalamic neurons are one established in vitro model for studying interneuronal and cellular signalling pathways underlying metabolism-related autonomous nervous functions. Here, we focus on brainstem neurons of the dorsal vagal (pre)motor nucleus (DMNX) and the nucleus tractus solitarius (NTS), which both represent pivotal relay areas for control of nutrition and digestion. We compare tonic (pre)motor dorsal vagal neurons (DVN) with similarly active glucagon-like peptide-1 (GLP-1) neurons that comprise a subgroup of NTS cells. This chapter has four aims: (1) to show that central neurons with different functions are capable of generating tonic discharge upon isolation in a brain slice; (2) to exemplify electrophysiological, optical and molecular tools applicable for analysing responses of such neurons to anoxia and aglycaemia; (3) to exemplify biophysical membrane processes involved in the latter metabolism-related or neuromodulator-induced variation of their activity; and (4) to point out methodological strategies for preserving their (sensory) functions and also pitfalls in that regard. We expect that some of our findings using improved in vitro conditions for studying metabolism-sensitive tonic brainstem neurons are applicable, if not necessary, for in vivo-like spontaneous activities and cellular properties of neural networks in slices from other brain regions.

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

Access this chapter

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 179.00
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. Loewi O (1922) Ueber humorale Uebertragbarkeit der Herznervenwirkung. II Mitteilung. Pflugers Arch 193:201–213

    CAS  Google Scholar 

  2. Powley TL (2000) Vagal circuitry mediating cephalic-phase responses to food. Appetite 34:184–188

    PubMed  CAS  Google Scholar 

  3. Groves DA, Brown VJ (2005) Vagal nerve stimulation: a review of its applications and potential mechanisms that mediate its clinical effects. Neurosci Biobehav Rev 29:493–500

    PubMed  Google Scholar 

  4. Yarom Y, Bracha O, Werman R (1985) Intracellular injection of acetylcholine blocks various potassium conductances in vagal motoneurons. Neuroscience 16:739–752

    PubMed  CAS  Google Scholar 

  5. Yarom Y, Sugimori M, Llinas R (1985) Ionic currents and firing patterns of mammalian vagal motoneurons in vitro. Neuroscience 16:719–737

    PubMed  CAS  Google Scholar 

  6. Travagli RA, Gillis RA, Rossiter CD, Vicini S (1991) Glutamate and GABA-mediated synaptic currents in neurons of the rat dorsal motor nucleus of the vagus. Am J Physiol 260:G531–G536

    PubMed  CAS  Google Scholar 

  7. Cowan AI, Martin RL (1992) Ionic basis of membrane potential changes induced by anoxia in rat dorsal vagal motoneurones. J Physiol 455:89–109

    PubMed  CAS  Google Scholar 

  8. Trapp S, Ballanyi K (1995) KATP channel mediation of anoxia-induced outward current in rat dorsal vagal neurons in vitro. J Physiol 487:37–50

    PubMed  CAS  Google Scholar 

  9. Trapp S, Ballanyi K, Richter DW (1994) Spontaneous activation of KATP current in rat dorsal vagal neurones. Neuroreport 5:1285–1288

    PubMed  CAS  Google Scholar 

  10. Sah P (1995) Properties of channels mediating the apamin-insensitive afterhyperpolarization in vagal motoneurons. J Neurophysiol 74:1772–1776

    PubMed  CAS  Google Scholar 

  11. Sah P (1995) Different calcium channels are coupled to potassium channels with distinct physiological roles in vagal neurons. Proc Biol Sci 260:105–111

    PubMed  CAS  Google Scholar 

  12. Sah P, Davies P (2000) Calcium-activated potassium currents in mammalian neurons. Clin Exp Pharmacol Physiol 27:657–663

    PubMed  CAS  Google Scholar 

  13. Loewy AD, Spyer KM (1990) Vagal preganglionic neurons. In: Loewy AD, Spyer KM (eds) Central regulation of autonomic functions. Oxford University Press, New York, pp 68–87

    Google Scholar 

  14. McTigue DM, Hermann GE, Rogers RC (1997) Effect of pancreatic polypeptide on rat dorsal vagal complex neurons. J Physiol 499:475–483

    PubMed  CAS  Google Scholar 

  15. Mussa BM, Sartor DM, Verberne AJ (2010) Dorsal vagal preganglionic neurons: differential responses to CCK1 and 5-HT3 receptor stimulation. Auton Neurosci 156:36–43

    PubMed  CAS  Google Scholar 

  16. Wang Y, Jones JF, Ramage AG, Jordan D (1995) Effects of 5-HT and 5-HT1A receptor agonists and antagonists on dorsal vagal preganglionic neurones in anaesthetized rats: an ionophoretic study. Br J Pharmacol 116:2291–2297

    PubMed  CAS  Google Scholar 

  17. Holmes GM, Browning KN, Tong M, Qualls-Creekmore E, Travagli RA (2009) Vagally mediated effects of glucagon-like peptide 1: in vitro and in vivo gastric actions. J Physiol 587:4749–4759

    PubMed  CAS  Google Scholar 

  18. Wan S, Browning KN, Coleman FH, Sutton G, Zheng H, Butler A, Berthoud HR, Travagli RA (2008) Presynaptic melanocortin-4 receptors on vagal afferent fibers modulate the excitability of rat nucleus tractus solitarius neurons. J Neurosci 28:4957–4966

    PubMed  CAS  Google Scholar 

  19. Wan S, Browning KN, Travagli RA (2007) Glucagon-like peptide-1 modulates synaptic transmission to identified pancreas-projecting vagal motoneurons. Peptides 28:2184–2191

    PubMed  CAS  Google Scholar 

  20. Travagli RA, Hermann GE, Browning KN, Rogers RC (2006) Brainstem circuits regulating gastric function. Annu Rev Physiol 68:279–305

    PubMed  CAS  Google Scholar 

  21. Browning KN, Travagli RA (2009) Modulation of inhibitory neurotransmission in brainstem vagal circuits by NPY and PYY is controlled by cAMP levels. Neurogastroenterol Motil 21:1309–e1126

    PubMed  CAS  Google Scholar 

  22. Ballanyi K, Doutheil J, Brockhaus J (1996) Membrane potentials and microenvironment of rat dorsal vagal cells in vitro during energy depletion. J Physiol 495:769–784

    PubMed  CAS  Google Scholar 

  23. Cone RD, Cowley MA, Butler AA, Fan W, Marks DL, Low MJ (2001) The arcuate nucleus as a conduit for diverse signals relevant to energy homeostasis. Int J Obes Relat Metab Disord 25(suppl 5):S63–S67

    PubMed  CAS  Google Scholar 

  24. Miki T, Seino S (2005) Roles of KATP channels as metabolic sensors in acute metabolic changes. J Mol Cell Cardiol 38:917–925

    PubMed  CAS  Google Scholar 

  25. Balfour RH, Trapp S (2007) Ionic currents underlying the response of rat dorsal vagal neurones to hypoglycaemia and chemical anoxia. J Physiol 579:691–702

    PubMed  CAS  Google Scholar 

  26. Balfour RH, Hansen AM, Trapp S (2006) Neuronal responses to transient hypoglycaemia in the dorsal vagal complex of the rat brainstem. J Physiol 570:469–484

    PubMed  CAS  Google Scholar 

  27. Appleyard SM, Marks D, Kobayashi K, Okano H, Low MJ, Andresen MC (2007) Visceral afferents directly activate catecholamine neurons in the solitary tract nucleus. J Neurosci 27:13292–13302

    PubMed  CAS  Google Scholar 

  28. Matsushita N, Okada H, Yasoshima Y, Takahashi K, Kiuchi K, Kobayashi K (2002) Dynamics of tyrosine hydroxylase promoter activity during midbrain dopaminergic neuron development. J Neurochem 82:295–304

    PubMed  CAS  Google Scholar 

  29. Appleyard SM, Bailey TW, Doyle MW, Jin YH, Smart JL, Low MJ, Andresen MC (2005) Proopiomelanocortin neurons in nucleus tractus solitarius are activated by visceral afferents: regulation by cholecystokinin and opioids. J Neurosci 25:3578–3585

    PubMed  CAS  Google Scholar 

  30. Cowley MA, Smart JL, Rubinstein M, Cerdan MG, Diano S, Horvath TL, Cone RD, Low MJ (2001) Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature 411:480–484

    PubMed  CAS  Google Scholar 

  31. Hisadome K, Reimann F, Gribble FM, Trapp S (2010) Leptin directly depolarizes preproglucagon neurons in the nucleus tractus solitarius: electrical properties of glucagon-like Peptide 1 neurons. Diabetes 59:1890–1898

    PubMed  CAS  Google Scholar 

  32. Reimann F, Habib AM, Tolhurst G, Parker HE, Rogers GJ, Gribble FM (2008) Glucose sensing in L cells: a primary cell study. Cell Metab 8:532–539

    PubMed  CAS  Google Scholar 

  33. Edwards FA, Konnerth A, Sakmann B, Takahashi T (1989) A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflugers Arch 414:600–612

    PubMed  CAS  Google Scholar 

  34. Ballanyi K (1999) Isolated tissues: in vitro preparations. In: Windhorst U, Johansson H (eds) Modern techniques in neuroscience research. Springer, Heidelberg, pp 307–326

    Google Scholar 

  35. Konnerth A (1990) Patch-clamping in slices of mammalian CNS. Trends Neurosci 13:321–323

    PubMed  CAS  Google Scholar 

  36. Haas H, Büsselberg D (1992) Recording chambers-slices. In: Kettenmann H, Grantyn R (eds) Practical electrophysiological methods. Wiley-Liss, New York, pp 16–19

    Google Scholar 

  37. Wilson RJ, Straus C, Remmers JE (1999) Efficacy of a low volume recirculating superfusion chamber for long term administration of expensive drugs and dyes. J Neurosci Methods 87:175–184

    PubMed  CAS  Google Scholar 

  38. Strupp M, Jund R, Schneider U, Grafe P (1991) Glucose availability and sensitivity to anoxia of isolated rat peroneal nerve. Am J Physiol 261:E389–E394

    PubMed  CAS  Google Scholar 

  39. Hille B (2001) Ion channels of excitable membranes, 3rd edn. Sinauer Associates, Inc., Sunderland

    Google Scholar 

  40. Neher E, Sakmann B (eds) (2009) Single-channel recording, 2nd edn. Springer, New York/Dordrecht/Heidelberg/London

    Google Scholar 

  41. Walz W (2007) Patch-clamp analysis. In: Walz W (ed) Advanced techniques, 2nd edn. Humana, Totowa

    Google Scholar 

  42. Lowry JP, Griffin K, McHugh SB, Lowe AS, Tricklebank M, Sibson NR (2010) Real-time electrochemical monitoring of brain tissue oxygen: a surrogate for functional magnetic resonance imaging in rodents. Neuroimage 52:549–555

    PubMed  Google Scholar 

  43. Blanton MG, Lo Turco JJ, Kriegstein AR (1989) Whole cell recording from neurons in slices of reptilian and mammalian cerebral cortex. J Neurosci Methods 30:203–210

    PubMed  CAS  Google Scholar 

  44. Moore AR, Zhou W, Jakovcevski I, Zecevic N, Antic S (2012) Physiological properties of human fetal cortex in vitro. In Isolated Central Nervous System Circuits (Ed K Ballanyi), Neuromethods Series Vol. 73 (Ed W Walz). Springer Science+Business Media, LLC, New York, NY, pp 125–158

    Google Scholar 

  45. Dodt HU, Zieglgansberger W (1994) Infrared videomicroscopy: a new look at neuronal structure and function. Trends Neurosci 17:453–458

    PubMed  CAS  Google Scholar 

  46. MacVicar BA (1984) Infrared video microscopy to visualize neurons in the in vitro brain slice preparation. J Neurosci Methods 12:133–139

    PubMed  CAS  Google Scholar 

  47. Ruangkittisakul A, Okada Y, Oku Y, Koshiya N, Ballanyi K (2009) Fluorescence imaging of active respiratory networks. Respir Physiol Neurobiol 168:26–38

    PubMed  Google Scholar 

  48. Yuste R, Konnerth A, Masters B (2006) Imaging in neuroscience and development, a laboratory manual. J Biomed Opt 11:19902

    Google Scholar 

  49. Neher E (1989) Combined fura-2 and patch-clamp measurements in rat peritoneal mast cells. In: Sellin LC, Libelius R, Theslaff S (eds) Neuromuscular junction. Elsevier, Science Publishers (Biomedical Division), Amsterdam, pp 65–75

    Google Scholar 

  50. Ruangkittisakul A, Panaitescu B, Secchia L, Bobocea N, Kantor C, Kuribayashi J, Iizuka M, Ballanyi K (2012) Isolated brainstem respiratory centers from perinatal rodents. In Isolated Central Nervous System Circuits (Ed K Ballanyi), Neuromethods Series Vol. 73 (Ed W Walz). Springer Science+Business Media, LLC, New York, NY, pp 61–124

    Google Scholar 

  51. Somjen GG (2002) Ion regulation in the brain: implications for pathophysiology. Neuroscientist 8:254–267

    PubMed  CAS  Google Scholar 

  52. Raupach T, Ballanyi K (2004) Intracellular pH and KATP channel activity in dorsal vagal neurons of juvenile rats in situ during metabolic disturbances. Brain Res 1017:137–145

    PubMed  CAS  Google Scholar 

  53. Müller M, Ballanyi K (2003) Dynamic recording of cell death in the in vitro dorsal vagal nucleus of rats in response to metabolic arrest. J Neurophysiol 89:551–561

    PubMed  Google Scholar 

  54. Kulik A, Brockhaus J, Pedarzani P, Ballanyi K (2002) Chemical anoxia activates ATP-sensitive and blocks Ca2+-dependent K+ channels in rat dorsal vagal neurons in situ. Neuroscience 110:541–554

    PubMed  CAS  Google Scholar 

  55. Müller M, Brockhaus J, Ballanyi K (2002) ATP-independent anoxic activation of ATP-sensitive K+ channels in dorsal vagal neurons of juvenile mice in situ. Neuroscience 109:313–328

    PubMed  Google Scholar 

  56. Kulik A, Trapp S, Ballanyi K (2000) Ischemia but not anoxia evokes vesicular and Ca2+-independent glutamate release in the dorsal vagal complex in vitro. J Neurophysiol 83:2905–2915

    PubMed  CAS  Google Scholar 

  57. Pedarzani P, Kulik A, Müller M, Ballanyi K, Stocker M (2000) Molecular determinants of Ca2+-dependent K+ channel function in rat dorsal vagal neurones. J Physiol 527:283–290

    PubMed  CAS  Google Scholar 

  58. Karschin A, Brockhaus J, Ballanyi K (1998) KATP channel formation by the sulphonylurea receptors SUR1 with Kir6.2 subunits in rat dorsal vagal neurons in situ. J Physiol 509:339–346

    PubMed  CAS  Google Scholar 

  59. Ballanyi K, Kulik A (1998) Intracellular Ca2+ during metabolic activation of KATP channels in spontaneously active dorsal vagal neurons in medullary slices. Eur J Neurosci 10:2574–2585

    PubMed  CAS  Google Scholar 

  60. Trapp S, Lückermann M, Brooks PA, Ballanyi K (1996) Acidosis of rat dorsal vagal neurons in situ during spontaneous and evoked activity. J Physiol 496:695–710

    PubMed  CAS  Google Scholar 

  61. Laughton WB, Powley TL (1987) Localization of efferent function in the dorsal motor nucleus of the vagus. Am J Physiol 252:R13–R25

    PubMed  CAS  Google Scholar 

  62. Baumgärtl H (1987) Systematic investigations of needle electrode properties in polarographic measurements of local tissue P02. In: Ehrly AM, Hauss J, Huch R (eds) Clinical oxygen pressure measurement: Tissue oxygen pressure and transcutaneous oxygen pressure. Springer

    Google Scholar 

  63. Grote J, Zimmer K, Schubert R (1981) Effects of severe arterial hypocapnia on regional blood flow regulation, tissue PO2 and metabolism in the brain cortex of cats. Pflugers Arch 391:195–199

    PubMed  CAS  Google Scholar 

  64. Abi-Saab WM, Maggs DG, Jones T, Jacob R, Srihari V, Thompson J, Kerr D, Leone P, Krystal JH, Spencer DD, During MJ, Sherwin RS (2002) Striking differences in glucose and lactate levels between brain extracellular fluid and plasma in conscious human subjects: effects of hyperglycemia and hypoglycemia. J Cereb Blood Flow Metab 22:271–279

    PubMed  CAS  Google Scholar 

  65. De Vries MG, Arseneau LM, Lawson ME, Beverly JL (2003) Extracellular glucose in rat ventromedial hypothalamus during acute and recurrent hypoglycemia. Diabetes 52:2767–2773

    PubMed  Google Scholar 

  66. Fray AE, Forsyth RJ, Boutelle MG, Fillenz M (1996) The mechanisms controlling physiologically stimulated changes in rat brain glucose and lactate: a microdialysis study. J Physiol 496:49–57

    PubMed  CAS  Google Scholar 

  67. Gramsbergen JB, Skjoth-Rasmussen J, Rasmussen C, Lambertsen KL (2004) On-line monitoring of striatum glucose and lactate in the endothelin-1 rat model of transient focal cerebral ischemia using microdialysis and flow-injection analysis with biosensors. J Neurosci Methods 140:93–101

    PubMed  CAS  Google Scholar 

  68. Lowry JP, O’Neill RD, Boutelle MG, Fillenz M (1998) Continuous monitoring of extracellular glucose concentrations in the striatum of freely moving rats with an implanted glucose biosensor. J Neurochem 70:391–396

    PubMed  CAS  Google Scholar 

  69. Silver IA, Erecinska M (1994) Extracellular glucose concentration in mammalian brain: continuous monitoring of changes during increased neuronal activity and upon limitation in oxygen supply in normo-, hypo-, and hyperglycemic animals. J Neurosci 14:5068–5076

    PubMed  CAS  Google Scholar 

  70. Ashford ML, Boden PR, Treherne JM (1990) Tolbutamide excites rat glucoreceptive ventromedial hypothalamic neurones by indirect inhibition of ATP-K+ channels. Br J Pharmacol 101:531–540

    PubMed  CAS  Google Scholar 

  71. Ashford ML, Boden PR, Treherne JM (1990) Glucose-induced excitation of hypothalamic neurones is mediated by ATP-sensitive K+ channels. Pflugers Arch 415:479–483

    PubMed  CAS  Google Scholar 

  72. Minami T, Oomura Y, Sugimori M (1986) Electrophysiological properties and glucose responsiveness of guinea-pig ventromedial hypothalamic neurones in vitro. J Physiol 380:127–143

    PubMed  CAS  Google Scholar 

  73. Wang R, Liu X, Hentges ST, Dunn-Meynell AA, Levin BE, Wang W, Routh VH (2004) The regulation of glucose-excited neurons in the hypothalamic arcuate nucleus by glucose and feeding-relevant peptides. Diabetes 53:1959–1965

    PubMed  CAS  Google Scholar 

  74. Song Z, Levin BE, McArdle JJ, Bakhos N, Routh VH (2001) Convergence of pre- and postsynaptic influences on glucosensing neurons in the ventromedial hypothalamic nucleus. Diabetes 50:2673–2681

    PubMed  CAS  Google Scholar 

  75. Burdakov D, Jensen LT, Alexopoulos H, Williams RH, Fearon IM, O’Kelly I, Gerasimenko O, Fugger L, Verkhratsky A (2006) Tandem-pore K+ channels mediate inhibition of orexin neurons by glucose. Neuron 50:711–722

    PubMed  CAS  Google Scholar 

  76. Dallaporta M, Perrin J, Orsini JC (2000) Involvement of adenosine triphosphate-sensitive K+ channels in glucose-sensing in the rat solitary tract nucleus. Neurosci Lett 278:77–80

    PubMed  CAS  Google Scholar 

  77. Dallaporta M, Himmi T, Perrin J, Orsini JC (1999) Solitary tract nucleus sensitivity to moderate changes in glucose level. Neuroreport 10:2657–2660

    PubMed  CAS  Google Scholar 

  78. Kaila K (1994) Ionic basis of GABAA receptor channel function in the nervous system. Prog Neurobiol 42:489–537

    PubMed  CAS  Google Scholar 

  79. Lückermann M, Trapp S, Ballanyi K (1997) GABA- and glycine-mediated fall of intracellular pH in rat medullary neurons in situ. J Neurophysiol 77:1844–1852

    PubMed  Google Scholar 

  80. Ballanyi K (2004) Protective role of neuronal KATP channels in brain hypoxia. J Exp Biol 207:3201–3212

    PubMed  CAS  Google Scholar 

  81. Ballanyi K (2004) Neuromodulation of the perinatal respiratory network. Curr Neuro­pharmacol 2:221–243

    CAS  Google Scholar 

  82. Haddad GG, Jiang C (1993) O2 deprivation in the central nervous system: on mechanisms of neuronal response, differential sensitivity and injury. Prog Neurobiol 40:277–318

    PubMed  CAS  Google Scholar 

  83. Lipton P (1999) Ischemic cell death in brain neurons. Physiol Rev 79:1431–1568

    PubMed  CAS  Google Scholar 

  84. Lu VB, Biggs JE, Stebbing MJ, Balasubramanyan S, Todd KG, Lai AY, Colmers WF, Dawbarn D, Ballanyi K, Smith PA (2009) Brain-derived neurotrophic factor drives the changes in excitatory synaptic transmission in the rat superficial dorsal horn that follow sciatic nerve injury. J Physiol 587:1013–1032

    PubMed  CAS  Google Scholar 

  85. Metzger F, Klapproth N, Kulik A, Sendtner M, Ballanyi K (2005) Optical assessment of motoneuron function in a “twenty-four-hour” acute spinal cord slice model from fetal rats. J Neurosci Methods 141:309–320

    PubMed  Google Scholar 

  86. Kulik A, Nishimaru H, Ballanyi K (2000) Role of bicarbonate and chloride in GABA- and glycine-induced depolarization and (Ca2+)i rise in fetal rat motoneurons in situ. J Neurosci 20:7905–7913

    PubMed  CAS  Google Scholar 

  87. Schuchmann S, Lückermann M, Kulik A, Heinemann U, Ballanyi K (2000) Ca2+- and metabolism-related changes of mitochondrial potential in voltage-clamped CA1 pyramidal neurons in situ. J Neurophysiol 83:1710–1721

    PubMed  CAS  Google Scholar 

  88. Kulik A, Haentzsch A, Lückermann M, Reichelt W, Ballanyi K (1999) Neuron-glia signaling via α1 adrenoceptor-mediated Ca2+ release in Bergmann glial cells in situ. J Neurosci 19:8401–8408

    PubMed  CAS  Google Scholar 

  89. Ruangkittisakul A, Schwarzacher SW, Secchia L, Ma Y, Bobocea N, Poon BY, Funk GD, Ballanyi K (2008) Generation of eupnea and sighs by a spatiochemically organized inspiratory network. J Neurosci 28:2447–2458

    PubMed  CAS  Google Scholar 

  90. Nakamura T, Michel S, Block G, Colwell C (2012) Neural circuits underlying circadian oscillations in mammals: clocks in a dish. In Isolated Central Nervous System Circuits (Ed K Ballanyi), Neuromethods Series Vol. 73 (Ed W Walz). Springer Science+Business Media, LLC, New York, NY, pp 183–210

    Google Scholar 

  91. Kantor C, Panaitescu B, Kuribayashi J, Ruangkittisakul A, Jovanovic I, Leung V, Lee TF, MacTavish D, Jhamandas J, Cheung PY, Ballanyi K (2012) Early network oscillations in cortex and locus coeruleus of horizontal brain slices from newborn mammals. In Isolated Central Nervous System Circuits (Ed K Ballanyi), Neuromethods Series Vol. 73 (Ed W Walz). Springer Science+Business Media, LLC, New York, NY, pp 315–356

    Google Scholar 

  92. De Curtis M, Lilbrizzi L, Uva L, Gnatkovsky V (2012) Neuronal networks in the in vitro isolated guinea pig brain. In Isolated Central Nervous System Circuits (Ed K Ballanyi), Neuromethods Series Vol. 73 (Ed W Walz). Springer Science+Business Media, LLC, New York, NY, pp 357–383

    Google Scholar 

  93. Biggs J, Lu V, Kim H, Lai A, Todd K, Ballanyi K, Colmers W, Smith P (2012) Defined medium organotypic cultures of spinal cord put ‘Pain in a Dish’. In Isolated Central Nervous System Circuits (Ed K Ballanyi), Neuromethods Series Vol. 73 (Ed W Walz). Springer Science+Business Media, LLC, New York, NY, pp 405–435

    Google Scholar 

  94. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450

    PubMed  CAS  Google Scholar 

  95. Tsien RY (1980) New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Bio­chemistry 19:2396–2404

    PubMed  CAS  Google Scholar 

  96. 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

    PubMed  CAS  Google Scholar 

  97. Homma R, Baker BJ, Jin L, Garaschuk O, Konnerth A, Cohen LB, Zecevic D (2009) Wide-field and two-photon imaging of brain activity with voltage- and calcium-sensitive dyes. Philos Trans R Soc Lond B Biol Sci 364:2453–2467

    PubMed  CAS  Google Scholar 

  98. Thomas JA, Buchsbaum RN, Zimniak A, Racker E (1979) Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18:2210–2218

    PubMed  CAS  Google Scholar 

  99. Duchen MR (1992) Ca2+-dependent changes in the mitochondrial energetics in single dissociated mouse sensory neurons. Biochem J 283:41–50

    PubMed  CAS  Google Scholar 

  100. Duchen M (1992) Fluorescence-monitoring cell chemistry in vivo. In: Monitoring neuronal activity-a practical approach. Stamford JA (ed) IRL Press at Oxford University Press, New York, pp. 231–260

    Google Scholar 

  101. Duchen MR (2004) Mitochondria in health and disease: perspectives on a new mitochondrial biology. Mol Aspects Med 25:365–451

    PubMed  CAS  Google Scholar 

  102. Emaus RK, Grunwald R, Lemasters JJ (1986) Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Biochim Biophys Acta 850:436–448

    PubMed  CAS  Google Scholar 

  103. Bevensee MO, Schwiening CJ, Boron WF (1995) Use of BCECF and propidium iodide to assess membrane integrity of acutely isolated CA1 neurons from rat hippocampus. J Neurosci Methods 58:61–75

    PubMed  CAS  Google Scholar 

  104. Laake JH, Haug FM, Wieloch T, Ottersen OP (1999) A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence. Brain Res Brain Res Protoc 4:173–184

    PubMed  CAS  Google Scholar 

  105. Loo DT, Rillema JR (1998) Measurement of cell death. Methods Cell Biol 57:251–264

    PubMed  CAS  Google Scholar 

  106. Voipio J, Ballanyi K (1997) Interstitial PCO2 and pH, and their role as chemostimulants in the isolated respiratory network of neonatal rats. J Physiol 499:527–542

    PubMed  CAS  Google Scholar 

  107. Ballanyi K, Völker A, Richter DW (1996) Functional relevance of anaerobic metabolism in the isolated respiratory network of newborn rats. Pflugers Arch 432:741–748

    PubMed  CAS  Google Scholar 

  108. Morawietz G, Ballanyi K, Kuwana S, Richter DW (1995) Oxygen supply and ion homeostasis of the respiratory network in the in vitro perfused brainstem of adult rats. Exp Brain Res 106:265–274

    PubMed  CAS  Google Scholar 

  109. Völker A, Ballanyi K, Richter DW (1995) Anoxic disturbance of the isolated respiratory network of neonatal rats. Exp Brain Res 103:9–19

    PubMed  Google Scholar 

  110. Brockhaus J, Ballanyi K, Smith JC, Richter DW (1993) Microenvironment of respiratory neurons in the in vitro brainstem-spinal cord of neonatal rats. J Physiol 462:421–445

    PubMed  CAS  Google Scholar 

  111. Ballanyi K, Kuwana S, Völker A, Morawietz G, Richter DW (1992) Developmental changes in the hypoxia tolerance of the in vitro respiratory network of rats. Neurosci Lett 148:141–144

    PubMed  CAS  Google Scholar 

  112. Ballanyi K, Mückenhoff K, Bellingham MC, Okada Y, Scheid P, Richter DW (1994) Activity-related pH changes in respiratory neurones and glial cells of cats. Neuroreport 6:33–36

    PubMed  CAS  Google Scholar 

  113. Ballanyi K, Kettenmann H (1990) Intracellular Na+ activity in cultured mouse oligodendrocytes. J Neurosci Res 26:455–460

    PubMed  CAS  Google Scholar 

  114. Ballanyi K, Grafe P, ten Bruggencate G (1987) Ion activities and potassium uptake mechanisms of glial cells in guinea-pig olfactory cortex slices. J Physiol 382:159–174

    PubMed  CAS  Google Scholar 

  115. Ballanyi K, Grafe P (1985) An intracellular analysis of gamma-aminobutyric-acid-associated ion movements in rat sympathetic neurones. J Physiol 365:41–58

    PubMed  CAS  Google Scholar 

  116. Ammann D (1986) Ion-selective microelectrodes. Springer Berlin, Heidelberg/New York/Tokyo

    Google Scholar 

  117. Grafe P, Rimpel J, Reddy MM, ten Bruggencate G (1982) Changes of intracellular sodium and potassium ion concentrations in frog spinal motoneurons induced by repetitive synaptic stimulation. Neuroscience 7:3213–3220

    PubMed  CAS  Google Scholar 

  118. Cowan AI, Martin RL (1995) Simultaneous measurement of pH and membrane potential in rat dorsal vagal motoneurons during normoxia and hypoxia: a comparison in bicarbonate and HEPES buffers. J Neurophysiol 74:2713–2721

    PubMed  CAS  Google Scholar 

  119. Sucher NJ, Deitcher DL (1995) PCR and patch-clamp analysis of single neurons. Neuron 14:1095–1100

    PubMed  CAS  Google Scholar 

  120. Trapp S, Aller MI, Wisden W, Gourine AV (2008) A role for TASK-1 (KCNK3) channels in the chemosensory control of breathing. J Neurosci 28:8844–8850

    PubMed  CAS  Google Scholar 

  121. Trapp S, Tucker S, Gourine AV (2011) Respiratory responses to hypercapnia and hypoxia in mice with genetic ablation of Kir5.1 (Kcnj16). Exp Physiol 96:451–459

    PubMed  CAS  Google Scholar 

  122. Hopwood SE, Trapp S (2005) TASK-like K+ channels mediate effects of 5-HT and extracellular pH in rat dorsal vagal neurones in vitro. J Physiol 568:145–154

    PubMed  CAS  Google Scholar 

  123. Ballanyi K, Ruangkittisakul A (2009) Structure-function analysis of rhythmogenic inspiratory pre-Botzinger complex networks in “calibrated” newborn rat brainstem slices. Respir Physiol Neurobiol 168:158–178

    PubMed  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  125. Ashcroft FM, Kakei M, Gibson JS, Gray DW, Sutton R (1989) The ATP- and tolbutamide-sensitivity of the ATP-sensitive K-channel from human pancreatic B cells. Diabetologia 32:591–598

    PubMed  CAS  Google Scholar 

  126. Kozlowski RZ, Hales CN, Ashford ML (1989) Dual effects of diazoxide on ATP-K+ currents recorded from an insulin-secreting cell line. Br J Pharmacol 97:1039–1050

    PubMed  CAS  Google Scholar 

  127. Niki I, Kelly RP, Ashcroft SJ, Ashcroft FM (1989) ATP-sensitive K-channels in HIT T15 beta-cells studied by patch- clamp methods, 86Rb efflux and glibenclamide binding. Pflugers Arch 415:47–55

    PubMed  CAS  Google Scholar 

  128. Trube G, Rorsman P, Ohno Shosaku T (1986) Opposite effects of tolbutamide and diazoxide on the ATP-dependent K+ channel in mouse pancreatic beta-cells. Pflugers Arch 407:493–499

    PubMed  CAS  Google Scholar 

  129. Gribble FM, Loussouarn G, Tucker SJ, Zhao C, Nichols CG, Ashcroft FM (2000) A novel method for measurement of submembrane ATP concentration. J Biol Chem 275:30046–30049

    PubMed  CAS  Google Scholar 

  130. Karschin C, Ecke C, Ashcroft FM, Karschin A (1997) Overlapping distribution of KATP channel-forming Kir6.2 subunit and the sulfonylurea receptor SUR1 in rodent brain. FEBS Lett 401:59–64

    PubMed  CAS  Google Scholar 

  131. Ashcroft FM, Gribble FM (1999) ATP-sensitive K+ channels and insulin secretion: their role in health and disease. Diabetologia 42:903–919

    PubMed  CAS  Google Scholar 

  132. Trapp S, Ashcroft FM (1997) A metabolic sensor in action: news from the ATP-sensitive K+-channel. News Physiol Sci 12:255–263

    CAS  Google Scholar 

  133. Browning KN, Coleman FH, Travagli RA (2005) Effects of pancreatic polypeptide on pancreas-projecting rat dorsal motor nucleus of the vagus neurons. Am J Physiol Gastrointest Liver Physiol 289:G209–G219

    PubMed  CAS  Google Scholar 

  134. Browning KN, Coleman FH, Travagli RA (2005) Characterization of pancreas-projecting rat dorsal motor nucleus of vagus neurons. Am J Physiol Gastrointest Liver Physiol 288:G950–G955

    PubMed  CAS  Google Scholar 

  135. Gribble FM, Ashfield R, Ammala C, Ashcroft FM (1997) Properties of cloned ATP-sensitive K+ currents expressed in Xenopus oocytes. J Physiol 498:87–98

    PubMed  CAS  Google Scholar 

  136. Tucker SJ, Gribble FM, Zhao C, Trapp S, Ashcroft FM (1997) Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea receptor. Nature 387:179–183

    PubMed  CAS  Google Scholar 

  137. Bantel C, Maze M, Trapp S (2010) Noble gas xenon is a novel adenosine triphosphate-sensitive potassium channel opener. Anesthesiology 112:623–630

    PubMed  CAS  Google Scholar 

  138. Inagaki N, Gonoi T, Clement JP 4th, Namba N, Inazawa J, Gonzalez G, Aguilar Bryan L, Seino S, Bryan J (1995) Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270:1166–1170

    PubMed  CAS  Google Scholar 

  139. Schuit FC, Huypens P, Heimberg H, Pipeleers DG (2001) Glucose sensing in pancreatic beta-cells: a model for the study of other glucose-regulated cells in gut, pancreas, and hypothalamus. Diabetes 50:1–11

    PubMed  CAS  Google Scholar 

  140. Sakura H, Ashcroft SJ, Terauchi Y, Kadowaki T, Ashcroft FM (1998) Glucose modulation of ATP-sensitive K-currents in wild-type, homozygous and heterozygous glucokinase knock-out mice. Diabetologia 41:654–659

    PubMed  CAS  Google Scholar 

  141. Efrat S, Tal M, Lodish HF (1994) The pancreatic beta-cell glucose sensor. Trends Biochem Sci 19:535–538

    PubMed  CAS  Google Scholar 

  142. Guillam MT, Hummler E, Schaerer E, Yeh JI, Birnbaum MJ, Beermann F, Schmidt A, Deriaz N, Thorens B (1997) Early diabetes and abnormal postnatal pancreatic islet development in mice lacking Glut-2. Nat Genet 17:327–330

    PubMed  CAS  Google Scholar 

  143. Thorens B (1992) Molecular and cellular physiology of GLUT-2, a high-Km facilitated diffusion glucose transporter. Int Rev Cytol 137:209–238

    PubMed  CAS  Google Scholar 

  144. Matschinsky FM (1996) Banting Lecture 1995. A lesson in metabolic regulation inspired by the glucokinase glucose sensor paradigm. Diabetes 45:223–241

    PubMed  CAS  Google Scholar 

  145. Terauchi Y, Sakura H, Yasuda K, Iwamoto K, Takahashi N, Ito K, Kasai H, Suzuki H, Ueda O, Kamada N et al (1995) Pancreatic beta-cell-specific targeted disruption of glucokinase gene. Diabetes mellitus due to defective insulin secretion to glucose. J Biol Chem 270:30253–30256

    PubMed  CAS  Google Scholar 

  146. Gonzalez JA, Jensen LT, Doyle SE, Miranda-Anaya M, Menaker M, Fugger L, Bayliss DA, Burdakov D (2009) Deletion of TASK1 and TASK3 channels disrupts intrinsic excitability but does not abolish glucose or pH responses of orexin/hypocretin neurons. Eur J Neurosci 30:57–64

    PubMed  CAS  Google Scholar 

  147. Guyon A, Tardy MP, Rovere C, Nahon JL, Barhanin J, Lesage F (2009) Glucose inhibition persists in hypothalamic neurons lacking tandem-pore K+ channels. J Neurosci 29:2528–2533

    PubMed  CAS  Google Scholar 

  148. Terzic A, Tung RT, Kurachi Y (1994) Nucleotide regulation of ATP sensitive potassium channels. Cardiovasc Res 28:746–753

    PubMed  CAS  Google Scholar 

  149. Trapp S, Lückermann M, Kaila K, Ballanyi K (1996) Acidosis of hippocampal neurones mediated by a plasmalemmal Ca2+/H+ pump. Neuroreport 7:2000–2004

    PubMed  CAS  Google Scholar 

  150. Erdemli G, Xu YZ, Krnjevic K (1998) Potassium conductance causing hyperpolarization of CA1 hippocampal neurons during hypoxia. J Neurophysiol 80:2378–2390

    PubMed  CAS  Google Scholar 

  151. Leblond J, Krnjevic K (1989) Hypoxic changes in hippocampal neurons. J Neuro­physiol 62:1–14

    PubMed  CAS  Google Scholar 

  152. Nowicky AV, Duchen MR (1998) Changes in (Ca2+)i and membrane currents during impaired mitochondrial metabolism in dissociated rat hippocampal neurons. J Physiol 507:131–145

    PubMed  CAS  Google Scholar 

  153. Vrang N, Hansen M, Larsen PJ, Tang-Christensen M (2007) Characterization of brainstem preproglucagon projections to the paraventricular and dorsomedial hypothalamic nuclei. Brain Res 1149:118–126

    PubMed  CAS  Google Scholar 

  154. Larsen PJ, Tang-Christensen M, Holst JJ, Orskov C (1997) Distribution of glucagon-like peptide-1 and other preproglucagon-derived peptides in the rat hypothalamus and brainstem. Neuroscience 77:257–270

    PubMed  CAS  Google Scholar 

  155. Trapp S, Hisadome K (2011) Glucagon-like peptide 1 and the brain: central actions-central sources? Auton Neurosci 161:14–19

    PubMed  CAS  Google Scholar 

  156. Llewellyn-Smith IJ, Reimann F, Gribble FM, Trapp S (2011) Preproglucagon neurons project widely to autonomic control areas in the mouse brain. Neuroscience 180:111–121

    PubMed  CAS  Google Scholar 

  157. Holst JJ (2007) The physiology of glucagon-like peptide 1. Physiol Rev 87:1409–1439

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (ST, KB), Wilhelm Sander Stiftung (KB), Medical Research Council UK (ST), the Canadian Institutes of Health Research (KB) and the Alberta Heritage Foundation for Medical Research (KB).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Stefan Trapp .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media New York

About this protocol

Cite this protocol

Trapp, S., Ballanyi, K. (2012). Autonomic Nervous System In Vitro: Studying Tonically Active Neurons Controlling Vagal Outflow in Rodent Brainstem Slices. In: Ballanyi, K. (eds) Isolated Central Nervous System Circuits. Neuromethods, vol 73. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-020-5_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-020-5_1

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-019-9

  • Online ISBN: 978-1-62703-020-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics