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Electrophysiological and Neurochemical Techniques to Investigate Sensory Neurons in Analgesia Research

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Analgesia

Part of the book series: Methods in Molecular Biology ((MIMB,volume 617))

Abstract

The primary afferent nociceptive neuron has recently attracted major research interest because of the cloning of very selectively expressed and well-conserved ion channel genes. All parts of the neuron, sensory terminals, axon and cell body, are accessible to validated research techniques in vitro using various isolated tissues or cells taken from laboratory animals. Single-unit recording and measuring stimulated calcitonin gene-related peptide (CGRP) release as well as patch-clamping and calcium imaging of cultured sensory neurons provide different kinds of information, and no model alone answers all questions. In combination, however, consistent results and complementary evidence form a solid basis for translational research to follow.

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References

  1. Novakovic SD, Tzoumaka E, McGivern JG, Haraguchi M, Sangameswaran L, Gogas KR, Eglen RM, Hunter JC (1998) Distribution of the tetrodotoxin-resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions. J Neurosci 18:2174–2187

    PubMed  CAS  Google Scholar 

  2. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D (1997) The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389:816–824

    Article  PubMed  CAS  Google Scholar 

  3. Chen CC, Akopian AN, Sivilotti L, Colquhoun D, Burnstock G, Wood JN (1995) A P2X puri­noceptor expressed by a subset of sensory neurons. Nature 377:428–431

    Article  PubMed  CAS  Google Scholar 

  4. Dib-Hajj SD, Cummins TR, Black JA, Waxman SG (2007) From genes to pain: Na v 1.7 and human pain disorders. Trends Neurosci 30:555–563

    Article  PubMed  CAS  Google Scholar 

  5. Namer B, Barta B, Orstavik K, Schmidt R, Carr R, Schmelz M, Handwerker HO (2009) Microneurographic assessment of C-fibre function in aged healthy subjects. J Physiol 587:419–428

    Article  PubMed  CAS  Google Scholar 

  6. Orstavik K, Namer B, Schmidt R, Schmelz M, Hilliges M, Weidner C, Carr RW, Handwerker H, Jorum E, Torebjork HE (2006) Abnor­mal function of C-fibers in patients with diabetic neuropathy. J Neurosci 26:11287–11294

    Article  PubMed  Google Scholar 

  7. Bessou P, Perl ER (1969) Response of cutaneous sensory units with unmyelinated fibers to noxious stimuli. J Neurophysiol 32:1025–1943

    PubMed  CAS  Google Scholar 

  8. Kumazawa T, Mizumura K, Sato J (1987) Response properties of polymodal receptors studied using in vitro testis superior spermatic nerve preparation of dogs. J Neurophysiol 57:702–711

    PubMed  CAS  Google Scholar 

  9. Reeh PW (1986) Sensory receptors in mammalian skin in an in vitro preparation. Neurosci Lett 66:141–147

    Article  PubMed  CAS  Google Scholar 

  10. Cain DM, Khasabov SG, Simone DA (2001) Response properties of mechanoreceptors and nociceptors in mouse glabrous skin: an in vivo study. J Neurophysiol 85:1561–1574

    PubMed  CAS  Google Scholar 

  11. Reeh PW (1988) Sensory receptors in a mammalian skin-nerve in vitro preparation. Prog Brain Res 74(271-6):271–276

    Article  PubMed  CAS  Google Scholar 

  12. Bernardini N, Roza C, Sauer SK, Gomeza J, Wess J, Reeh PW (2002) Muscarinic M2 rece­ptors on peripheral nerve endings: a mole­cular target of antinociception. J Neurosci 22:RC229

    PubMed  Google Scholar 

  13. Kress M, Averbeck B (1997) Release of calcitonin gene-related peptide from sensory neurons in culture. Pflügers Archiv - Europ J Physiol 433:360.

    Google Scholar 

  14. Kress M, Riedl B, Reeh PW (1995) Effects of oxygen radicals on nociceptive afferents in the rat skin in vitro. Pain 62:87–94

    Article  PubMed  CAS  Google Scholar 

  15. Kress M, Rödl J, Reeh PW (1996) Stable analogs of cyclic AMP but not cyclic GMP sensitize unmyelinated primary afferents in the rat skin to mechanical and heat stimuli but not to inflammatory mediators, in vitro. Neuro­science 74:609–617

    Article  PubMed  CAS  Google Scholar 

  16. Mogil JS, Miermeister F, Seifert F, Strasburg K, Zimmermann K, Reinold H, Austin JS, Bernardini N, Chesler EJ, Hofmann HA et al (2005) Variable sensitivity to noxious heat is mediated by differential expression of the CGRP gene. Proc Natl Acad Sci U S A 102:12938–12943

    Article  PubMed  CAS  Google Scholar 

  17. Petho G, Derow A, Reeh PW (2001) Bradykinin-induced nociceptor sensitization to heat is mediated by cyclooxygenase pro­ducts in isolated rat skin. Eur J NeuroSci 14:210–218

    Article  PubMed  CAS  Google Scholar 

  18. Reeh PW, Petho G (2000) Nociceptor excitation by thermal sensitization - a hypothesis. Prog Brain Res 129:39–50

    Article  PubMed  CAS  Google Scholar 

  19. Ringkamp M, Schmelz M, Kress M, Allwang M, Ogilvie A, Reeh PW (1994) Activated human platelets in plasma excite nociceptors in rat skin, in vitro. Neurosci Lett 170:103–106

    Article  PubMed  CAS  Google Scholar 

  20. Steen KH, Wegner H, Reeh PW (1999) The pH response of rat cutaneous nociceptors correlates with extracellular [Na+] and is increased under amiloride. Eur J NeuroSci 11:2783–2792

    Article  PubMed  CAS  Google Scholar 

  21. Steen KH, Reeh PW, Kreysel HW (1995) Topical acetylsalicylic, salicylic acid and indomethacin supress pain from experimental tissue acidosis in human skin. Pain 62:339–347

    Article  PubMed  CAS  Google Scholar 

  22. Alloui A, Zimmermann K, Mamet J, Duprat F, Noel J, Chemin J, Guy N, Blondeau N, Voilley N, Rubat-Coudert C et al (2006) TREK-1, a K+ channel involved in polymodal pain perception. EMBO J 25:2368–2376

    Article  PubMed  CAS  Google Scholar 

  23. Zimmermann K, Leffler A, Fischer MM, Messlinger K, Nau C, Reeh PW (2005) The TRPV1/2/3 activator 2-aminoethoxydiphenyl borate sensitizes native nociceptive neurons to heat in wildtype but not TRPV1 deficient mice. Neuroscience 135:1277–1284

    Article  PubMed  CAS  Google Scholar 

  24. Kirchhoff C, Jung S, Reeh PW, Handwerker HO (1990) Carrageenan inflammation increases bradykinin sensitivity of rat cutaneous nociceptors. Neurosci Lett 111:206–210

    Article  PubMed  CAS  Google Scholar 

  25. Koltzenburg M, Kress M, Reeh PW (1992) The nociceptor sensitization by bradykinin does not depend on sympathetic neurons. Neuroscience 46:465–473

    Article  PubMed  CAS  Google Scholar 

  26. Spitzer MJ, Reeh PW, Sauer SK (2008) Mechanisms of potassium- and capsaicin-induced axonal calcitonin gene-related peptide release: involvement of L- and T-type calcium channels and TRPV1 but not sodium channels. Neuroscience 151:836–842

    Article  PubMed  CAS  Google Scholar 

  27. Averbeck B, Peisler MIIRPW (2003) Inflammatory mediators do not stimulate CGRP release if prostaglandin synthesis is blocked by S(+)-flurbiprofen in isolated rat skin. Inflamm Res 52(12):519–523

    Article  PubMed  CAS  Google Scholar 

  28. Averbeck B, Reeh PW, Michaelis M (2001) Modulation of CGRP and PGE2 release from isolated rat skin by alpha-adrenoceptors and kappa-opioid-receptors. Neuroreport 12:2097–2100

    Article  PubMed  CAS  Google Scholar 

  29. Huang YM, Neher E (1996) Ca2+-dependent exocytosis in the somata of dorsal root ganglion neurons. Neuron 17:135–145

    Article  PubMed  CAS  Google Scholar 

  30. Safronov BV, Bischoff U, Vogel W (1996) Single voltage-gated K+ channels and their functions in small dorsal root ganglion neurones of rat. J Physiol 493(Pt 2):393–408

    PubMed  CAS  Google Scholar 

  31. Scholz A, Gruss M, Vogel W (1998) Properties and functions of calcium-activated K+ channels in small neurones of rat dorsal root ganglion studied in a thin slice preparation. J Physiol 513(Pt 1):55–69

    Article  PubMed  CAS  Google Scholar 

  32. Woodbury CJ, Zwick M, Wang S, Lawson JJ, Caterina MJ, Koltzenburg M, Albers KM, Koerber HR, Davis BM (2004) Nociceptors lacking TRPV1 and TRPV2 have normal heat responses. J Neurosci 24:6410–6415

    Article  PubMed  CAS  Google Scholar 

  33. Zimmermann K, Leffler A, Babes A, Cendan CM, Carr RW, Kobayashi J, Nau C, Wood JN, Reeh PW (2007) Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures. Nature 447:855–858

    Article  PubMed  CAS  Google Scholar 

  34. Reid G, Flonta ML (2001) Physiology. Cold current in thermoreceptive neurons. Nature 413:480

    Article  PubMed  CAS  Google Scholar 

  35. Dittert I, Vlachova V, Knotkova H, Vitaskova Z, Vyklicky L, Kress M, Reeh PW (1998) A technique for fast application of heated solutions of different composition to cultured neurones. J Neurosci Methods 82:195–201

    Article  PubMed  CAS  Google Scholar 

  36. Dittert I, Benedikt J, Vyklicky L, Zimmermann K, Reeh PW, Vlachova V (2006) Improved superfusion technique for rapid cooling or heating of cultured cells under patch-clamp conditions. J Neurosci Methods 151: 178–185

    Article  PubMed  Google Scholar 

  37. Ellman GL, Courtney KD, Andres V Jr, Feather-Stone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95

    Article  PubMed  CAS  Google Scholar 

  38. Bretag AH (1969) Synthetic interstitial fluid for isolated mammalian tissue. Life Science 8:319–329

    Article  CAS  Google Scholar 

  39. Zimmermann K, Hager UA, Hein A, Kaczmarek JS, Turnquist BP, Clapham DE, Reeh PW (2009) Phenotyping sensory nerve endings in vitro in the mouse. Nature Protocols 4(2):174–196

    Article  PubMed  CAS  Google Scholar 

  40. Sakmann B, Neher E (1995) Single-channel recording. Plenum, New York

    Book  Google Scholar 

  41. Anonymus (2008) The axon guide: a guide to electrophysiology and biophysics laboratory techniques. Molecular Devices, Sunnyvale, CA., USA

    Google Scholar 

  42. Knight DE (1990) Disposable filters may damage your cells. Nature 343:218

    Article  PubMed  CAS  Google Scholar 

  43. Bottenstein JE, Sato GH (1979) Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci U S A 76:514–517

    Article  PubMed  CAS  Google Scholar 

  44. Bottenstein JE, Skaper SD, Varon SS, Sato GH (1980) Selective survival of neurons from chick embryo sensory ganglionic dissociates utilizing serum-free supplemented medium. Exp Cell Res 125:183–190

    Article  PubMed  CAS  Google Scholar 

  45. Purves RD (1981) Microelectrode methods for intracellular recording and ionophoresis. Academic, London

    Google Scholar 

  46. Viana F, de la Peña E, Belmonte C (2002) Specificity of cold thermotransduction is determined by differential ionic channel expression. Nat Neurosci 5:254–260

    Article  PubMed  CAS  Google Scholar 

  47. Reid G, Flonta ML (2002) Ion channels activated by cold and menthol in cultured rat dorsal root ganglion neurones. Neurosci Lett 324:164–168

    Article  PubMed  CAS  Google Scholar 

  48. McKemy DD, Neuhausser WM, Julius D (2002) Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416:52–58

    Article  PubMed  CAS  Google Scholar 

  49. Freshney RI (2005) Cuture of animal cells: a manual of basic technique. Wiley-Blackwell, Oxford

    Book  Google Scholar 

  50. Zaidi M, Girgis SI, MacIntyre I (1988) Development and performance of a highly sensitive carboxyl-terminal-specific radioimmunoassay of calcitonin gene-related peptide. Clin Chem 34:655–660

    PubMed  CAS  Google Scholar 

  51. Rae J, Cooper K, Gates P, Watsky M (1991) Low access resistance perforated patch recordings using amphotericin B. J Neurosci Methods 37:15–26

    Article  PubMed  CAS  Google Scholar 

  52. Kyrozis A, Reichling DB (1995) Perforated-patch recording with gramicidin avoids ­artifactual changes in intracellular chloride concentration. J Neurosci Methods 57:27–35

    Article  PubMed  CAS  Google Scholar 

  53. Lindau M, Fernandez JM (1986) A patch-clamp study of histamine-secreting cells. J Gen Physiol 88:349–368

    Article  PubMed  CAS  Google Scholar 

  54. Reid G, Babes A, Pluteanu F (2002) A cold- and menthol-activated current in rat dorsal root ganglion neurones: properties and role in cold transduction. J Physiol 545:595–614

    Article  PubMed  CAS  Google Scholar 

  55. Nassenstein C, Kwong KK, Taylor-Clark TE, Kollarik M, Macglashan DW, Braun A, Undem BJ (2008) TRPA1 expression and function in vagal afferent nerves innervating mouse lungs. J Physiol 586(6):1595–1604

    Article  PubMed  CAS  Google Scholar 

  56. Fajardo O, Meseguer V, Belmonte C, Viana F (2008) TRPA1 channels mediate cold temperature sensing in mammalian vagal sensory neurons: pharmacological and genetic evidence. J Neurosci 28:7863–7875

    Article  PubMed  CAS  Google Scholar 

  57. Price TJ, Flores CM (2007) Critical evaluation of the colocalization between calcitonin gene-related peptide, substance P, transient receptor potential vanilloid subfamily type 1 immunoreactivities, and isolectin B4 binding in primary afferent neurons of the rat and mouse. J Pain 8:263–272

    PubMed  CAS  Google Scholar 

  58. Kress M, Izydorczyk I, Kuhn A (2001) N- and L- but not P/Q-type calcium channels contribute to neuropeptide release from rat skin in vitro. Neuroreport 12:867–870

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Physiology and Biophysics, University of Bucharest, Bucharest, Romania

    Alexandru Babes

  2. Institute of Physiology and Pathophysiology, University of Erlangen – Nuremberg, Erlangen, Germany

    Michael J. M. Fischer, Susanne K. Sauer, Katharina Zimmermann & Peter W. Reeh

  3. Department of Physiology, University College, Cork, Ireland

    Gordon Reid

Authors
  1. Alexandru Babes
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  2. Michael J. M. Fischer
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  3. Gordon Reid
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  4. Susanne K. Sauer
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  5. Katharina Zimmermann
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  6. Peter W. Reeh
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Corresponding author

Correspondence to Peter W. Reeh .

Editor information

Editors and Affiliations

  1. Dept. Pathology, Monmouth Medical Center, Second Ave. 300, Long Branch, 07750, USA

    Arpad Szallasi

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Babes, A., Fischer, M.J.M., Reid, G., Sauer, S.K., Zimmermann, K., Reeh, P.W. (2010). Electrophysiological and Neurochemical Techniques to Investigate Sensory Neurons in Analgesia Research. In: Szallasi, A. (eds) Analgesia. Methods in Molecular Biology, vol 617. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-323-7_19

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  • DOI: https://doi.org/10.1007/978-1-60327-323-7_19

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-322-0

  • Online ISBN: 978-1-60327-323-7

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