Advertisement

Single-Cell Neuronal Dissociation for Electrophysiological Studies

  • Yu-Long LiEmail author
Protocol
  • 3.9k Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Since the patch-clamp technique was initially applied about 35 years ago, it has been thought to be a powerful method for studying electrical properties of ion channels and cell excitability in the varied excitable cells including peripheral and central neurons. Although we understand that many factors can affect the patch-clamp recording, the viability of the peripheral and central neurons is an essential point for the success of the patch-clamp experiments. In this chapter, I introduce (1) acute dissociation procedures of the peripheral neurons from ganglia and the central neurons from brain (such as equipment, materials, preparation of tissues, enzymatic treatment of the tissues, mechanical agitation of the tissue fragments, and preservation of the isolated cells), (2) explanatory notes for the whole procedure of the acute neuronal isolation, and (3) advantages and disadvantages of the isolated peripheral and central neurons for the electrophysiological studies.

Keywords

Action potential Cell culture Cell isolation Central nervous system Electrophysiology Ion channel Patch clamp Peripheral nervous system 

References

  1. Aitken PG, Breese GR, Dudek FF, Edwards F, Espanol MT, Larkman PM, Lipton P, Newman GC, Nowak TS Jr, Panizzon KL (1995) Preparative methods for brain slices: a discussion. J Neurosci Methods 59:139–149CrossRefPubMedGoogle Scholar
  2. Akaike N, Lee KS, Brown AM (1978) The calcium current of Helix neuron. J Gen Physiol 71:509–531CrossRefPubMedGoogle Scholar
  3. Aras MA, Hartnett KA, Aizenman E (2008) Assessment of cell viability in primary neuronal cultures. Curr Protoc Neurosci Chapter 7:Unit 7.18.1–7.18.15Google Scholar
  4. Brewer GJ (1997) Isolation and culture of adult rat hippocampal neurons. J Neurosci Methods 71:143–155CrossRefPubMedGoogle Scholar
  5. Brewer GJ, Torricelli JR (2007) Isolation and culture of adult neurons and neurospheres. Nat Protoc 2:1490–1498CrossRefPubMedGoogle Scholar
  6. Giordano G, Hong S, Faustman EM, Costa LG (2011) Measurements of cell death in neuronal and glial cells. Methods Mol Biol 758:171–178CrossRefPubMedGoogle Scholar
  7. Goslin K, Banker G (1989) Experimental observations on the development of polarity by hippocampal neurons in culture. J Cell Biol 108:1507–1516CrossRefPubMedGoogle Scholar
  8. Huettner JE, Baughman RW (1986) Primary culture of identified neurons from the visual cortex of postnatal rats. J Neurosci 6:3044–3060PubMedGoogle Scholar
  9. Ishizuka S, Hattori K, Akaike N (1984) Separation of ionic currents in the somatic membrane of frog sensory neurons. J Membr Biol 78:19–28CrossRefPubMedGoogle Scholar
  10. Jiang N, Shi P, Li H, Lu S, Braseth L, Cuadra AE, Raizada MK, Sumners C (2009) Phosphate-activated glutaminase-containing neurons in the rat paraventricular nucleus express angiotensin type 1 receptors. Hypertension 54:845–851CrossRefPubMedPubMedCentralGoogle Scholar
  11. Kay AR, Krupa DJ (2001) Acute isolation of neurons from the mature mammalian central nervous system. Curr Protoc Neurosci Chapter 6:Unit 6.5.1–6.5.7Google Scholar
  12. Kay AR, Wong RK (1986) Isolation of neurons suitable for patch-clamping from adult mammalian central nervous systems. J Neurosci Methods 16:227–238CrossRefPubMedGoogle Scholar
  13. Kostyuk PG, Krishtal OA, Pidoplichko VI, Shakhovalov Y (1979) Kinetics of calcium inward current activation. J Gen Physiol 73:675–680CrossRefPubMedGoogle Scholar
  14. Kuehl-Kovarik MC, Partin KM, Magnusson KR (2003) Acute dissociation for analyses of NMDA receptor function in cortical neurons during aging. J Neurosci Methods 129:11–17CrossRefPubMedGoogle Scholar
  15. Lamas JA, Selyanko AA, Brown DA (1997) Effects of a cognition-enhancer, linopirdine (DuP 996), on M-type potassium currents (IK(M)) and some other voltage- and ligand-gated membrane currents in rat sympathetic neurons. Eur J Neurosci 9:605–616CrossRefPubMedGoogle Scholar
  16. Leal-Cardoso H, Koschorke GM, Taylor G, Weinreich D (1993) Electrophysiological properties and chemosensitivity of acutely isolated nodose ganglion neurons of the rabbit. J Auton Nerv Syst 45:29–39CrossRefPubMedGoogle Scholar
  17. Li XM, Li JG, Yang JM, Hu P, Li XW, Wang Y, Qin LN, Gao TM (2004a) An improved method for acute isolation of neurons from the hippocampus of adult rats suitable for patch-clamping study. Sheng Li Xue Bao 56:112–117PubMedGoogle Scholar
  18. Li YL, Sun SY, Overholt JL, Prabhakar NR, Rozanski GJ, Zucker IH, Schultz HD (2004b) Attenuated outward potassium currents in carotid body glomus cells of heart failure rabbit: involvement of nitric oxide. J Physiol 555:219–229CrossRefPubMedGoogle Scholar
  19. Li YL, Tran TP, Muelleman R, Schultz HD (2008) Blunted excitability of aortic baroreceptor neurons in diabetic rats: involvement of hyperpolarization-activated channel. Cardiovasc Res 79:715–721CrossRefPubMedGoogle Scholar
  20. Lipton P, Aitken PG, Dudek FE, Eskessen K, Espanol MT, Ferchmin PA, Kelly JB, Kreisman NR, Landfield PW, Larkman PM et al (1995) Making the best of brain slices: comparing preparative methods. J Neurosci Methods 59:151–156CrossRefPubMedGoogle Scholar
  21. Mattson MP, Dou P, Kater SB (1988) Outgrowth-regulating actions of glutamate in isolated hippocampal pyramidal neurons. J Neurosci 8:2087–2100PubMedGoogle Scholar
  22. Neher E, Sakmann B (1976) Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260:799–802CrossRefPubMedGoogle Scholar
  23. Oyelese AA, Eng DL, Richerson GB, Kocsis JD (1995) Enhancement of GABAA receptor-mediated conductances induced by nerve injury in a subclass of sensory neurons. J Neurophysiol 74:673–683CrossRefPubMedPubMedCentralGoogle Scholar
  24. Stoddart MJ (2011) Cell viability assays: introduction. Methods Mol Biol 740:1–6CrossRefPubMedGoogle Scholar
  25. Summers BA, Overholt JL, Prabhakar NR (2002) CO(2) and pH independently modulate L-type Ca(2+) current in rabbit carotid body glomus cells. J Neurophysiol 88:604–612CrossRefPubMedGoogle Scholar
  26. Tan ZY, Lu Y, Whiteis CA, Benson CJ, Chapleau MW, Abboud FM (2007) Acid-sensing ion channels contribute to transduction of extracellular acidosis in rat carotid body glomus cells. Circ Res 101:1009–1019CrossRefPubMedGoogle Scholar
  27. Tu H, Zhang L, Tran TP, Muelleman RL, Li YL (2010) Reduced expression and activation of voltage-gated sodium channels contributes to blunted baroreflex sensitivity in heart failure rats. J Neurosci Res 88:3337–3349CrossRefPubMedPubMedCentralGoogle Scholar
  28. Ye JH, Zhang J, Xiao C, Kong JQ (2006) Patch-clamp studies in the CNS illustrate a simple new method for obtaining viable neurons in rat brain slices: glycerol replacement of NaCl protects CNS neurons. J Neurosci Methods 158:251–259CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Emergency MedicineUniversity of Nebraska Medical CenterOmahaUSA

Personalised recommendations