Patch‐Clamp Recording from Neuronal Dendrites

Nicholas P. Poolos1, Terrance D. Jones1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.19
DOI:  10.1002/0471142301.ns0619s29
Online Posting Date:  November, 2004
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Abstract

Pyramidal neurons of the central nervous system have extensively arborized apical dendrites that contribute importantly to the signaling properties of the neuron. Recent advances in electrophysiological techniques have allowed recording from neuronal dendrites. These techniques depend on using infrared optics to visualize dendritic processes in the unstained brain slice preparation, on pipet positioning with high resolution micromanipulators, and on stringent techniques for brain slice preparation that preserved healthy dendritic processes, even in tissue from mature animals. The procedures underlying these techniques are described in this unit.

Keywords: dendrites; patch‐clamp recording; brain slice; hippocampus; IR‐DIC microscopy

     
 
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Table of Contents

  • Basic Protocol 1: Dendritic Recording
  • Basic Protocol 2: Simultaneous Dendritic and Somatic Recording
  • Support Protocol 1: Preparation of Brain Slices
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Dendritic Recording

  Materials
  • Brain slice (see protocol 3)
  • Extracellular solution (see recipe)
  • Internal pipet solution—e.g., for whole‐cell or cell‐attached recordings (see reciperecipes)
  • Experimental chamber (see Fig. B)
  • 34‐G platinum wire semicircle held with nylon threads (see Fig. B)
  • Upright microscope with IR‐DIC optics (e.g., Zeiss Axioskop 2 FSplus, Olympus BX‐51), 40× to 63× water‐immersion objective, IR‐sensitive video or CCD camera (e.g., Dage Instruments Newvicon, Hamamatsu C7500‐50), and high‐contrast monitor (e.g., Dage Instruments HR120)
  • Patch pipets (unit 6.3), preferably coated with Sylgard and fire‐polished with a microforge (e.g., Narishige MF 830) just before use
  • 5‐ml syringes with tubing appropriate for pipet
  • Flexible needle (e.g., Kwick‐fill, World Precision Instruments)
  • Electrophysiology setup (units 6.1& 6.6) with high‐resolution electromechanical, hydraulic, or piezoelectric micromanipulator (e.g., Sutter MP‐225)
  • Pressure gauge (e.g., Dwyer Magnelic 2050)
  • Additional reagents and equipment for patch‐pipet recording in brain slices using the DIC technique (unit 6.7)

Basic Protocol 2: Simultaneous Dendritic and Somatic Recording

  Materials
  • Anesthetic: e.g., ketamine/xylazine/acepromazine
  • Experimental animal (e.g., rat)
  • Perfusion solution (see recipe), ice cold and bubbled with carbogen (95% O 2/5% CO 2)
  • Vetbond (cyanoacrylate glue, 3M)
  • Extracellular solution (see recipe), bubbled with carbogen
  • Absorbent underpad (Fisher)
  • No. 10 scalpel
  • Large dissecting forceps, toothed
  • Surgical scissors
  • Large hemostat
  • 18‐ and 25‐G hypodermic needles
  • Intravenous (i.v.) perfusion set and delivery system (e.g., 60‐ml syringe reservoir with appropriate tubing)
  • Pyrex dish, round, 3‐in. diameter with 1‐in. sides
  • Iridectomy scissors, carbide‐edged
  • Bone rongeurs
  • Weighing minispatula with a 30° bend in the blade
  • Filter paper disk, 2‐in. diameter
  • Petri dish, chilled
  • Vibrating tissue slicer (e.g., Vibratome 1500; Electron Microscopy Sciences OTS 4000)
  • Incubation chamber: 150‐ml Pyrex beaker fitted with an acrylic ring and 35‐mm‐i.d. plastic petri dish to support gauze platform (see Fig. A)
  • 35°C water bath
  • Cutting blade, preferrably glass (Pelco knife maker, Ted Pella) or artificial sapphire (Delaware Diamond Knives)
  • Dumont tweezers
  • Additional reagents and equipment for anesthetizing animals ( appendix 4B)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.
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Figures

Videos

Literature Cited

   Benardo, L.S., Masukawa, L.M., and Prince, D.A. 1982. Electrophysiology of isolated hippocampal pyramidal dendrites. J. Neurosci. 2:1614‐1622.
   Carpenter, M.B. 1985. Core Text of Neuroanatomy. Williams & Wilkins, Baltimore, Md.
   Hausser, M., Spruston, N., and Stuart, G.J. 2000. Diversity and dynamics of dendritic signaling. Science 290:739‐744.
   Hoffman, D.A., Magee, J.C., Colbert, C.M., and Johnston, D. 1997. K+ channel regulation of signal propagation in dendrites of hippocampal pyramidal neurons. Nature 387:869‐875.
   Lee, J.Y., Kim, Y.H., and Koh, J.Y. 2001. Protection by pyruvate against transient forebrain ischemia in rats. J. Neurosci. 21:RC171.
   MacGregor, D.G., Higgins, M.J., Jones, P.A., Maxwell, W.L., Watson, M.W., Graham, D.I., and Stone, T.W. 1996. Ascorbate attenuates the systemic kainate‐induced neurotoxicity in the rat hippocampus. Brain Res. 727:133‐144.
   Moyer, J.R. Jr. and Brown, T.H. 1998. Methods for whole‐cell recording from visually preselected neurons of perirhinal cortex in brain slices from young and aging rats. J. Neurosci. Methods 86:35‐54.
   Poolos, N.P. and Kocsis, J.D. 1990. Dendritic action potentials activated by NMDA receptor‐mediated EPSPs in CA1 hippocampal pyramidal cells. Brain Res. 524:342‐346.
   Poolos, N.P., Migliore, M., and Johnston, D. 2002. Pharmacological upregulation of h‐channels reduces the excitability of pyramidal neuron dendrites. Nat. Neurosci. 5:767‐774.
   Spruston, N. and Johnston, D. 1992. Perforated patch‐clamp analysis of the passive membrane properties of three classes of hippocampal neurons. J. Neurophysiol. 67:508‐529.
   Stuart, G.J. and Sakmann, B. 1994. Active propagation of somatic action potentials into neocortical pyramidal cell dendrites. Nature 367:69‐72.
   Stuart, G.J., Dodt, H.U., and Sakmann, B. 1993. Patch‐clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. Pflugers Arch. 423:511‐518.
   Velumian, A.A., Zhang, L., Pennefather, P., and Carlen, P.L. 1997. Reversible inhibition of IK, IAHP, Ih and ICa currents by internally applied gluconate in rat hippocampal pyramidal neurones. Pflugers Arch. 433:343‐350.
   Zhu, J.J. 2000. Maturation of layer 5 neocortical pyramidal neurons: Amplifying salient layer 1 and layer 4 inputs by Ca2+ action potentials in adult rat tuft dendrites. J. Physiol. 3:571‐587.
Key References
   Stuart et al., 1993. See above.
  First application of IR‐DIC optical techniques to patch‐clamp recording in brain slices.
   Moyer and Brown, 1998. See above.
  Detailed examination of tissue slicing parameters that affect neuronal health when using mature animals.
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