Patch‐Pipet Recording in Brain Slices

Greg Stuart1

1 John Curtin School of Medical Research, Australian National University, Canberra, Australia
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.7
DOI:  10.1002/0471142301.ns607s02
Online Posting Date:  May, 2001
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Abstract

Patch‐clamp recording in brain slices provides a powerful approach for investigating the intrinsic electrical properties of neurons and glia and analyzing synaptic interactions between neurons. There are two main methods presently in use to obtain patch‐clamp recordings from neuronal or glial cell bodies and their processes in brain slices, both described in this unit. In the blind technique described in this unit, the approach to a cell is performed blindly and recordings are obtained relying purely on electrical cues. Alternatively, the movement of the recording pipet through the brain slice and its placement onto the cell membrane can be performed as described under visual control; this method is usually performed with the aid of differential interference contrast (DIC) optics and is referred to as the DIC technique. Increased resolution of cells and their processes can be obtained by combining DIC optics with infrared illumination and video microscopy techniques. Using either of these methods it is possible to reliably apply the full power of the patch‐clamp technique to the study of neurons and glial cells in brain slices.

     
 
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Table of Contents

  • Basic Protocol 1: The Blind Technique
  • Basic Protocol 2: The DIC Technique
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Blind Technique

  Materials
  • Extracellular artificial cerebrospinal fluid (aCSF) solution (see recipe), oxygenated with carbogen gas (95% O 2/5% CO 2)
  • Intracellular patch‐pipet solution (see recipe)
  • Experimental chamber (either immersion or interface type) for holding brain slices during recording
  • Basic patch‐clamp setup and associated equipment, including amplifier (headstage and pipet holder), manipulator, and computer (units 6.1 & 6.6)
  • Patch pipets (unit 6.3)
  • Dissecting microscope

Basic Protocol 2: The DIC Technique

  Materials
  • Extracellular artificial cerebrospinal fluid (aCSF) solution (see recipe) oxygenated with carbogen gas (95% O 2/5% CO 2)
  • Intracellular patch‐pipet solution (see recipe)
  • Experimental chamber with glass bottom
  • Microscope: upright, preferably of “fixed‐stage” type, with DIC or Hoffman optics and high‐aperture (>0.7) water‐immersion lens and condenser (e.g., Zeiss or Olympus)
  • Infrared band‐pass filter (e.g., Omega Optical PIN 770 WB 40)
  • Infrared‐sensitive video camera (e.g., Newvicon C2400‐07)
  • Black and white video monitor
  • Pipet manipulator (e.g., Sutter)
  • Patch pipets (unit 6.3)
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Figures

Videos

Literature Cited

Literature Cited
   Blanton, M.G., Loturco, J.J., and Kriegstein, A.R. 1989. Whole cell recording from neurons in slices of reptilian mammalian cerebral cortex. J. Neurosci. Methods 30:203‐210.
   Borst, J.G.G., Helmchen, F., and Sakmann, B. 1995. Pre‐ and postsynaptic whole‐cell recordings in the medial nucleus of the trapezoid body of the rat. J. Physiol 489:825‐840.
   Dodt, H.‐U. and Zieglgänsberger, W. 1990. Visualizing unstained neurons in living brain slices by infrared DIC‐videomicroscopy. Brain Res. 537:333‐336.
   Edwards, F.A., Konnerth, A., Sakmann, B., and Takahashi, T. 1989. A thin slice preparation for patch clamp recordings from neurons of the mammalian central nervous system. Pfluegers Arch. Eur. J. Physiol. 414:600‐612.
   Ferster, D. and Jagadeesh, B. 1992. EPSP‐IPSP interactions in cat visual cortex studied with in vivo whole‐cell patch recording. J. Neurosci 12:1262‐1274.
   Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J. 1981. Improved patch‐clamp techniques for high‐resolution current recording from cells and cell‐free membrane patches. Pfluegers Arch. Eur. J. Physiol. 391:85‐100.
   Pei, X., Volgushev, M., Vidyasagar, T.R. and Creutzfeldt, O.D. 1991. Whole cell recording and conductance measurements in cat visual cortex in‐vivo. Neuroreport 2:485‐488.
   Pongracz, F., Firestein, S., and Shepherd, G.M. 1991. Electrotonic structure of olfactory sensory neurons analyzed by intracellular and whole cell patch techniques. J. Neurophysiol 65:747‐758.
   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. and Spruston, N. 1995. Probing dendritic function with patch pipettes. Curr. Opin. Neurobiol 5:389‐394.
   Stuart, G.J., Dodt, H.‐U. and Sakmann, B. 1993. Patch‐clamp recordings from the soma and dendrites of neurones in brain slices using infrared video microscopy. Pfluegers Arch. Eur. J. Physiol 423:511‐518.
   Stuart, G.J. and Sakmann, B. 1994. Active propagation of somatic action potentials into neocortical pyramidal cell dendrites. Nature 367:69‐72
   Takahashi, T. 1978. Intracellular recording from visually identified motoneurons in rat spinal cord slices. J. Proc. R. Soc. Lond. (Biol.) 202:417‐421.
Key References
   Blanton et al., 1989. See above.
  Original paper describing the blind patch technique to make patch‐pipet recordings from neurons in brain slices.
   Stuart et al., 1993. See above.
  Original paper describing the use of infrared DIC optics to make patch‐pipet recordings under visual control from neurons and their processes in brain slices.
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