Electrophysiological Studies of Neurotoxicants on Central Synaptic Transmission in Acutely Isolated Brain Slices

Yukun Yuan1, William D. Atchison1

1 Michigan State University, East Lansing, Michigan
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.11
DOI:  10.1002/0471140856.tx1111s17
Online Posting Date:  November, 2003
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Abstract

Effects of neurotoxic chemicals on central synaptic function can be assessed using electrophysiological recording techniques in freshly isolated brain slices. The three most commonly used electrophysiological recording methods in brain slices are extracellular microelectrode recording, intracellular microelectrode recording, and whole‐cell patch clamp recording. This unit presents the basic procedures and applications of extra‐ and intracellular recordings in hippocampal slices and whole‐cell recording in cerebellar slices.

Keywords: Brain slices; synaptic transmission; extracellular recording; intracellular recording; whole‐cell recording

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Extracellular Microelectrode Recordings in Hippocampal or Cerebellar Slices
  • Basic Protocol 2: Intracellular Recording in Hippocampal Slices
  • Basic Protocol 3: Whole‐Cell Current Recordings in Purkinje or Granule Cells in Cerebellar Slices
  • Support Protocol 1: Preparation of Acutely Isolated Hippocampal Slices
  • Support Protocol 2: Preparation of Acutely Isolated Cerebellar Slices
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Extracellular Microelectrode Recordings in Hippocampal or Cerebellar Slices

  Materials
  • 95% O 2/5% CO 2 saturated artificial cerebrospinal fluid (ACSF, see recipe)
  • 95% O 2/5% CO 2 gas mixture source
  • 300‐ to 400‐µm healthy hippocampal slices (see protocol 4)
  • 3 M NaCl, optional
  • Perfusion system (see Fig. ):
    • 60‐ml syringes
    • Perfusion valve control (e.g., VC‐6 valve control system, Warner Instruments)
    • PE‐160 tubing
    • 2000‐ml flask with tubing (Pyrex)
    • Vacuum pump (Cole‐Palmer Instruments)
    • Recording chamber (interface or submersion chamber, e.g., a modified RC26 chamber assembled with a slice support, Warner Instruments)
  • Ag/AgCl ground electrode (e.g., REF‐1L reference cell or E206 Ag/AgCl pellet electrode, Warner Instruments)
  • Basic electrophysiological setup:
    • Microelectrode amplifier (e.g., Axoclamp‐2 microelectrode clamp)
    • Digidata 1200 interface (Axon Instruments)
    • PC computer system installed with pClamp 8.x software
    • Two manually‐operated manipulators (e.g., MP1 Narishige manipulator, Narishige Scientific Instruments)
    • Stimulator (Grass S88, Grass)
    • Stimulus isolation unit (Grass SIU5, Grass)
  • Oscilloscope (Gould Instrument Systems).
  • Wide‐bore Pasteur pipet (simply break the narrow tip end and insert it into a small rubber suction bulb)
  • U‐shaped platinum‐nylon mesh
  • Fiber‐optic light (Dolan Jenner Industrials)
  • Dissection microscope (e.g., Wild M5A)
  • Extracellular recording pipets pulled from borosilicated glass capillaries (1.0‐mm o.d., 0.5‐mm i.d.; tip impedance of 5 to 15 MΩ when filled with ACSF or 3 to 4 M NaCl)
  • Glass microelectrode puller (e.g., Model P‐97, Sutter Instruments)
  • Pipet filler: MicroFil MF28G needle (WPI)
  • Solution in‐line heater SH‐27B and TC‐344B chamber system heater controller (Warner Instruments) or other alternative heating system (depending on the type of chamber being used), this may be required if experiments will be performed at 30° to 35°C
  • Concentric bipolar metal electrode or monopolar tungsten electrode (3 MΩ, FHC; alternatively, a broken‐tip glass pipet filled with ACSF can also be used as the stimulation electrode)
NOTE: The oscilloscope can be replaced with the computer and monitor if appropriate software is available.

Basic Protocol 2: Intracellular Recording in Hippocampal Slices

  Materials
  • 95% O 2/5% CO 2 saturated artificial cerebrospinal fluid (ACSF, see recipe)
  • 95% O 2/5% CO 2 gas mixture source
  • 300‐ to 400‐µm healthy hippocampal slices (see protocol 4)
  • 3 to 4 M KCl or potassium acetate
  • Neurotransmitters of interest
  • 500 mM glutamate in 100 mM NaCl (pH 8.0)
  • Perfusion system (see Fig. ):
    • 60‐ml syringes
    • Perfusion valve control (e.g., VC‐6 valve control system, Warner Instruments)
    • PE‐160 tubing
    • 2000‐ml flask with tubing (Pyrex)
    • Vacuum pump (Cole‐Palmer Instruments)
    • Recording chamber (interface or submersion chamber, e.g., a modified RC26 chamber assembled with a slice support, Warner Instruments)
  • Ag/AgCl ground electrode (e.g., REF‐1L reference cell or E206 Ag/AgCl pellet electrode, Warner Instruments)
  • Wide‐bore Pasteur pipet (simply break the narrow tip end and insert it into a small rubber suction bulb)
  • U‐shaped platinum frame with attached nylon mesh
  • Microscope
  • Basic electrophysiological setup:
    • Microelectrode amplifier (e.g., Axoclamp‐2 microelectrode clamp)
    • Digidata 1200 Interface (Axon Instruments)
    • PC or Macintosh computer system installed with pClamp 8.x software, MiniAnalysis 5.2.8 (Synaptosoft), or other available software
    • Two manipulators (one must have fine‐movement control, preferably a remote, fine‐movement control system—motorized, hydraulic, or piezoelectric control—e.g., MP1 Narishige manipulator, Narishige Scientific Instruments)
    • Grass S88 stimulator and Grass SIU5 stimulus isolation unit (Grass)
  • Intracellular recording pipets pulled from borosilicated glass capillaries (impedance of 80 to 120 MΩ for CA1 neurons when filled with 3 to 4 M KCl or potassium acetate)

Basic Protocol 3: Whole‐Cell Current Recordings in Purkinje or Granule Cells in Cerebellar Slices

  Materials
  • Thin 150‐ to 200‐µm cerebellar slice (see protocol 5)
  • ACSF (see recipe), ice cold and saturated with 95% O 2/5% CO 2
  • Intracellular pipet solution: the composition of pipet solution is highly dependent on the experimental purpose or design (see reciperecipes for internal pipet solutions and KGlu‐based pipet solution, and Critical Parameters)
  • Glutamate receptor blockers such as 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 10 to 20 µM) for kainate/AMPA receptors and D,L‐2‐amino‐5‐phosphonopentanoic acid (APV, 50 to 100 µM) for NMDA receptors or CNQX, APV, and tetrodotoxin (TTX, 0.5 to 1 µM)
  • Electrophysiological setup:
    • Axopatch 200B and Digidata 1200B interface (Axon Instrument); or a comparable circuit, microcomputer system installed with pClamp 8.x software and MiniAnalysis program 5.2.8 (Synaptosoft) or similar software for data analyses
    • Two hydraulic or step‐driven control micromanipulators such as the Burleigh 5000‐150 series manipulators (Burleigh Instruments)
    • Grass S88 stimulator and SIU5 stimulus isolation unit (Grass)
  • Platinum frame with attached nylon mesh
  • Microscope setup: Nikon E600FN (Zeiss Axioskop 2 FS or Olympus BX50WI) upright microscope equipped with Nomarski optics (at least 40× water‐immersion objective, 2 mm long working distance), infrared CCD video camera system, and a black‐and‐white monitor (Sony). Ideally, mount the microscope on a fixed and stable platform, e.g., Gibraltar platform with an X‐Y stage base, and mount all manipulators and chamber on the same platform.
  • Recording chamber (submersion chamber, e.g., an RC26 assembled with an SS‐3 slice support, Warner Instruments)
  • Ag/AgCl ground electrode (e.g., REF‐1L reference cell or E206 Ag/AgCl pellet electrode, Warner Instruments)
  • Pipet filler: MicroFil MF28G needle (WPI) or home‐made pipet filler pulled from a 1‐ml plastic syringe
  • Pipets coated with Sylgard 184 for whole‐cell recording (1.5‐µm o.d., 0.75‐µm i.d. glass pipets) for Purkinje cells or (1.5‐µm o.d., 1.0‐µm i.d. glass pipets) for granule cells (see Critical Parameters)
  • 10‐ml syringes
  • Stimulating electrode: a concentric bipolar metal electrode or monopolar tungsten electrode (3 MΩ, FHC; alternatively, a broken tip glass pipet filled with recipeACSF can be used as the stimulation electrode)

Support Protocol 1: Preparation of Acutely Isolated Hippocampal Slices

  Materials
  • 95% O 2/5% CO 2–saturated slicing solution (see recipe), 4°C
  • 95% O 2/5% CO 2 gas mixture cylinder
  • ACSF (see recipe)
  • Rubber cement
  • 10‐ to 30‐day‐old rats (either gender; Sprague‐Dawley or Charles River)
  • 500‐ml glass beaker
  • Whatman no. 1 or no. 2 filter paper
  • Tissue chopper
  • Single‐edged and high‐quality double‐edged razor blades
  • Rat guillotine
  • Large surgical scissors
  • Small sharp dissecting scissors
  • Rongeurs
  • 50‐ or 75‐mm plastic petri dishes
  • Blunt plastic knives (remove sharp edges and smooth with fine sandpaper)
  • Fine, soft artist's paintbrush
  • Home‐made slice holding chamber (Fig. )

Support Protocol 2: Preparation of Acutely Isolated Cerebellar Slices

  Materials
  • 95% O 2/5% CO 2–saturated slicing solution (see recipe), 4°C
  • 95% O 2/5% CO 2 gas mixture cylinder
  • ACSF (see recipe)
  • 5% agar gel block or Sylgard 184 block (see recipe)
  • Cyanoacrylate glue (super‐glue)
  • 10‐ to 20‐day‐old rats (either gender; Sprague‐Dawley or Charles River)
  • 500‐ml beaker
  • Single‐edged and high‐quality double‐edged razor blades
  • Automatic oscillating tissue slicer (e.g., OTS‐3000‐05, FHC) or equivalent
  • Rat guillotine
  • Large surgical scissors
  • Small, sharp dissecting scissors
  • Rongeurs
  • Whatman no. 1 or no. 2 filter papers
  • 50‐ or 75‐mm plastic petri dishes
  • No. 5 fine‐tip tweezers
  • Wide‐bore Pasteur pipet
  • Home‐made slice‐holding chamber (Fig. )
  • 35° to 37°C water bath
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Figures

Videos

Literature Cited

   Blanton, M.G., Lo Turco, J.J., and Kriegstein, A.R. 1989. Whole‐cell recording from neurons in slices of reptilian and mammalian cerebral cortex. J. Neurosci. Meth. 30:203‐210.
   Blitzer, R.D. and Landau, E.M. 1994. Whole‐cell patch recording in brain slices. Meth. Enzymol. 238:375‐384.
   Edwards, F.A., Konnerth, A., Sakmann, B., and Takahashi, T. 1989. A thin slice preparation for patch recordings from neurons of the mammalian central nervous system. Pfl¨gers Arch. Eur. J. Physiol. 414:600‐612.
   Egert, U., Heck, D., and Aertsen, A. 2002. Two‐dimensional monitoring of spiking networks in acute brain slices. Exp. Brain Res. 142:268‐274.
   Finkel, A. and Bookman, R. 1997. The electrophysiology setup. In Current Protocols in Neuroscience (J.N. Crawley, C.R. Gerfen, R. Mckay, M.A. Rogawski, D.R. Sibley, P. Skolnick, and S. Wray, eds.) pp. 6.1.1‐6.1.6. John Wiley & Sons. New York.
   Hendersen, G. 1993. Pharmacological analysis of synaptic transmission in brain slices. In Electrophysiology (D.I. Wallis, ed.) pp. 89‐107, Oxford, New York.
   Johnston, D. and Brown, T.H. 1984. Biophysics and microphysiology of synaptic transmission in hippocampus. In Brain Slice (R. Dingeldine, ed.) pp. 51‐86. Plenum Press, New York.
   Konnerth, A., Llano, I., and Armstrong, C.M. 1990. Synaptic currents in cerebellar Purkinje cells. Proc. Natl. Acad. Sci. U.S.A. 87:2662‐2665.
   Langmoen, I.A. and Andersen, P. 1981. The hippocampal slice in vitro. A description of the technique and some examples of the opportunities it offers. In Electrophysiology of Isolated Mammalian CNS Preparations (J. Kerkut and H. Wheal, eds.) pp. 15‐50. Academic Press, London.
   Oka, H., Shimono, K., Ogawa, R., Sugihara, H., and Taketani, M. 1999. A new planar multielectrode array for extracellular recording: Application to hippocampal acute slice. J. Neurosci. Meth. 93:61‐67.
   Penner, R. 1995. A practical guide to patch clamping. In Single‐Channel Recording (B. Sakmann and E. Neher, eds.) pp. 3‐30. Plenum, New York.
   Plant, T.D., Eilers, J., and Konnerth, A. 1995. Patch‐clamp technique in brain slices. In Neuromethods, Vol. 26: Patch‐Clamp Application and Protocols (A.A. Boulton, G.B. Baker, and W. Walz, eds.) pp. 233‐257. Human Press, Totowa, New Jersey.
   Rae, J.L. and Levis, R.A. 1997. Fabrication of patch pipets. In Current Protocols in Neuroscience (J.N. Crawley, C.R. Gerfen, R. Mckay, M.A. Rogawski, D.R. Sibley, P. Skolnick, and S. Wray, eds.) pp. 6.3.1‐6.3.31. John Wiley & Sons. New York.
   Sakmann, B. and Stuart, G. 1995. Patch‐pipet recordings from the soma, dendrites, and axon of neurons in brain slices. In Single‐Channel Recording (B. Sakmann and E. Neher, eds.) pp. 199‐211. Plenum, New York.
   Schwartzkroin, P.A. 1981. To slice or not to slice. In Electrophysiology of Isolated Mammalian CNS Preparations (J. Kerkut and H. Wheal eds.) pp. 15‐50. Academic Press, London.
   Stoppini, L., Duport, S., and Corrèges, P. 1997. A new extracellular multirecording system for electrophysiological studies: Application to hippocampal organotypic cultures. J. Neurosci. Meth. 72:23‐33.
   Stuart, G., Dolt, H.‐U., and Sakmann, B. 1993. Patch‐clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. Pflügers Arch. Eur. J. Physiol. 423:511‐518.
   Yamamoto, C. and McIlwain, H. 1966. Electrical activities in thin sections from mammalian brain maintained in chemically‐defined media in vitro. J. Neurochem. 13:1333‐1343.
Key References
   The Axon Guide for Electrophysiology and Biophysics Laboratory Techniques. Axon Instruments. Union City, California.
  A practical laboratory guide covering a broad range of topics, from the biological basis of bioelectricity and a description of the basic experimental setup, to the principles of operation of the most advanced hardware and software currently available.
   Langmoen et al., 1981. See above.
  An early and comprehensive description of the preparation of extracellular and intracellular recording techniques in hippocampal slices.
   Stuart et al., 1993. See above.
  This is the original paper describing the blow‐and‐seal technique for making whole‐cell patch recording from a single cell in thin brain slices.
Internet Resources
  http://axon.com
  A Web site for downloading a free copy of The Axon Guide for Electrophysiology & Biophysics: Laboratory Techniques and the AxoBit Newsletter describing some specific methods and frequently asked questions.
  http://www.ltp-program.com
  A Web site for downloading a free copy of the LTP2.22A program and manual. This is useful if considering studies of long‐term potentiation (LTP) in the experimental design.
  http://www.synaptosoft.com
  A Web site for downloading a MiniAnalysis program demo.
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