Electrophysiological Recordings from Neonatal Neocortical Brain Slices

Michael Daw1, John Isaac2

1 University of Bristol, Bristol, 2 NINDS/NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.23
DOI:  10.1002/0471142301.ns0623s38
Online Posting Date:  January, 2007
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Abstract

Many brain processes and the expression of many neuronal proteins are developmentally regulated; therefore, studying cellular and molecular processes in tissue from very young animals is vital to understanding the development of the brain. Working on tissue from very young animals (first postnatal week) presents a number of specific problems. This unit describes techniques to overcome these problems to produce in vitro slices from neonatal rodents and to reliably achieve high‐quality patch‐clamp recordings from neurons in neonatal brain slices.

Keywords: patch clamp; neonatal; cortex; thalamocortical; rat; mouse; development

     
 
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Table of Contents

  • Basic Protocol 1: Extracellular Field Recordings
  • Support Protocol 1: Neonatal Brain Slice Preparation
  • Alternate Protocol 1: Whole‐Cell Recording
  • Alternate Protocol 2: “Blind” Patch Recording Using Low Magnification
  • Alternate Protocol 3: Perforated Patch‐Clamp Recordings Using Antibiotics
  • Reagents and Solutions
  • Commentary
  • Key References
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Extracellular Field Recordings

  Materials
  • Neocortical slices prepared in high Mg2+ aCSF (see protocol 2)
  • Standard artificial cerebrospinal fluid (aCSF; see recipe)
  • 4 M NaCl, optional
  • Recording chamber
  • Perfusion system including a peristaltic pump and a vacuum pump
  • Temperature controller with in‐chamber sensor (e.g., NPI)
  • Metal grid for securing brain slice
  • Bipolar stimulating electrodes (unit 6.13)
  • Recording pipets (filamented borosilicate glass capillaries; unit 6.3)
  • Patch‐clamp amplifier with headstage(s) and electrode holder(s)
  • IR DIC microscope with low (4×) and high (40× submerged) power objective lenses, CCD camera, and monitor
  • Micromanipulators (e.g., Burleigh, Luigs and Neumann)
  • Stimulus isolation unit (e.g., digitimer)
  • Additional reagents and equipment for setting up electrophysiology equipment (unit 6.1), constructing recording pipets (unit 6.3), and recording and analyzing synaptic currents and potentials (unit 6.10)

Support Protocol 1: Neonatal Brain Slice Preparation

  Materials
  • Laboratory neonatal animal (e.g., Wistar or Sprague‐Dawley rats; CD1 or C57 mice)
  • Anesthetic (e.g., pentobarbitone)
  • High‐Mg2+ aCSF (see recipe), chilled and bubbled with 95% O 2/5% CO 2
  • Cyanoacrylate adhesive
  • Syringe and small‐gauge (e.g., 25‐G) hypodermic needle
  • Surgical instruments for decapitation and dissection:
    • Large scissors
    • Scalpel
    • Small, pointed scissors
    • Watchmaker's tweezers
    • Spatula
  • Filter paper
  • Vibrating microslicer (e.g., DSK, Leica, Vibratome)
  • Modified Pasteur pipet (see annotation to step )
  • Dissecting microscope

Alternate Protocol 1: Whole‐Cell Recording

  • Whole‐cell solution (see recipe)
  • Antivibration table (e.g., Newport)
  • 0.2‐µm filter

Alternate Protocol 2: “Blind” Patch Recording Using Low Magnification

  • Manometer consisting of two vertical measuring pipets

Alternate Protocol 3: Perforated Patch‐Clamp Recordings Using Antibiotics

  • 20 mg/ml gramicidin A in DMSO
  • Whole‐cell solution (see recipe)
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Figures

Videos

Literature Cited

Key References
   Akaike, N. and Harata, N. 1994. Nystatin perforated patch recording and its applications to analyses of intracellular mechanisms. Jpn. J. Physiol. 44:433‐473.
  The first paper describing the use of gramicidin A for perforated patch recording, and also includes a comparison of the effectiveness of gramicidin A, amphotericin B and nystatin.
   Falke, L.C., Gillis, K.D., Pressel, D.M., and Misler, S. 1989. ‘Perforated patch recording’ allows long‐term monitoring of metabolite‐induced electrical activity and voltage‐dependent Ca2+ currents in pancreatic islet B cells. FEBS Lett. 251:167‐172.
  One of the first papers describing the perforated patch technique.
   Kyrozis, A. and Reichling, D.B. 1995. Perforated‐patch recording with gramicidin avoids artifactual changes in intracellular chloride concentration. J. Neurosci. Methods 57:27‐35.
  This paper confirms the assumption that gramicidin A perforated patch recording preserves endogenous intracellular Cl− concentration.
   Rae, J., Cooper, K., Gates, P., and Watsky, M. 1991. Low access resistance perforated patch recordings using amphotericin B. J. Neurosci. Methods 37:15‐26.
  The first paper describing the use of amphotericin B for perforated patch.
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