Rapid Solution Application Methods

Cha‐Min Tang1

1 University of Maryland School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.9
DOI:  10.1002/0471142301.ns0100s05
Online Posting Date:  May, 2001
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Abstract

Solution changes to deliver solutes of different compositions are required in virtually every cellular electrophysiological experiment. Also, in many neurobiology experiments, it is necessary to make rapid step changes in the concentration of a test compound in order to outpace receptor desensitization or to mimic the brief lifetime of a fast synaptic response. The goal of this unit is to aid the investigator in choosing the rapid solution application method that is most appropriate for the experimental situation and to describe the methods for fabricating two relatively simple devices for making rapid changes between different solutions.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: A Concentration Stepper for Whole‐Cell Recording Using a Theta Glass Perfusion Tip
  • Basic Protocol 2: A Concentration Clamp for Excised Membrane Patches Using Multipartitioned Glass
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: A Concentration Stepper for Whole‐Cell Recording Using a Theta Glass Perfusion Tip

  Materials
  • Sylgard (Dow Corning silicone Elastomer 184; World Precision Instruments)
  • 5‐min epoxy (Devcon Plexus)
  • Test solutions
  • Target cells of interest
  • Dye (e.g., methylene blue, phenol red)
  • Thick‐walled theta glass tubing with a single thin septum (R & D Scientific Glass)
  • Electrode puller for thick glass
  • Diamond‐tipped glass cutter
  • Nonmetallic polishing surface
  • Dissecting microscope
  • Polyimide‐coated fused silica tubing (J & W Scientific or Polymicro Technologies): 0.70‐mm o.d./0.53‐mm i.d and 0.43‐mm o.d./0.32‐mm i.d.
  • Oven with 50° to 80°C capacity
  • Thick‐walled borosilicate electrode glass tubing, 1.5‐mm o.d. (World Precision Instruments)
  • Solenoid valves (LFAA1201718H; The Lee Company)
  • Tygon tubing, 0.09‐in. (2.3‐mm) o.d., 0.05‐in. (1.3‐mm) i.d. (VWR)
  • Blunt 18‐G needles attached to 10‐ml syringes with the plungers removed
  • Toggle switches
  • 12‐volt lantern battery
  • Pulse generator (optional)
  • Three‐axis micromanipulator

Basic Protocol 2: A Concentration Clamp for Excised Membrane Patches Using Multipartitioned Glass

  Materials
  • Hydrofluoric acid (HF; Aldrich)
  • UV‐cured liquid adhesive, e.g., Crystal Clear (Loctite) or NOA81 (Thorlabs)
  • 5‐min epoxy (Devcon Plexus)
  • Test solutions
  • Target cells of interest
  • Multipartitioned glass theta tubing (R & D Scientific Glass)
  • Diamond‐tipped glass cutter
  • Polyimide‐coated fused silica tubing (J & W Scientific or Polymicro Technologies): 0.20‐mm o.d./0.10‐mm i.d. and 0.43‐mm o.d./0.32‐mm i.d.
  • Thick‐walled borosilicate electrode glass tubing, 1.5‐mm o.d. (World Precision Instruments)
  • Strong UV light source, e.g., mercury lamp from a fluorescent microscope
  • Thin metal bolts and screws
  • Flat piece of Plexiglas (2 to 3 mm thick, ≈25 × 50–mm, depending on size of piezoelectric translator)
  • Piezoelectric translator (Burleigh Instruments, PZS‐200; for performance criteria, see Critical Parameters)
  • Low‐voltage piezoelectric driver (Burleigh Instruments, PZ‐100, or Physik Instrumente)
  • Computer interface with software (e.g., PClamp, Axon Instruments)
  • Tygon tubing, 0.09‐in. (2.3‐mm) o.d, 0.05‐in. (1.3‐mm) i.d. (VWR)
  • Pneumatic pressure system (Medical Systems, PPS‐2 and PTM‐2)
  • Additional reagents and equipment for fabricating connectors (see protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
   Boll, W. and Lux, H.D. 1985. Action of organic antagonists on neuronal calcium currents. Neurosci. Lett. 56:335‐339.
   Brett, R.S., Dilger, J.P., Adams, P.R., and Lancaster, B. 1986. A method for the rapid exchange of solutions bathing excised membrane patches. Biophys. J. 50:987‐992.
   Clements, J. 1993. Rapid solution exchange using a piezoelectric translator. AxoBits (a publication of Axon Instruments) 13:9‐11.
   Fenwick, E.M., Marty, A., and Neher, E. 1982. A patch‐clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine. J. Physiol. 331:577‐597.
   Franke, C., Hatt, H., and Dudel, J. 1987. Liquid filament switch for ultra‐fast exchanges of solutions at excised patches of synaptic membrane of crayfish muscle. Neurosci. Lett. 77:199‐204.
   Johnson, J.W. and Ascher, P. 1987. Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325:529‐531.
   Jonas, P. 1995. Fast application of agonists to isolated membrane patches. In Single‐Channel Recording (B. Sakmann and E. Neher, eds.) pp. 231‐243. Plenum, New York.
   Jones, K.A. and Baughman, R.W. 1991. Both NMDA and non‐NMDA subtypes of glutamate receptors are concentrated at synapases in cerebral cortical neurons in culture. Neuron 7:593‐603.
   Krishtal, O.A. and Pidoplichko, V.I. 1980. A receptor for protons in the nerve cell membrane. Neuroscience 5:2325‐2327.
   The Lee Company. 1994. The Lee Company Electro‐Fluidic Systems Handbook, 6th ed., p. 215. The Lee Company, Westbrook, Conn.
   Maconochie, D.J. and Knight, D.E. 1989. A method for making solution changes in the submillisecond range at the tip of a patch pipet. Pflügers Arch. 414:589‐596.
   Tang, C.‐M., Dichter, M., and Morad, M. 1989. Quisqualate activates a rapidly inactivating high conductance ionic channel in hippocampal neurons. Science 243:1474‐1477.
   Tong, G. and Jahr, C.E. 1994. Multivesicular release from excitatory synapses of cultured hippocampal neurons. Neuron 12:51‐59.
   Vyklicky, L. Jr., Benveniste, M., and Mayer, M.L. 1990. Modulation of N‐methyl‐D‐aspartic acid receptor desensitization by glycine in cultured mouse hippocampal neurons. J. Physiol. 428:313‐331.
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