Microdialysis in Rodents

Agustin Zapata1, Vladimir I. Chefer1, Toni S. Shippenberg1

1 Integrative Neuroscience Section, NIH/NIDA Intramural Research Program, Baltimore, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.2
DOI:  10.1002/0471142301.ns0702s47
Online Posting Date:  April, 2009
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Microdialysis is an in vivo sampling technique that permits the quantification of various substances (e.g., neurotransmitters, peptides, electrolytes) in blood and tissue. It is also used to infuse substances into the brain and spinal cord. This unit describes methods for the construction and stereotaxic implantation of microdialysis probes into discrete brain regions of the rat and mouse. Procedures for the conduct of conventional and quantitative microdialysis experiments in the awake and anesthetized rodent are also provided. Curr. Protoc. Neurosci. 47:7.2.1‐7.2.29. © 2009 by John Wiley & Sons, Inc.

Keywords: microdialysis probe construction; stereotaxic surgery; liquid swivel; dialysate sample; concentric dialysis probe

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Dialysis Probe/Guide Cannula Assembly: Construction of a Concentric Probe (Design 1)
  • Alternate Protocol 1: Construction of a Side‐by‐Side Probe (Design 2)
  • Alternate Protocol 2: Construction of a Horizontal Probe
  • Basic Protocol 2: Implantation and Tethering of Dialysis Probe/Guide Cannula Assembly in the Rat: Implantation of Concentric or Loop Probe/Guide Assemblies
  • Alternate Protocol 3: Implantation of a Horizontal Probe
  • Basic Protocol 3: Surgical Implantation of Dialysis Probe/Guide Cannula Assembly in the Mouse
  • Basic Protocol 4: Microdialysis In Vitro
  • Basic Protocol 5: In Vivo Microdialysis
  • Support Protocol 1: In Vivo Determination of Extracellular Concentration
  • Support Protocol 2: Determining Extracellular Concentration (Cext)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of Dialysis Probe/Guide Cannula Assembly: Construction of a Concentric Probe (Design 1)

  Materials
  • Epoxy, rapid drying
  • 70% ethanol
  • 36‐G stainless steel tubing
  • Razor blades
  • Jeweler's pliers
  • Fine forceps
  • Dremel tool (optional; Small Parts)
  • Dissecting microscope
  • Sandpaper (optional)
  • Dialysis fiber (regenerated cellulose hollow fiber; Fisher)
  • Stainless steel tubing or pin small enough to fit inside dialysis fiber
  • Microbore tubing to fit over 28‐G internal guide cannula (Plastics One)
  • 28‐G internal cannula (Plastics One)
  • Sonicator
  • 40°C oven
  • Vernier calipers
  • 29‐G stainless steel tubing (optional)
  • Additional reagents and equipment for perfusing probes in vitro (see protocol 7)

Alternate Protocol 1: Construction of a Side‐by‐Side Probe (Design 2)

  • Polyimide resin
  • Superglue
  • Push‐pull perfusion connector to fit 26‐G guide cannula (Plastics One)
  • 26‐G guide cannula, concave
  • 24‐G Silastic tubing
  • Fused silica tubing (100‐µm o.d., 0.40‐µm i.d.; Polymicro Technologies)
  • Teflon tubing
  • Tape
  • Plastic syringe caps
  • 26‐G needle
  • Needle with plastic hub
  • Additional reagents and equipment for perfusing probes in vitro (see protocol 7)

Alternate Protocol 2: Construction of a Horizontal Probe

  • Dialysis fiber (340‐µm o.d.; Amicon)
  • Tungsten wire (125‐µm diameter, prestraightened)
  • Stainless steel tube (0.65‐mm o.d.; Small Parts)
  • Waterproof pen
  • Stereotaxic atlas
  • Additional reagents and equipment for perfusing probes in vitro (see protocol 7)

Basic Protocol 2: Implantation and Tethering of Dialysis Probe/Guide Cannula Assembly in the Rat: Implantation of Concentric or Loop Probe/Guide Assemblies

  Materials
  • Rat
  • Sodium pentobarbital
  • Atropine sulfate
  • Betadine disinfectant (povidone‐iodine)
  • 75% ethanol
  • Hydrogen peroxide (optional)
  • Dental acrylic (e.g., Plastics One)
  • Stereotaxic atlas for rat
  • Stereotaxic frame equipped with carrier for guide cannula or dialysis probe/guide assembly (David Kopf Instruments, Stoelting, or Harvard Instruments)
  • Shaver
  • Guide cannula assembly (e.g., David Kopf Instruments, Stoelting, or Harvard Instruments)/dialysis probe (see protocol 1 or protocol 2)
  • Scalpel
  • Serrefine forceps or hemostats
  • Q‐tips or cotton swabs
  • Spatula
  • Dental or hand drill
  • Jeweler's forceps and screwdriver
  • 3/16‐in. bone screws, 0 to 80 stainless steel (Small Parts, Plastics One)
  • Heating blanket and thermometer
  • Liquid swivel and tether (see )
NOTE: All surgical instruments must be sterile and aseptic procedures should be used unless otherwise specified.

Alternate Protocol 3: Implantation of a Horizontal Probe

  • Saline
  • 1× artificial cerebrospinal fluid (aCSF; see recipe) or Ringer's solution
  • Epoxy
  • Electrode carriers, modified to position probe in lateral axis
  • Horizontal dialysis probe (see protocol 3)
  • Stereotaxic ear bars (David Kopf Instruments), modified to raise position of the head in the apparatus
  • Sutures
  • Gauze
  • 2.5‐mm drill bit
  • 22‐G stainless steel tubing (cannula) and removable plastic caps (e.g., Plastics One)
  • Polyethylene (PE) tubing to fit 22‐G tubing
  • Additional reagents and equipment for perfusing probes in vitro (see protocol 7)
NOTE: All surgical instruments must be sterile and procedures are to be performed in a sterile manner unless otherwise specified.

Basic Protocol 3: Surgical Implantation of Dialysis Probe/Guide Cannula Assembly in the Mouse

  Materials
  • Anesthetic: urethane (nonsurvival), sodium pentobarbital (survival)
  • Bonding resin (dental acrylic, Denmat)
  • Shaver
  • Stereotaxic frame (David Kopf Instruments, Stoelting) adapted for use with small rodents (e.g., Kopf mouse adapter set)
  • Stage to elevate body of mouse to level of ear bars (e.g., inverted pipet‐tip box)
  • Blunt forceps
  • Instruments for small animal surgery
  • Dental or hand (Dremel) drill
  • Miniature pin vice
  • Guide cannula (CMA Microdialysis, Plastics One)/probe assembly
  • Stereotaxic atlas for mouse (e.g., Slotnick and Leonard, ; Paxinos and Franklin, )
  • Electrode carrier (David Kopf Instruments, Stoelting)
  • Slotted peg for tether attachment
  • Additional reagents and equipment for probe implantation in the rat (see protocol 4)
NOTE: All surgical instruments must be sterile and procedures are to be performed in an aseptic manner unless otherwise specified.

Basic Protocol 4: Microdialysis In Vitro

  Materials
  • Analyte of interest (e.g., neurotransmitter, peptide or drug; usually in powder form)
  • Perfusate, e.g., 1× artificial cerebrospinal fluid (aCSF; see recipe) or Ringer's solution (see unit 7.1 for discussion of perfusates)
  • 37°C platform shaking incubator
  • Microsyringe pump and glass Luer‐Lock gas‐tight microsyringes (Hamilton)
  • Microdialysis probe (see protocol 1 and Alternate Protocols protocol 21 and protocol 32)
  • Dissecting microscope
  • Collection vials (e.g., 0.2‐ml microcentrifuge tubes)

Basic Protocol 5: In Vivo Microdialysis

  Materials
  • Perfusate: e.g., 1× artificial cerebrospinal fluid (aCSF; see recipe)
  • Rat or mouse with implanted dummy cannula (see protocol 4 or protocol 63)
  • Large flasks
  • Microsyringe pump and glass Luer‐Lock gas‐tight microsyringes (Hamilton)
  • Liquid swivel (see )
  • Microdialysis probe (see protocol 1 or protocol 2)
  • Collection vials
  • Testing cage
  • Dissecting microscope
NOTE: All surgical instruments must be sterile and procedures are to be performed in an aseptic manner unless otherwise specified.

Support Protocol 1: In Vivo Determination of Extracellular Concentration

  Materials
  • Perfusate, e.g., 1× artificial cerebrospinal fluid (aCSF; see recipe) or Ringer's solution
  • Analyte of interest
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Adell, A. and Artigas, F. 1998. A microdialysis study of the in vivo release of 5‐HT in the median raphe nucleus of the rat. Br. J. Pharmacol. 125:1361‐1367.
   Benveniste, H. and Huttemeier, P.C. 1990. Microdialysis: Theory and application. Prog. Neurobiol. 35:195‐215.
   Blair, R., Fishman, B., Amit, Z., and Weeks, J.R. 1980. A simple double channel swivel for infusions of fluids into unrestrained animals. Pharmacol. Biochem. Behav. 12:463‐466.
   Chefer, V.I., Zakharova, I., and Shippenberg, T.S. 2003. Enhanced responsiveness to novelty and cocaine is associated with decreased basal dopamine uptake and release in the nucleus accumbens: Quantitative microdialysis in rats under transient conditions. J. Neurosci. 23:3076‐3084.
   Chefer, V.I., Zapata, A., Shippenberg, T.S., and Bungay, P. 2006. Quantitative no‐net‐flux microdialysis permits detection of increases and decreases in dopamine uptake in mouse nucleus accumbens J. Neurosci. Meth. 155:187‐193.
   Harte, M. and O'Connor, W.T. 2005. Evidence for a selective prefrontal cortical GABA(B) receptor‐mediated inhibition of glutamate release in the ventral tegmental area: A dual probe microdialysis study in the awake rat. Neuroscience 130:215‐222.
   Hsiao, J.K., Ball, B.A., Morrison, P.F., Mefford, I.N., and Bungay, P. 1990. Effects of different semipermeable membranes on in vitro and in vivo performance of microdialysis probes. J. Neurochem. 54:1449‐1452.
   Imperato, A. and Di Chiara, G. 1984. Trans‐striatal dialysis coupled to reverse‐phase high performance liquid chromatography with electrochemical detection: A new method for study of the in vivo release of endogenous dopamine and metabolites. J. Pharmacol. Exp. Ther. 4:966‐977.
   Jacobson, I., Sandberg, M., and Hamberger, A. 1985. Mass transfer in brain dialysis devices—A new method for the estimation of extracellular amino acids concentration. J. Neurosci. Meth. 15:263‐268.
   Kendrick, K.M. 1990. Microdialysis measurement of in vivo neuropeptide release. J. Neurosci. Meth. 34:35‐46.
   Konig, J.F. and Klippel, R.A. 1963. The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Williams and Wilkins, Baltimore.
   Lonnroth, P., Jansson, P.A., and Smith, U. 1987. A microdialysis method allowing characterization of intercellular water space in humans. Am. J. Physiol. 253:E228‐E231.
   Lonnroth, P., Jansson, P.A., Fredholm, B.B., and Smith, U. 1989. Microdialysis of intercellular adenosine concentration in subcutaneous tissue in humans. Am. J. Physiol. 256:E250‐E255.
   Maidment, N.T., Brumbaugh, D.R., Rudolph, V.D., Erdelyi, E., and Evans, C.J. 1989. Microdialysis of extracellular endogenous opioid peptides from rat brain in vivo. Neuroscience 33:549‐557.
   Matochik, J.A., Sipos, M.L., Nyby, J.G., and Barfield, R.J. 1994. Intracranial androgenic activation of male‐typical behaviors in house mice: Motivation vs. performance. Behav. Brain Res. 60:141‐149.
   Olson, R.J. and Justice, J.B. Jr. 1993. Quantitative microdialysis under transient conditions. Anal. Chem. 65:1017‐1025.
   Parsons, L.H. and Justice, J.B. Jr. 1994. Quantitative approaches to in vivo brain microdialysis. Crit. Rev. Neurobiol. 8:189‐220.
   Paxinos, G. and Watson, C. 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego.
   Paxinos, G. and Franklin, K.B.J. 2001. The Mouse Brain in Stereotaxic Coordinates. Academic Press, San Diego.
   Pellegrino, L. and Cushman, A.J. 1979. A Stereotaxic Atlas of the Rat Brain. Plenum, New York.
   Pettit, H.O. and Justice, J.B. 1991. Procedures for microdialysis with smallbore HPLC. In Microdialysis in the Neurosciences (T.E. Robinson and J.B. Justice, Jr., eds.) pp. 117‐154. Elsevier Science Publishing, New York.
   Robinson, T.E. and Whishaw, I.Q. 1988. Normalization of extracellular dopamine in striatum following recovery from a partial unilateral 6‐OHDA lesion of the substantia nigra: A microdialysis study in a freely moving rat. Brain Res. 450:209‐224.
   Slotnick, B.M. and Leonard, C.M. 1975. A Stereotaxic Atlas of the Albino Mouse Forebrain. DHEW Publication (ADM) 75‐100, U.S. Government Printing Office, Rockville, Maryland.
   Snider, R.S. and Lee, J.C. 1961. A Stereotaxic Atlas of the Monkey Brain (Macaca mulatta). University of Chicago Press, Toronto.
   Thompson, A.C., Justice, J.B. Jr., and McDonald, J.K. 1995. Quantitative microdialysis of neuropeptide Y. J. Neurosci. Meth. 60:189‐198.
   Thompson, A.C., Matochik, J.A., Acri, J.B., Pani, A.K., and Shippenberg, T.S. 1996. Characterization of striatal dopamine in the mouse using conventional and quantitative microdialysis methods. In Seventh International Conference on In Vivo Methods (J.L. Gonzalez‐Mora, R. Berges, and M. Mas, eds.) p. 21. University of Laguja, Santa Cruz de Tenerife, Spain.
   Ungerstedt, U., Herrera‐Marchintz, M., Jungnelius, U., Stahle, L., Tossman, U., and Zetterstrom, T. 1982. Dopamine synaptic mechanisms reflected in studies combining behavioral recordings and brain dialysis. In Advances in Dopamine Research (M. Kotisaka, ed.) pp. 219‐231. Pergamon Press, Elmsford, N.Y.
   Wages, S.A., Church, W.H., and Justice, J.B. 1986. Sampling considerations for on‐line microbore liquid chromatography of brain dialysis. Anal. Chem. 58:1649‐1656.
   Zapata, A., Capdevila, J.L., and Trollas, R. 1998. Region‐specific and calcium‐dependent increase in dialysate choline levels by NMDA. J. Neurosci. 18:3597‐3605.
   Zetterstrom, T., Sharp, T., Marsden, C.A., and Ungerstedt, U. 1984. Effects of neuroleptic drugs on striatal dopamine release and metabolism in the awake rat: Studies by intracerebral dialysis. Eur. J. Pharmacol. 106:27‐37.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library